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| Biotechnol Lett, 2003 Mar, 25(5), 409 - 12 An extracellular Bacillus sp . chitinase for the production of chitotriose as a major chitinolytic product; Woo CJ et al.; An extracellular chitinase of Bacillus sp . WY22 was purified by 9.6-fold . It had a Mr of 35 kDa, an apparent Km value for colloidal chitin of 3 mg ml(-1) and was optimally active at 37 degrees C and pH 5.5 over 1 h . The enzyme could also hydrolyse swollen chitin, glycol chitin and chitosan with relative activities of 76%, 34% and 23% compared with colloidal chitin . It formed chitotriose as a major product from colloidal chitin and glycol chitin. Biotechnol Lett, 2003 Jan, 25(2), 155 - 9 Cloning and characterization of an exoinulinase from Bacillus polymyxa; Kwon HJ et al.; A gene encoding an exoinulinase (inu) from Bacillus polymyxa MGL21 was cloned and sequenced . It is composed of 1455 nucleotides, encoding a protein (485 amino acids) with a molecular mass of 55,522 Da . Inu was expressed in Escherichia coli and the His-tagged exoinulinase was purified . The purified enzyme hydrolyzed sucrose, levan and raffinose, in addition to inulin, with a sucrose/inulin ratio of 2 . Inulinase activity was optimal at 35 degrees C and pH 7, was completely inactivated by 1 mM Ag+ or Hg2+ . The Km and Vmax values for inulin hydrolysis were 0.7 mM and 2500 microM min(-1) mg(-1) protein . The enzyme acted on inulin via an exo-attack to produce fructose mainly. Folia Microbiol (Praha), 2003, 48(3), 427 - 34 Role of T cells in the adjuvant effect of Bacillus firmus on the immune system of mice: intranasal and intratracheal immunization study with ovalbumin; Mlckova P et al.; Functions of T cells were determined after intranasal and intratracheal immunization of mice with ovalbumin (Ova) and Bacillus firmus (Bf), a Gram-positive nonpathogenic bacterium of the external environment, or delipidated Bf (dBf) as adjuvants, with the aim to elucidate the mechanism of support of Ova-specific antibody production caused by Bf that had been observed in an identical experiment . Neither Bf nor dBf in a mixture with Ova stimulated Ova-specific T-cell response tested as antigen-specific blast transformation . By contrast, a mild polyclonal stimulation was observed in splenocytes from mice given dBf . In vitro incubation of splenocytes with 100 micrograms (but not 10 micrograms) of Bf or dBf led to a highly significant inhibition of proliferation below the control level in all groups of animals . Supernatants of splenocyte cultures were further tested for cytokine production . IL-10 and IFN-gamma were released after in vitro challenge with dBf and in some cases also with Bf . Analysis of sera demonstrated that administration of Ova + adjuvant brought about an increase in anti-Ova IgG1, IgG2a and IgG2b whereas treatment with Ova alone caused a rise in IgG1 only . The role of Bf or dBf in the enhancement of antigen-specific antibody production could be in influencing macrophages and inducing cytokine milieu composed of IL-10, IFN-gamma and other factors that leads to a bystander stimulation of specifically activated Ova-B cell receptor (Ova-BCR)-bearing cells. Adv Appl Microbiol, 2000, 47, 157 - 99 Advances in phytase research; Mullaney EJ et al.; Since its discovery in 1907, a complex of technological developments has created a potential $500 million market for phytase as an animal feed additive . During the last 30 years, research has led to increased use of soybean meal and other plant material as protein sources in animal feed . One problem that had to be overcome was the presence of antinutritional factors, including phytate, in plant meal . Phytate phosphorus is not digested by monogastric animals (e.g., hogs and poultry), and in order to supply enough of this nutrient, additional phosphate was required in the feed ration . Rock phosphate soon proved to be a cost-effective means of supplying this additional phosphorus, and the excess phytin phosphorus could be disposed of easily with the animals' manure . However, this additional phosphorus creates a massive environmental problem when the land's ability to bind it is exceeded . Over the last decade, numerous feed studies have established the efficacy of a fungal phytase, A . niger NRRL 3135, to hydrolyze phytin phosphorus in an animal's digestive tract, which benefits the animal while reducing total phosphorus levels in manure . The gene for phytase has now been cloned and overexpressed to provide a commercial source of phytase . This monomeric enzyme, a type of histidine acid phophatase (HAP), has been characterized and extensively studied . HAPs are also found in other fungi, plants, and animals . Several microbial and plant HAPs are known to have significant phytase activity . A second A . niger phytase (phyB), a tetramer, is known and, like phyA, has had its X-ray crystal structure determined . The model provided by this crystal structure research has provided an enhanced understanding of how these molecules function . In addition to the HAP phytase, several other phytases that lack the unique HAP active site motif RHGXRXP have been studied . The best known group of the non-HAPs is phytase C (phyC) from the genus Bacillus . While a preliminary X-ray crystallographic analysis has been initiated, no enzymatic mechanism has been proposed . Perhaps the pivotal event in the last century that created the need for phytase was the development of modern fertilizers after the Second World War . This fostered a transformation in agriculture and a tremendous increase in feed-grain production . These large quantities of cereals and meal in turn led to the transition of one segment of agriculture into "animal agriculture," with their its animal production capability . The huge volumes of manure spawned by these production units in time exceeded both the capacity of their crops and crop lands to utilize or bind the increased amount of phosphorus . Nutrient runoff from this land has now been linked to a number of blooms of toxin-producing microbes . Fish kills associated with these blooms have attracted public and governmental concern, as well as greater interest in phytase as a means to reduce this phosphorus pollution . Phytase research efforts now are focused on the engineering of an improved enzyme . Improved heat tolerance to allow the enzyme to survive the brief period of elevated temperature during the pelletization process is seen as an essential step to lower its cost in animal feed . Information from the X-ray crystal structure of phytase is also relevant to improving the pH optimum, substrate specificity, and enzyme stability . Several studies on new strategies that involve synergistic interactions between phytase and other hydrolytic enzymes have shown positive results . Further reduction in the production cost of phytase is also being pursued . Several studies have already investigated the use of various yeast expression systems as an alternative to the current production method for phytase using overexpression in filamentous fungi . Expression in plants is underway as a means to commercially produce phytase, as in biofarming in which plants such as alfalfa are used as "bioreactors," and also by developing plant cultivars that would produce enough transgenic phytase so that additional supplementation of their grain or meals is not necessary . Ultimately, transgenic poultry and hogs may produce their own digestive phytase . Another active area of current phytase research is expanding its usage . One area that offers tremendous opportunity is increasing the use of phytase in aquaculture . Research is currently centered on utilizing phytase to allow producers in this industry to switch to lower-cost plant protein in their feed formulations . Development of a phytase for this application could significantly lower production costs . Other areas for expanded use range from the use of phytase as a soil amendment, to its use in a bioreactor to generate specific myo-inositol phosphate species . The transformation of phytase into a peroxidase may lead to another novel use for this enzyme . As attempts are made to widen the use of phytase, it is also important that extended exposure and breathing its dust be avoided as prudent safety measures to avoid possible allergic responses . In expanding the use of phytase, another important consideration has been achieved . Conservation of the world's deposits of rock phosphate is recognized as important for future generations . Phosphorus is a basic component of life like nitrogen, but, unlike nitrogen, phosphorus does not have a cycle to constantly replenish its supply . It is very likely that the use of phytase will expand as the need to conserve the world's phosphate reserves increases. Mar Biotechnol (NY), 2003 Mar-Apr, 5(2), 116 - 8 Isolation of an organic-solvent-tolerant cholesterol-transforming Bacillus species, BC1, from coastal sediment; Sardessai Y et al.; Steroid transformation is of great importance in the pharmaceutical industry . The major limiting factor in this process is the extremely poor solubility of steroids in aqueous media, which lowers their transformation rate and increases costs . This problem can be overcome by using organic-solvent-tolerant bacteria (OSTB), which can carry out the desired bioconversions in an organic-solvent-saturated system . OSTB are a relatively novel group of extremophilic microbes that have developed various adaptations to withstand solvent toxicity . The aim of this study was to isolate marine bacteria producing organic-solvent-stable cholesterol-transforming enzymes . A Bacillus species, BC1, isolated from Arabian Sea sediment was found to degrade cholesterol and exhibit excellent solvent tolerance particularly to chloroform . OSTB have tremendous potential in industrial processes involving nonaqueous biocatalysis and transformation in the presence of an organic phase. Acta Crystallogr D Biol Crystallogr, 2003 Aug, 59(Pt 8), 1472 - 3 Epub 2003 Jul 23. Crystallization and preliminary X-ray diffraction data for the carboxylesterase Est30 from Bacillus stearothermophilus; Liu P et al.; Crystals have been grown of the carboxylesterase Est30 from Bacillus stearothermophilus by hanging-drop vapor diffusion using ammonium sulfate as precipitant . The crystals diffracted to better than 2.0 A resolution . X-ray diffraction data were reduced in space group C222(1), with unit-cell parameters a = 55.83, b = 58.15, c = 179.65 A . R(merge) was 0.038 for 17 449 independent reflections with a completeness of 85.1% . V(M) was calculated to be 2.43 A(3) Da(-1), which suggested that there was one molecule of Est30 in the asymmetric unit . These crystals are suitable for structure determination. Acta Crystallogr D Biol Crystallogr, 2003 Aug, 59(Pt 8), 1414 - 21 Epub 2003 Jul 23. Structure of MrsD, an FAD-binding protein of the HFCD family; Blaesse M et al.; MrsD from Bacillus sp . HIL-Y85/54728 is a member of the HFCD (homo-oligomeric flavin-containing Cys decarboxylases) family of flavoproteins and is involved in the biosynthesis of the lantibiotic mersacidin . It catalyses the oxidative decarboxylation of the C-terminal cysteine residue of the MrsA precursor peptide of mersacidin, yielding a (Z)-enethiol intermediate as the first step in the formation of the unusual amino acid S-{(Z)-2-aminovinyl}-methyl-D-cysteine . Surprisingly, MrsD was found to bind FAD, in contrast to the three other characterized members of the HFCD family, which bind FMN . To determine the molecular discriminators of FAD binding within the HFCD family, the crystal structure of MrsD was analyzed at a resolution of 2.54 A . Crystals of space group F432 contain one MrsD monomer in the asymmetric unit . However, a Patterson search with EpiD-derived models failed . Based on the consideration that the dodecameric MrsD particle of tetrahedral symmetry resembles the quaternary structure of EpiD, rotational and translational parameters were derived from the geometric consideration that the MrsD dodecamer is generated from a monomer by crystallographic symmetry around the position (1/4, 1/4, 1/4) of the unit cell . A structural comparison with the FMN-binding members of the HFCD family EpiD and AtHAL3a shows conserved sequence motifs in contact with the flavin's pyrimidine ring but divergent environments for the dimethylbenzene ring of the isoalloxazine moiety . The position of the ribityl chain differs in MrsD from that found in EpiD and AtHAL3a . However, the FMN-phosphate binding sites are also highly conserved in their exact positions . In all three cases, the flavin cofactor is bound to a structurally conserved region of the Rossmann-fold monomer, exposing its Re side for catalysis . The adenosyl phosphate of FAD is anchored in a well defined binding site and the adenosine moieties are oriented towards the interior of the hollow particle, where three of them pack against each other around the threefold axis of a trimeric facet. Eur Urol, 2003 Aug, 44(2), 222 - 5; discussoion 225 Management and prognosis of transitional cell carcinoma superficial recurrence in muscle-invasive bladder cancer after bladder preservation; Pieras E et al.; PURPOSE: To assess the bladder preservation rate and cancer-specific survival after conservative treatment of superficial relapses in invasive tumors after bladder preservation . MATERIAL AND METHODS: Fifty-one patients with invasive bladder tumor (T2) were treated using transurethral resection (TUR) followed by three cycles of systemic chemotherapy (carboplatin-vinblastine) . After three weeks, an endoscopic reappraisal was made including deep TUR of the site of the original tumor and multiple cold cup biopsies . Forty-two patients retained their bladder (33 complete responses and 9 partial responses) . RESULTS: With a median follow-up of 63 months, 18 patients recurred as superficial TCC tumor (43%) . Fourteen patients with high grade superficial recurrence were treated with TUR and Bacillus Calmette-Guerin (BCG) instillations; two patients (G2-3 T1) with TUR as well as endovesical mytomicine, and two patients with low grade recurrence with only TUR . With a median follow-up of 44 months after TUR of first superficial relapse, there was only one case with progression of the disease without any evidence of bladder tumor . Two cystectomies were made due to carcinoma in situ (Cis) persistence and high grade superficial recurrence . Eighty-three percent of the patients who had superficial recurrence retained their bladders, with 94% cancer-specific survival . CONCLUSIONS: A very strict follow-up is mandatory due to the high rate of superficial relapses (43%) . Cis is the most frequent type of superficial recurrence . Superficial recurrences in bladder preservation may be treated with TUR and BCG instillations when they are high grade and and/or associated with Cis . Superficial recurrences do not imply a worse prognosis for bladder preservation or cancer-specific survival. J Mol Biol, 2003 Aug 1, 331(1), 101 - 21 Evidence of a thermal unfolding dimeric intermediate for the Escherichia coli histone-like HU proteins: thermodynamics and structure; Ramstein J et al.; The Escherichia coli histone-like HU protein pool is composed of three dimeric forms: two homodimers, EcHUalpha(2) and EcHUbeta(2), and a heterodimer, EcHUalphabeta . The relative abundance of these dimeric forms varies during cell growth and in response to environmental changes, suggesting that each dimer plays different physiological roles . Here, differential scanning calorimetry and circular dichroism (CD) were used to study the thermal stability of the three E.coli HU dimers and show that each of them has its own thermodynamic signature . Unlike the other HU proteins studied so far, which melt through a single step (N(2)<-->2D), this present thermodynamic study shows that the three E.coli dimers melt according to a two-step mechanism (N(2)<-->I(2)<-->2D) . The native dimer, N(2), melts partially into a dimeric intermediate, I(2), which in turn yields the unfolded monomers, D . In addition, the crystal structure of the EcHUalpha(2) dimer has been solved . Comparative thermodynamic and structural analysis between EcHUalpha(2) and the HU homodimer from Bacillus stearothermophilus suggests that the E.coli dimer is constituted by two subdomains of different energetic properties . The CD study indicates that the intermediate, I(2), corresponds to an HU dimer having partly lost its alpha-helices . The partially unfolded dimer I(2) is unable to complex with high-affinity, single-stranded break-containing DNA . These structural, thermodynamic and functional results suggest that the N(2)<-->I(2) equilibrium plays a central role in the physiology of E.coli HU . The I(2) molecular species seems to be the EcHUbeta(2) preferential conformation, possibly related to its role in the E.coli cold-shock adaptation . Besides, I(2) might be required in E.coli for the HU chain exchange, which allows the heterodimer formation from homodimers. Infect Immun, 2003 Aug, 71(8), 4647 - 56 Stimulation of neutrophil granulocytes with Mycobacterium bovis bacillus Calmette-Guérin induces changes in phenotype and gene expression and inhibits spontaneous apoptosis; Suttmann H et al.; Polymorphonuclear neutrophil granulocytes (PMN) have been implicated in the early inflammatory response against mycobacteria besides monocytes/macrophages . Yet, little is known about the interaction of mycobacteria with PMN . We investigated the potential of Mycobacterium bovis bacillus Calmette-Guerin (BCG) to stimulate and influence PMN phenotype, gene expression profile and spontaneous apoptosis . Flow cytometric analyses revealed an upregulation of the function-associated molecules Fc gamma receptor III (Fc gamma R III) and II (CD16 and CD32) as well as MAC-1 (CD11b and CD18) on BCG-stimulated PMN . As determined by cDNA microarrays and multiplex reverse transcriptase PCR, stimulation with BCG alters the expression of various genes for proinflammatory cytokines/chemokines or receptors in PMN . We detected an upregulation or de novo synthesis of interleukin 1 alpha (IL-1 alpha), IL-1 beta, IL-8, macrophage inflammatory protein 1 alpha (MIP-1 alpha), MIP-1 beta, GRO-alpha, transforming growth factor beta, MCP-1, IL-2 receptor gamma (IL-2R gamma), IL-10R alpha, and IL-6R . Genes for IL-9, IL-12 alpha, IL-15, IL-5R alpha, and IL-13R alpha(1) were found to be downregulated or switched off . Furthermore, Giemsa and annexin V-propidium iodide double staining demonstrated an inhibition of spontaneous PMN apoptosis following BCG stimulation . Changes in phenotype and inhibition of apoptosis did not depend on direct mycobacterial stimulation alone, but were a result of an autocrine-paracrine stimulation mechanism . Our findings support the hypothesis that PMN become activated at the site of mycobacterial infections and that this activation might set the stage for a subsequent antimycobacterial immune response. Infect Immun, 2003 Aug, 71(8), 4238 - 49 Simultaneous blocking of human Toll-like receptors 2 and 4 suppresses myeloid dendritic cell activation induced by Mycobacterium bovis bacillus Calmette-Guérin peptidoglycan; Uehori J et al.; The Mycobacterium bovis bacillus Calmette-Guerin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4 . Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-alpha) production and CD83/CD86 up-regulation . The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-alpha production ability was completely impaired . Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-kappa B activation as determined by a reporter assay . Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-kappa B activation by 80% . Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent . BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs . Similar but less profound suppression of BCG CWS-mediated DC maturation was observed . Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2 . Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-kappa B in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation. J Immunol, 2003 Aug 1, 171(3), 1602 - 9 Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of bacille Calmette-Guérin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara; Goonetilleke NP et al.; Heterologous prime-boost immunization strategies can evoke powerful T cell immune responses and may be of value in developing an improved tuberculosis vaccine . We show that recombinant modified vaccinia virus Ankara, expressing Mycobacterium tuberculosis Ag 85A (M.85A), strongly boosts bacille Calmette-Guerin (BCG)-induced Ag 85A specific CD4(+) and CD8(+) T cell responses in mice . A comparison of intranasal (i.n.) and parenteral immunization of BCG showed that while both routes elicited comparable T cell responses in the spleen, only i.n . delivery elicited specific T cell responses in the lung lymph nodes, and these responses were further boosted by i.n . delivery of M.85A . Following aerosol challenge with M . tuberculosis, i.n . boosting of BCG with either BCG or M.85A afforded unprecedented levels of protection in both the lungs (2.5 log) and spleens (1.5 log) compared with naive controls . Protection in the lung correlated with the induction of Ag 85A-specific, IFN-gamma-secreting T cells in lung lymph nodes . These findings support further evaluation of mucosally targeted prime-boost vaccination approaches for tuberculosis. Biomedica, 2003 Jun, 23(2), 131 - 3 {Image analysis to quantify S100-positive dendritic cells in leprosy-affected skin}; Camargo LH et al.; A morphometric analysis of skin dendritic cells was done on biopsies of patients with different forms of leprosy . An anti S100 antibody was used to determine dendritic cell quantity and extension . Patients with a better immune response to the bacillus showed a greater number of dendritic cells in the cases of dimorphic tuberculoid leprosy and tuberculoid leprosy . This result contrasted with that from patients with dimorphic lepromatous leprosy and lepromatous leprosy. J Basic Microbiol, 2003, 43(4), 341 - 7 In vitro growth response of bread-spoilage Bacillus strains to selected natural antimicrobials; Pattison TL et al.; This study assessed the in vitro responses of Bacillus (B.) strains isolated from ropey bread to natural antimicrobials under optimum growth conditions . The responses of six Bacillus strains {B . subtilis (2), B . licheniformis (2) and B . pumilus (2)} to acetic acid (AA), lactic acid (LA), calcium lactate (CL) and a lactate-containing cocktail (LCC), singly and in combinations were determined and compared to calcium propionate (CP) . Isolates were each inoculated into flasks containing Nutrient Broth (NB) and the respective antimicrobial treatments and pHs were left unadjusted . A duplicate set of flasks, also containing NB and the respective antimicrobials, but adjusted to pH's corresponding to those of baked brown bread containing the same antimicrobials was also inoculated . Growth curves were obtained spectrophotometrically and used to estimate lag times . The organic acids used in this study {0.1% (v/v) AA and 0.25% (v/v) LA} singly and in combination with each other and with CL, CP or LCC, completely inhibited the growth of all six Bacillus strains, but only at non-adjusted pHs . The efficacies of LA, AA and CL notably decreased when the pH of the test media containing the respective preservatives was adjusted to the corresponding in situ (bread) values . However, the natural antimicrobials were still as effective as CP in retarding growth of the six Bacillus strains at the in situ (bread) pH values. Trop Doct, 2003 Jul, 33(3), 154 - 6 Drug resistant tuberculosis in diabetes mellitus: a retrospective study from south India; Subhash HS et al.; This study was conducted in a tertiary care teaching hospital in south India to evaluate the association of drug resistant tuberculosis (TB) in diabetic subjects . There were: 361 subjects with positive mycobacterial culture and susceptibility tests results over a 3-year period; 267 (74%) acid-fast bacillus smear positive; and 94 (26%) smear negative cases . One hundred and seventy-seven (49%) had resistant isolates to any one first line antiTB drugs (resistant group) and 184 (51%) had isolates sensitive to all drugs (non-resistant group) . In the resistant and non-resistant subjects the mean duration of TB symptoms was, respectively, 22 months and 4.5 months, past history of TB 126 (71%) and 48 (26%), past antiTB drug therapy 126 (71%) and 47 (25%), inadequate anti TB drug therapy 42 (24%) and 23 (13%), HIV positive six and 13 subjects . There were 72 diabetic subjects {35 and 37, respectively} with a duration of diabetes 5.8 +/- 7.5 years and 3.7 +/- 5.0 years in the resistant and non-resistant groups . Twenty-six per cent of the diabetic subjects (19/72) had multi-drug resistantTB . Drug resistance to first line anti-TB drugs was not found to be associated with diagnosis or duration of diabetes mellitus. J Infect Dis, 2003 Aug 1, 188(3), 364 - 70 Epub 2003 Jul 14. Rapid diagnosis of active tuberculosis by detecting antibodies from lymphocyte secretions; Raqib R et al.; In the present study, we investigated the tuberculosis (TB) diagnostic performance of an assay on the basis of detection of TB-specific antibodies from peripheral blood mononuclear cells (PBMCs), to determine whether antibodies in lymphocyte secretions obtained from PBMCs would better reflect active disease than antibodies in serum . PBMCs from patients with and without TB cultured in various concentrations for different times were assessed . Immunoglobulin G (IgG) specific for antigen (bacille Calmette-Guerin {BCG} vaccine and purified protein derivative {PPD}) was measured in lymphocyte secretions . Patients with active TB had higher BCG- or PPD-specific IgG antibody responses than patients without TB or healthy subjects (P=.001) . This method can be used as a quick diagnostic aid to facilitate rapid detection of TB cases. Clin Exp Immunol, 2003 Aug, 133(2), 182 - 92 Immune biology of macaque lymphocyte populations during mycobacterial infection; Lai X et al.; Immune responses of lymphocyte populations during early phases of mycobacterial infection and reinfection have not been well characterized in humans . A non-human primate model of Mycobacterium bovis bacille Calmette-Guerin (BCG) infection was employed to characterize optimally the immune responses of mycobacteria-specific T cells . Primary BCG infection induced biphasic immune responses, characterized by initial lymphocytopenia and subsequent expansion of CD4+, CD8+ and gammadelta T cell populations in the blood, lymph nodes and the pulmonary compartment . The potency of detectable T cell immune responses appears to be influenced by the timing and route of infection as well as challenge doses of BCG organisms . Systemic BCG infection introduced by intravenous challenge induced a dose-dependent expansion of circulating CD4+, CD8+ and gammadelta T cells whereas, in the pulmonary compartment, the systemic infection resulted in a predominant increase in numbers of gammadelta T cells . In contrast, pulmonary exposure to BCG through the bronchial route induced detectable expansions of CD4+, CD8+ and gammadelta T cell populations in only the lung but not in the blood . A rapid recall expansion of these T cell populations was seen in the macaques reinfected intravenously and bronchially with BCG . The expanded alphabeta and gammadelta T cell populations exhibited their antigen specificity for mycobacterial peptides and non-peptide phospholigands, respectively . Finally, the major expansion of T cells was associated with a resolution of active BCG infection and reinfection . The patterns and kinetics of CD4+, CD8+ and gammadelta T cell immune responses during BCG infection might contribute to characterizing immune protection against tuberculosis and testing new tuberculosis vaccines in primates. J Electron Microsc (Tokyo), 2003, 52(2), 153 - 9 Scanning electron microscopy of food-poisoning bacterium Bacillus cereus using a variable-pressure SEM; Nishimura M et al.; A variable-pressure scanning electron microscopy (VP-SEM) with a cooling stage permits long hours of observation of water-containing specimens in their natural or close to natural state, without the conventional specimen preparations of fixation, dehydration, drying and metal coating . It reduces water vaporization and beam damage by keeping the specimens at a low temperature . We observed Bacillus cereus colonies on nutrient agar, which would shrink significantly if any conventional specimen preparation technique were used . We also studied the growing process of the bacteria on raw and steamed rice using the VP-SEM without conventional preparation techniques . Original specimens were directly mounted onto specimen holders and their backscattered electron images observed under the following conditions: specimen stage temperature, -10 degrees C; specimen chamber vacuum level, 30-70 Pa; and accelerating voltage, 15-20 kV . We recognized that the VP-SEM minimized deformation of the colonies due to shrinkage of the nutrient agar, and successfully imaged the morphology of the colonies and bacteria without a decline in bacteria number, which is apt to occur during fixation and dehydration . Also, the growth process of the bacteria on raw or steamed rice could be observed promptly, since there is no specimen preparation step. J Bacteriol, 2003 Aug, 185(15), 4442 - 9 Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp . strain TA2.A1; Cook GM et al.; We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp . strain TA2.A1 . The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c . The subunits were identified by N-terminal protein sequencing and mass spectroscopy . A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity . ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold . This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1) . After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi) . A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis . ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect . Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species . The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis. Syst Appl Microbiol, 2003 Jun, 26(2), 254 - 61 Molecular typing of Bacillus thuringiensis serovars by RAPD-PCR; Gaviria Rivera AM et al.; One hundred and twenty-six strains of Bacillus thuringiensis representing 57 serovars were allocated to 58 genomic types using random amplified polymorphic DNA (RAPD)-PCR patterns . Serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, morrisoni, pakistani, sotto, thuringiensis and tolworthi each encompassed identical or closely related strains . Despite this genomic homogeneity, most of these serovars also included at least one variant strain . Serovars aizawai, canadensis, entomocidus and sotto biotype dendrolimus, on the other hand, were genomically heterogeneous . Of the 57 serovars examined, 31 contained at least one strain with a closely related or identical RAPD pattern to a strain from a different serovar . We conclude that while the species is genomically diverse, the homogeneous serovars represent clonal lineages of successful insect pathogens. Indian J Med Res, 2003 Jan, 117, 1 - 9 New drug targets for Mycobacterium tuberculosis; Chopra P et al.; In spite of the availability of effective chemotherapy and Bacille-Calmette-Guerin (BCG) vaccine, tuberculosis remains a leading infectious killer world-wide . Many factors such as, human immunodeficiency virus (HIV) co-infection, drug resistance, lack of patient compliance with chemotherapy, delay in diagnosis, variable efficacy of BCG vaccine and various other factors contribute to the mortality due to tuberculosis . In spite of the new advances in understanding the biology of Mycobacterium tuberculosis, and availability of functional genomic tools, such as microarray and proteomics, in combination with modern approaches, no new drug has been developed in the past 30 yr . Therefore, there is an urgent need to identify new drug targets in mycobacteria and eventually, develop new drugs . The release of the complete genome sequence of M . tuberculosis has facilitated a more rational, and directional approach to search for new drug targets . In general, gene products involved in mycobacterial metabolism, persistence, transcription, cell wall synthesis and virulence would be possible targets for the development of new drugs . The exploitation of host cell signaling pathways for the benefit of the pathogen is a phenomenon that deserves to be looked into with a new perspective in the current scenario to combat M . tuberculosis . Reversible phosphorylation and dephosphorylation, which are carried out by specific protein kinases and phosphatases have been shown to modify the host proteins and help in the establishment of disease by several pathogenic bacteria . In this review, we discuss some possible drug targets for M . tuberculosis. J Chromatogr A, 2003 May 23, 998(1-2), 103 - 8 kappa-Carrageenan as a new smart macroaffinity ligand for the purification of pullulanase; Roy I et al.; kappa-Carrageenan is a polysaccharide from red seaweed which gets precipitated by K+ ions and dissolves again in water . This smart, K(+)-responsive polymer was found to selectively bind pullulanase activity from Bacillus acidopullulyticus . Gel filtration on Sephadex G-200 showed the formation of the polymer-pullulanase complex at the pre-precipitation stage . On the other hand, phospholipase D, an enzyme which did not co-precipitate with kappa-carrageenan, did not form any complex with the polymer . Thus, K+ ions could be used to selectively precipitate the pullulanase activity . Then, 92% enzyme activity could be eluted with 1 M maltose solution . The single step protocol resulted in 50-fold purification, with a single band on sodium dodecylsulfate-polyacrylamide gel electrophoresis. Urol Nurs, 2003 Jun, 23(3), 189 - 91, 199; quiz 192 Intravesical Bacillus Calmette-Guerin for treating bladder cancer; Boyd LA; Bladder cancer continues to be a leading cause of malignant neoplasm in men . Since 1976, Bacillus Calmette-Guerin (BCG) has been a recommended treatment for superficial bladder malignancy . BCG treatment indications, administration, side effects, patient education, and nursing implications are discussed. Ann N Y Acad Sci, 2003 Jun, 990, 267 - 78 Clinical impact of persistent Bartonella bacteremia in humans and animals; Chomel BB et al.; Bartonella spp . are emerging vector-borne pathogens that cause persistent, often asymptomatic bacteremia in their natural hosts . As our knowledge progresses, it appears that chronic infection may actually predispose the host to mild, insidious nonspecific manifestations or induce, in selected instances, severe diseases . Persistent asymptomatic bacteremia is most common in animals that serve as the main reservoir for the specific Bartonella . In humans, these organisms are B . bacilliformis and B . quintana . Other Bartonella species, for which humans are not the natural reservoir, tend to cause persistent bacteremia only in immunodeficient individuals . In some of these individuals, endothelial cell proliferation may create lesions such as bacillary angiomatosis or bacillary peliosis . In cats, bacteremia of variable level and continuity may last for years . Some strains of B . henselae may induce clinical manifestations, including fever, mild neurological signs, reproductive disorders, whereas others do not induce clinically obvious disease . Reproductive disorders have also been reported in mice experimentally infected with B . birtlesii . Finally, canids constitute the most interesting naturally occurring animal model for the human disease . Like immunocompetent people, healthy dogs only occasionally demonstrate long-term bacteremia when infected with Bartonella spp . However, some dogs develop severe clinical manifestations, such as endocarditis, and the pathologic spectrum associated with Bartonella spp . infection in domestic dogs is rapidly expanding and resembles the infrequently reported clinical entities observed in humans . In coyotes, persistent bacteremia is more common than in domestic dogs . It will be of interest to determine if coyotes develop clinical or pathological indications of infection. J Infect, 2003 Aug, 47(2), 139 - 47 Infection of human monocytes with Mycobacterium bovis BCG induces production of CC-chemokines; Mendez-Samperio P et al.; DESIGN: CC-chemokines are potent leukocyte activators and chemoattractants, which have an important role in granuloma formation, function critical for the immune responses to mycobacterial infection . This study investigated whether infection of human monocytes with Mycobacterium bovis bacillus Calmette-Guerin (BCG) elicits secretion of RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta . METHODS: RANTES, MIP-1alpha and MIP-1beta synthesis was measured by the presence of protein secretion in the cell culture supernatant as determined by enzyme-linked immunosorbent assay . To investigate the mechanism of M . bovis BCG stimulation of RANTES, we carried out inhibition assays with antibodies to CD40 and we used an intracellular calcium chelator BAPTA-AM . RESULTS: Infection of human monocytes with M . bovis BCG induced RANTES, MIP-1alpha and MIP-1beta secretion in a dose-dependent manner . This stimulation of CC-chemokines production was not attributed to LPS contamination . M . bovis-induced RANTES secretion was dependent upon bacterial uptake and on tumor necrosis factor (TNF)-alpha . Interestingly, the production of RANTES by M . bovis BCG-infected monocytes occurs through a mechanism that requires intracellular calcium and was significantly inhibited (P<0.05) with antibodies to CD40 . CONCLUSIONS: These results suggest that the ability of M . bovis BCG to produce CC-chemokines might lead to protection in the acquired immune response of mycobacterial infection and at the same time indicate that M . bovis BCG-induced RANTES secretion is mediated by CD40 and dependent on the intracellular calcium influx. J Mol Biol, 2003 Jul 25, 330(5), 1189 - 201 Effects of domain dissection on the folding and stability of the 43 kDa protein PGK probed by NMR; Reed MA et al.; The characterization of early folding intermediates is key to understanding the protein folding process . Previous studies of the N-domain of phosphoglycerate kinase (PGK) from Bacillus stearothermophilus combined equilibrium amide exchange data with a kinetic model derived from stopped-flow kinetics . Together, these implied the rapid formation of an intermediate with extensive native-like hydrogen bonding . However, there was an absence of protection in the region proximal to the C-domain in the intact protein . We now report data for the intact PGK molecule, which at 394 residues constitutes a major extension to the protein size for which such data can be acquired . The methods utilised to achieve the backbone assignment are described in detail, including a semi-automated protocol based on a simulated annealing Monte Carlo technique . A substantial increase in the stability of the contact region is observed, allowing protection to be inferred on both faces of the beta-sheet in the intermediate . Thus, the entire N-domain acts concertedly in the formation of the kinetic refolding intermediate rather than there existing a distinct local folding nucleus. J Appl Microbiol, 2003, 95(2), 310 - 6 Comparison of the expression of Bacillus thuringiensis full-length and N-terminally truncated vip3A gene in Escherichia coli; Chen J et al.; AIMS: Studies were performed to demonstrate the function of the putative signal peptide of Vip3A proteins in Escherichia coli . METHODS AND RESULTS: The full-length vip3A-S184 gene was isolated from a soil-isolated Bacillus thuringiensis, and the vip3AdeltaN was constructed by deleting 81 nucleotides at the 5'-terminus of vip3A-S184 . Both were transformed and expressed in E . coli . About 19.2% of Vip3A-S184 proteins secreted soluble proteins and others formed inclusion bodies in the periplasmic space . In contrast, the Vip3AdeltaN was insoluble and formed inclusion bodies in the cytoplasm . Bioassay indicated that Vip3A-S184 showed different toxicity against Spodoptera exigua, Helicoverpa armigera and S . litura, but Vip3AdeltaN showed no toxicity to either of them because of the deletion of the first 27 amino acids at the N-terminus . CONCLUSIONS: The results suggest that the deleted N-terminal sequences were essential for the secretion of Vip3A-S184 protein in E . coli and might be required for toxicity . SIGNIFICANCE AND IMPACT OF THE STUDY: The function of the putative signal peptide of Vip3A protein in E . coli was investigated . These would be helpful to make clear the unknown secretion pathway of Vip3A protein in B . thuringiensis and determine the receptor-binding domain or toxic fragment of Vip3A-S184 protein. J Appl Microbiol, 2003, 95(2), 267 - 72 Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: production and some properties; Joo HS et al.; AIMS: An investigation was carried out on an oxidative and SDS-stable alkaline protease secreted by Bacillus clausii of industrial significance . METHODS AND RESULTS: Maximum enzyme activity was produced when the bacterium was grown in the medium containing (g l-1): soyabean meal, 15; wheat flour, 10; liquid maltose, 25; K2HPO4, 4; Na2HPO4, 1; MgSO4.7H2O, 0.1; Na2CO3, 6 . The enzyme has an optimum pH of around 11 and optimum temperature of 60 degrees C . The alkaline protease showed extreme stability towards SDS and oxidizing agents, which retained its activity above 75 and 110% on treatment for 72 h with 5% SDS and 10% H2O2, respectively . Inhibition profile exhibited by phenylmethylsulphonyl fluoride suggested that the protease from B . clausii belongs to the family of serine proteases . CONCLUSIONS: Bacillus clausii produced high levels of an extracellular protease having high stability towards SDS and H2O2 . SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline protease from B . clausii I-52 is significant for an industrial perspective because of its ability to function in broad pH and temperature ranges in addition to its tolerance and stability in presence of an anionic surfactant, like SDS and oxidants like peroxides and perborates . The enzymatic properties of the protease also suggest its suitable application as additive in detergent formulations. Clin Microbiol Rev, 2003 Jul, 16(3), 357 - 64 Vaccinations for adult solid-organ transplant recipients: current recommendations and protocols; Duchini A et al.; Recipients of solid-organ transplantation are at risk of severe infections due to their life-long immunosuppression . Despite emerging evidence that vaccinations are safe and effective among immunosuppressed patients, most vaccines are still underutilized in these patients . The efficacy, safety, and protocols of several vaccines in this patient population are poorly understood . Timing of vaccination appears to be critical because response to vaccinations is decreased in patients with end-stage organ disease and in the first 6 months after transplantation . For these reasons, the primary immunizations should be given before transplantation, as early as possible during the course of disease . Vaccination strategy should include vaccination of household contacts and health care workers at transplant centers unless contraindicated . No conclusive data are available on the use of immunoadjuvants and screening for protective titers . Most vaccines appear to be safe in solid-organ transplantation recipients, but live vaccines should be avoided until further studies are available . The risk of rejection appears minimal . Recommended vaccines include pneumovax, hepatitis A and B, influenza, and tetanus-diphtheria . We outline specific protocols and recommendations in this particular patient population . Specific contraindications exist for other vaccines, such as yellow fever, oral polio vaccine, bacillus Calmette-Guerin, and vaccinia . We conclude that solid-organ recipients will benefit from consistent immunization practices . Further studies are recommended to improve established protocols in this patient population. Phys Rev Lett . 2003 Jun 27;90(25 Pt 1):258102 . Epub 2003 Jun 23. Self-organized pattern formation of a bacteria colony modeled by a reaction diffusion system and nucleation theory; Wakano JY et al.; Self-organized pattern formation is observed in bacterial colony growth . The recently reported knotted-branching pattern of the Bacillus circulans colony consists of the trajectories of aggregates which grow, move, and reproduce simultaneously . We modeled these processes by combining a reaction diffusion system of nutrient dynamics, nucleation theory for aggregate generation, and individual based dynamics of motion and growth of aggregates . The branching pattern produced by computer simulation shows great similarity with experiments . Response to the initial nutrient concentration is also consistent with the experiments. Clin Infect Dis, 2003 Jul 15, 37(2), e27 - 8 Epub 2003 Jul 09. Tuberculosis due to Mycobacterium bovis after alemtuzumab administration; Abad S et al.; We describe a patient with relapsing B chronic lymphocytic leukemia who developed systemic bacille Calmette-Guerin infection (BCGitis) after administration of alemtuzumab (Campath-1H). J Biol Chem, 2003 Sep 19, 278(38), 36396 - 402 Epub 2003 Jul 10. Functional analysis of RF2a, a rice transcription factor; Dai S et al.; RF2a is a bZIP transcription factor that regulates expression of the promoter of rice tungro bacilliform badnavirus . RF2a is predicted to include three domains that contribute to its function . The results of transient assays with mutants of RF2a from which one or more domains were removed demonstrated that the acidic domain was essential for the activation of gene expression, although the proline-rich and glutamine-rich domains each played a role in this function . Studies using fusion proteins of different functional domains of RF2a with the 2C7 synthetic zinc finger DNA-binding domain showed that the acidic region is a relatively strong activation domain, the function of which is dependent on the context in which the domain is placed . Data from transgenic plants further supported the conclusion that the acidic domain was important for maintaining the biological function of RF2a . RF2a and TBP (TATA-binding protein) synergistically activate transcription in vitro (Zhu, Q., Ordiz, M . I., Dabi, T., Beachy, R . N., and Lamb, C . (2002) Plant Cell 14, 795-803) . In vitro and in vivo assays showed that RF2a interacts with TBP through the glutamine-rich domain but not the acidic domain . Functional analysis of such interactions indicates that the acidic domain activates transcription through mechanisms other than via the direct recruitment of TBP. J Urol, 2003 Aug, 170(2 Pt 1), 605 - 10 Bacillus Calmette-Guerin initiates intracellular signaling in a transitional carcinoma cell line by cross-linking alpha 5 beta 1 integrin; Chen F et al.; PURPOSE: The adherence of bacillus Calmette-Guerin (BCG) to the surface of transitional carcinoma tumor cells initiates nuclear factor (NF)-kappa B signal transduction pathways that modulate the expression of proteins important in the antitumor response to BCG . We tested the hypothesis that BCG initiates NF-kappa B signaling as a consequence of cross-linking alpha 5 beta 1 integrin receptors present on the tumor cell surface . MATERIALS AND METHODS: The effect of alpha 5 beta 1 antibody mediated cross-linking on interleukin (IL)-6 mRNA expression, IL-6 promoter activation and activation of a specific NF-kappa B reporter construct was determined . A series of reporter constructs containing nonfunctional mutations in the AP-1, NF-IL-6 and NF-kappa B sites were used to determine the relative importance of these response elements in alpha 5 beta 1 cross-linking mediated activation of the IL-6 promoter . A final series of experiments assessed the role of alpha 5 beta 1 receptor occupancy by fibronectin (FN) in initiating antibody or BCG mediated signaling . RESULTS: Anti alpha 5 and anti beta 1 mediated cross-linking of alpha 5 beta 1 integrin initiated NF-kappa B signaling, IL-6 promoter activation and IL-6 mRNA expression . Deletion mutants demonstrated that alpha 5 beta 1 cross-link initiated, IL-6 promoter transactivation required intact NF-kappa B and AP-1 response elements . Receptor occupancy by FN was required for BCG but not for antibody initiated signaling . CONCLUSIONS: Cross-linking the alpha 5 beta 1 receptor present on the surface of human transitional carcinoma cells lines initiates signal transduction in a manner identical to that observed for BCG . We propose a model in which multiple FN binding sites present on BCG interact with alpha 5 beta 1 receptor bound FN molecules to cross-link alpha 5 beta 1 receptors and initiate intracellular signaling. Clin Diagn Lab Immunol, 2003 Jul, 10(4), 564 - 72 Infection with Mycobacterium bovis BCG diverts traffic of myelin oligodendroglial glycoprotein autoantigen-specific T cells away from the central nervous system and ameliorates experimental autoimmune encephalomyelitis; Sewell DL et al.; Infectious agents have been proposed to influence susceptibility to autoimmune diseases such as multiple sclerosis . We induced a Th1-mediated central nervous system (CNS) autoimmune disease, experimental autoimmune encephalomyelitis (EAE) in mice with an ongoing infection with Mycobacterium bovis strain bacillus Calmette-Guerin (BCG) to study this possibility . C57BL/6 mice infected with live BCG for 6 weeks were immunized with myelin oligodendroglial glycoprotein peptide (MOG(35-55)) to induce EAE . The clinical severity of EAE was reduced in BCG-infected mice in a BCG dose-dependent manner . Inflammatory-cell infiltration and demyelination of the spinal cord were significantly lessened in BCG-infected animals compared with uninfected EAE controls . ELISPOT and gamma interferon intracellular cytokine analysis of the frequency of antigen-specific CD4(+) T cells in the CNS and in BCG-induced granulomas and adoptive transfer of MOG(35-55)-specific green fluorescent protein-expressing cells into BCG-infected animals indicated that nervous tissue-specific (MOG(35-55)) CD4(+) T cells accumulate in the BCG-induced granuloma sites . These data suggest a novel mechanism for infection-mediated modulation of autoimmunity . We demonstrate that redirected trafficking of activated CNS antigen-specific CD4(+) T cells to local inflammatory sites induced by BCG infection modulates the initiation and progression of a Th1-mediated CNS autoimmune disease. J Econ Entomol, 2003 Jun, 96(3), 957 - 68 Transgenic Bacillus thuringiensis corn hybrid performance against univoltine ecotype European corn borer (Lepidoptera: Crambidae) in South Dakota; Catangui MA; The performances of Bt-corn hybrids against univoltine ecotype European corn borer larvae were evaluated in South Dakota from 1997 to 1999 . The corn hybrids were exposed to natural seasonal fluctuations of a univoltine ecotype European corn borer population . Larval injury parameters, grain yields, and gross incomes were quantified during the 3-yr study . In general, the Bt-corn hybrids had significantly higher yields than the untreated non-Bt isolines in 1997 and 1998 when corn borer pressures were high . However, most of the Bt-corn hybrids did not produce significant yield advantages in 1999 when the European corn borer pressure was low . Some of the Bt-corn hybrids even produced significantly lower yields than their untreated non-Bt counterparts in 1999 . The performances of non-Bt isolines that were treated with permethrin granules at whorl stage were similar to Bt-corn hybrids in 1998 when the corn borer pressure was high and similar to the untreated non-Bt isolines in 1999 when the corn borer pressure was low . Injury-free corn produced by Bt-corn hybrids did not necessarily translate into higher yields in some hybrid groups . Grain moisture at harvest, which can result in moisture penalty or dockage, was significantly higher in most Bt-corn hybrids . Gross incomes of the Bt-corn hybrids were generally higher than the untreated non-Bt isolines when the corn borer infestation was high, but were either similar to or lower than the untreated non-Bt isolines when the corn borer infestation was low. J Econ Entomol, 2003 Jun, 96(3), 941 - 9 Economic analysis of planting dates to manage European corn borer (Lepidoptera: Crambidae) with Bt corn; Pilcher CD et al.; Planting dates of transgenic Bacillus thuringiensis Berliner (Bt) corn were adjusted to determine the utility in managing European corn borer, Ostrinia nubilalis (Hubner) . Transgenic Bt (events 176 and Bt11) corn and non-Bt corn were planted at three different times to use the early- and late- planted corn as a potential trap crop for ovipositing European corn borer moths . Grain moisture and yields were recorded to determine the economic benefits of Bt corn planted on the different dates, based on European corn borer populations and corn damage data collected before harvest . Data were recorded from three locations in southwestern, central, and northeastern Iowa for three summers (1996-1998) . Economic benefits are discussed in relation to EILs and yield results . Adjusting the planting dates of Bt and non-Bt corn provided variable economic differences among planting dates in northern Iowa; however, greater economic benefits were realized when Bt corn was planted late during the planting sequence in central and southwestern Iowa . These results suggest that planting corn should be conducted in a timely manner and, if delayed or required to plant late, planting Bt corn would likely provide greater economic benefits . Although yield and economic variability were high, using Bt corn in combination with planting date adjustments may be a viable option for managing European corn borer. J Econ Entomol, 2003 Jun, 96(3), 931 - 4 Effect of MON810 Bt field corn on Helicoverpa zea (Lepidoptera: Noctuidae) cannibalism and its implications to resistance development; Horner TA et al.; Pairs of Helicoverpa zea (Boddie) larvae reared on diet-incorporated MON810 transgenic leaf tissue of field corn (Zea mays L.) were observed in the laboratory to characterize effects of sublethal levels of Bacillus thuringiensis variety kurstaki (Bt) Cry1Ab endotoxins on cannibalistic behavior and mortality . Feeding on sublethal levels of Bt corn reduced the frequency of cannibalistic behaviors exhibited by H . zea when uneven instars were paired together . Exposure to the Bt endotoxin had no significant effect on when cannibalistic mortality occurred or the level of mortality as a result of cannibalism . Assuming that H . zea larvae reared on nonBt corn tissue behaved in a similar way that resistant larvae would if feeding on Bt tissue, sublethal effects of Cry1Ab intoxication may reduce the chances of successful cannibalism by susceptible larvae and thus play a disproportionate role in the survival of multiple ear infestations . Furthermore, cannibalistic encounters could result in partially resistant larvae feeding on nontoxic food, thus temporarily providing an escape from exposure to the Bt endotoxin . These behavior alterations could increase the selective differential between susceptible individuals and those carrying resistance genes. J Econ Entomol, 2003 Jun, 96(3), 914 - 24 Development, survival and fitness performance of Helicoverpa zea (Lepidoptera: Noctuidae) in MON810 Bt field corn; Horner TA et al.; Helicoverpa zea (Boddie) development, survival, and feeding injury in MON810 transgenic ears of field corn (Zea mays L.) expressing Bacillus thuringiensis variety kurstaki (Bt) Cry1Ab endotoxins were compared with non-Bt ears at four geographic locations over two growing seasons . Expression of Cry1Ab endotoxin resulted in overall reductions in the percentage of damaged ears by 33% and in the amount of kernels consumed by 60% . Bt-induced effects varied significantly among locations, partly because of the overall level and timing of H . zea infestations, condition of silk tissue at the time of egg hatch, and the possible effects of plant stress . Larvae feeding on Bt ears produced scattered, discontinuous patches of partially consumed kernels, which were arranged more linearly than the compact feeding patterns in non-Bt ears . The feeding patterns suggest that larvae in Bt ears are moving about sampling kernels more frequently than larvae in non-Bt ears . Because not all kernels express the same level of endotoxin, the spatial heterogeneity of toxin distribution within Bt ears may provide an opportunity for development of behavioral responses in H . zea to avoid toxin . MON810 corn suppressed the establishment and development of H . zea to late instars by at least 75% . This level of control is considered a moderate dose, which may increase the risk of resistance development in areas where MON810 corn is widely adopted and H . zea overwinters successfully . Sublethal effects of MON810 corn resulted in prolonged larval and prepupal development, smaller pupae, and reduced fecundity of H . zea . The moderate dose effects and the spatial heterogeneity of toxin distribution among kernels could increase the additive genetic variance for both physiological and behavioral resistance in H . zea populations . Implications of localized population suppression are discussed. J Econ Entomol, 2003 Jun, 96(3), 909 - 13 Effect of maysin on wild-type, deltamethrin-resistant, and Bt-resistant Helicoverpa armigera (Lepidoptera: Noctuidae); Rector BG et al.; Larvae of the Old World corn earworm, Helicoverpa armigera (Hubner), were fed diets containing lyophilized silks from maize genotypes expressing varying levels of maysin, a flavone glycoside known to be toxic to the New World corn earworm, Helicoverpa zea Boddie . Three different H . armigera colonies were tested: a wild-type colony (96-S), a colony selected for resistance to deltamethrin (Del-R), and a colony selected for resistance to the Cry1Ac protoxin of Bacillus thuringiensis (Bt-R) . A colony of H . zea was also tested as a control . High-maysin silk diets significantly slowed the growth and arrested the development of larvae from all H . armigera colonies compared with low-maysin silk diets, maysin-lacking silk diets, and no-silk control diets . The effects on the H . armigera and H . zea colonies were similar across maysin levels, although H . zea is a larger insect than H . armigera and this overall size difference was observed . Among the H . armigera colonies, maysin effects were generally similar, although 7-d-old Del-R larvae were significantly smaller than 7-d-old Bt-R and 96-S larvae for one no-silk control and two maysin-containing silk treatments . The toxic effect of maysin on the Bt-R and Del-R colonies suggests that physiological mechanisms of H . armigera resistance to Cry1Ac and deltamethrin do not confer cross-resistance to maysin. J Econ Entomol, 2003 Jun, 96(3), 879 - 84 Susceptibility of leafrollers (Lepidoptera: Tortricidae) from organic and conventional orchards to azinphosmethyl, spinosad, and Bacillus thuringiensis; Smirle MJ et al.; Populations of obliquebanded leafroller, Choristoneura rosaceana Harris, and three-lined leafroller, Pandemis limitata Robinson, were obtained from seven sites in the Okanagan and Similkameen Valleys of British Columbia and assayed for their responses to three insecticides using a leaf disk bioassay . Lethal concentration ratios (LCRs) were calculated for all populations compared with a susceptible laboratory colony of C . rosaceana; significant variation was detected in response to all three insecticides . LCRs were 0.86-15.52 for azinphosmethyl, 0.38-2.37 for spinosad (Success), and 0.58-4.89 for Bacillus thuringiensis (Foray) . Correlation analysis indicated no cross-resistance among the three insecticides . Leafroller populations obtained from apple orchards managed with organic production practices were more susceptible to azinphosmethyl than leafrollers obtained from conventionally managed sites . Conversely, the highest levels of tolerance to B . thuringiensis were observed in the populations from organic sites, possibly reflecting usage patterns; B . thuringiensis is one of the few insecticides allowed under organic production guidelines . All populations were highly susceptible to spinosad, which may be a useful tool for resistance management programs if used judiciously. J Econ Entomol, 2003 Jun, 96(3), 755 - 62 Field and laboratory evaluations of transgenic cottons expressing one or two Bacillus thuringiensis var . kurstaki Berliner proteins for management of noctuid (Lepidoptera) pests; Chitkowski RL et al.; Field studies were conducted from 1999 to 2001 to evaluate the efficacy of the transgenic cotton, Gossypium hirsutum (L.), genotype, Bollgard II (Monsanto 15985), which expresses two Bacillus thuringiensis Berliner (Bt) proteins (Cry1Ac + Cry2Ab) that are active against lepidopterous pests . Bollgard II was compared with Bollgard (DP50B), which expresses only one Bt protein (Cry1Ac), and, in all tests, the conventional variety, DP50, was used as a non-Bt control . Larval populations of the bollworm, Helicoverpa zea (Boddie), and the soybean looper, Pseudoplusia includens (Walker), were significantly lower in Bollgard II than in Bollgard and conventional cotton, and the proportion of fruit damaged by H . zea was also lower . Fall armyworm, Spodoptera frugiperda (J . E . Smith), populations were lower in Bollgard II than in Bollgard, although not significantly . Field tests were supplemented with laboratory bioassays in 2001 to compare mortality of S . frugiperda, and beet armyworms, Spodoptera exigua (Hubner), feeding on these genotypes . Mortality of both species was significantly greater on Bollgard II plant material than on either Bollgard or conventional cotton . This study demonstrated that the dual-toxin Bollgard II genotype is highly effective against lepidopterous pests that are not adequately controlled by the current single-toxin Bollgard varieties . If toxin expression in future Bollgard II varieties remains consistent with that of Monsanto 15985, supplemental insecticides will be reduced, and may be eliminated for lepidopterous pests in South Carolina. J Econ Entomol, 2003 Jun, 96(3), 699 - 705 Distribution of bollworm, Helicoverpa zea (Boddie), injured reproductive structures on genetically engineered Bacillus thuringiensis var . kurstaki Berliner cotton; Gore J et al.; Bollworm, Helicoverpa zea (Boddie), larvae are commonly observed feeding in genetically engineered Bollgard cotton . Although no information is currently available characterizing the levels of injury bollworms cause, aproximately 25% of the Bollgard acreage in the United States receives at least one insecticide application annually targeting bollworm populations . Studies were conducted to determine the levels of fruiting form injury that can occur from bollworm larvae feeding on white flowers of two types of genetically engineered cotton . The two types of genetically engineered cotton included the original Bollgard that produces one protein (Cry1Ac) from Bacillus thuringiensis variety kurstaki Berliner and Bollgard II that produces two proteins (Cry1Ac + Cry2Ab) from B . thuringiensis kurstaki . In one study, individual larvae (24 +/- 6 h old) were placed in first position white flowers of Deltapine 5415 (non-Bollgard) and Deltapine NuCOTN 33B (Bollgard) . Larval infestations were made on 50 plants for each of 5 d during 2000 and 2001 . Each plant was visually examined at 3 d and every 2 d thereafter, until larvae were no longer recovered . Larvae injured a total of 46.6 fruiting forms per 50 plants on non-Bollgard cotton, compared with only 18.9 fruiting forms per 50 plants on Bollgard cotton . Mean larval injury per insect was 4.3 fruiting forms on non-Bollgard cotton compared with 2.7 fruiting forms on Bollgard cotton . In a second study, individual larvae (24 +/- 6 h old) were placed in first position white flowers of Deltapine 50 (non-Bollgard), Deltapine 50B (Bollgard), and an experimental Bollgard II line . Larval infestations were made on 10 plants per day for each of six consecutive days during 2001 . Larvae injured a total of 25.0 fruiting forms per 10 plants on non-Bollgard, 11.5 on Bollgard, and 6.4 on Bollgard II cottons . Mean larval injury per insect was 6.6 fruiting forms on non-Bollgard, 3.5 on Bollgard, and 0.8 on Bollgard II cottons . These data indicate that supplemental insecticide applications may be necessary to prevent yield losses on Bollgard cotton . In contrast, injury to Bollgard II cotton was minimal and may not require additional insecticide applications for bollworms. J AOAC Int, 2003 May-Jun, 86(3), 529 - 33 Interactions of antimicrobials in milk and their detection by the disk diffusion method and Delvotest SP; Kukurova I et al.; The combination of more than 2 different microbials might show interactions with various effects (synergistic, additive, antagonistic, or indifferent) on target microorgnisms . An objective of this paper was to evaluate the possible interactions of several antimicrobials--those used most frequently in the treatment of mastitis in clinical veterinary practice (beta-lactam antibiotics, aminoglycosides, peptides, other antibiotics, and sulfonamides)--and their consequences on detection limits . In the model experiment with milk artificially altered by means of Delvotest SP and the disk diffusion method with Bacillus stearothermophilus var . calidolactis C 953, we observed the synergistic effect between all the antimicrobials tested . The results show that Delvotest SP is more sensitive (approximately 7.5- to 40-fold) than the disk diffusion method in estimating the detection limits of cephalosporin antibiotics. Int J Clin Oncol, 2003 Jun, 8(3), 168 - 73 Bacillus Calmette-Guerin-refractory superficial bladder cancers: focus on pretreatment episodes; Okamura T et al.; BACKGROUND: Reasons for development of bacillus Calmette-Guerin(BCG)-refractory superficial bladder cancers are unknown . In the present study, a series of cases was therefore analyzed, focusing on the influence of treatment before BCG application . PATIENTS AND METHODS: A total of 96 patients with superficial bladder cancers received six weekly intravesical instillations of BCG followed in some cases by a further six applications at monthly intervals . If tumors recurred, a further course of treatment in association with surgery or some other therapy was chosen, depending on the patient and the tumor condition . RESULTS: Thirty-three cases (34.4%) demonstrated tumor recurrence within 24 to 146 months, including 9 with progression . Pretreatments had been performed in 19 of these cases (57.6%) whereas they had been conducted for only 10 (15.9%) of the BCG-effective cases . Of the total 96 patients, 29 received pretreatment with open surgery, systemic chemotherapy, intravesical instillation or oral administration of anticancer drugs, or immunotherapy . Sixty-six percent of these proved BCG refractory, in contrast to only 20.9% in the no-pretreatment group . Furthermore, 7 of the 9 patients demonstrating progression had undergone pretreatment . CONCLUSIONS: The data suggest that intravesical instillation of BCG is more effective when no prior treatment has been attempted, and that best results may be achieved if BCG is chosen as the initial therapy for superficial bladder cancer . When pretreatment has been performed and pT1 lesions recur, however, immediate total cystectomy should be advised. Toxicol Lett, 2003 Aug 28, 143(3), 317 - 22 Identification of organic fractions of diesel exhaust particulate (DEP) which inhibit nitric oxide (NO) production from a murine macrophage cell line; Saxena QB et al.; Diesel exhaust particulates (DEPs) can constitute a large component of the particulate air pollution in urban areas and is a health concern . The effects of DEP on nitric oxide (NO) production by a murine macrophage cell line (RAW264.7) in response to interferon-gamma (INFgamma), lipopolysaccharide, (LPS) and Bacillus Calmette-Guerin (BCG) were studied . The DEP was fractionated into organic and inorganic fractions (carbonaceous core) . The organic portion was further divided into asphaltene, saturates, less polar aromatics, more polar aromatics and resins-containing fractions . Each fraction was tested for the ability to suppress NO production from BCG-stimulated macrophages . DEP crude organic extract, more polar aromatic hydrocarbon, and resin fractions dose-dependently inhibited BCG-stimulated NO production . It is concluded that the responsiveness of the macrophages to stimuli, such as BCG, is suppressed by DEP and that this activity is most predominant in the polar aromatic hydrocarbons and resins-containing fractions. Tunis Med, 2003 Apr, 81(4), 235 - 8 {Prognostic study of liver abscess}; Nouira R et al.; The objective of this work is to study factors of prognostic of mortality of abscesses of the liver . We have treated between 1990 and 2000 in our service, 38 patient for abscess of the liver . The symptoms are dominated by the pain of the right hypochondria (37 cases) and the fever (34 cases) . An unique abscess has been recovered in 25 cases . Some multiple localizations have been observed in 12 cases . 21 patients have been operated . The bacteriological study at all patients revealed the presence of germ in 27 cases . In 6 cases, there were two germs . It was a bacillus negative gram in 26 cases and a cocci positive gram in 7 cases . Six complications have been observed at the operated patients . In 5 cases, it was a septic shock having leads to the death . After survey univariate and multivariate the only factor of bad prognostic recovered is the septic shock . The aetiology was identified in only 9 cases; it was abscess cholangiotis. Extremophiles, 2003 Oct, 7(5), 415 - 21 Epub 2003 Jul 05. Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries; Rees HC et al.; A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples . Clones expressing a cellulase activity were identified and sequenced . A new cellulase gene was identified . Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake . Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced . These were encoded by novel genes as judged by DNA sequence comparisons . Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples . Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil . A number of new cellulase and lipase/esterase genes were discovered in these libraries . Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment . Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library. Biosci Biotechnol Biochem, 2003 Jun, 67(6), 1327 - 34 A novel enzyme of Bacillus sp . 217C-11 that produces inulin from sucrose; Wada T et al.; We found a bacterium that converts sucrose to a useful material, using about 6,000 samples of bacteria isolated from soil . This bacterium, Bacillus sp . 217C-11, was identified according to Bergey's manual, and produced a highly efficient enzyme that converted sucrose into inulin . So, the enzyme was purified to homogeneity through five chromatographic steps, to identify its enzymatic properties . The molecular mass of the enzyme was estimated to be 45,000, and this enzyme was a monomer protein (by SDS-PAGE) . The optimum pH and temperature of this enzyme were 7-8 and 45-50 degrees C, respectively . The enzyme reacted only with sucrose, but did not with other disaccharides, fructooligosaccharides and inulin . This paper will show that our enzyme is a novel one, which is different from the other well-known enzymes concerned in inulin production. Biosci Biotechnol Biochem, 2003 Jun, 67(6), 1239 - 44 Production and characterization of biosurfactants from Bacillus licheniformis F2.2; Thaniyavarn J et al.; A biosurfactant-producing strain, Bacillus licheniformis F2.2, was isolated from a fermented food in Thailand . The strain was capable of producing a new biosurfactant, BL1193, as well as two kinds of popular lipopeptide biosurfactants, plipastatin and surfactin . Mass spectrometry and FT-IR analysis indicated that BL1193 had a molecular mass of 1,193 Da with no peptide portion in the molecule . While plipastatin and surfactin were abundantly produced in a nutrient YPD medium, BL1193 was produced only in a synthetic DF medium containing no amino acids . According to an oil displacement activity test, the specific activity of BL1193 (6.53 kBS units/mg) is equivalent to that of surfactin (5.78-6.83 kBS units/mg). Biol Pharm Bull, 2003 Jul, 26(7), 920 - 6 His151 and His296 are the acid-base catalytic residues of Bacillus cereus sphingomyelinase in sphingomyelin hydrolysis; Obama T et al.; Bacillus cereus sphingomyelinase belongs to the Mg(2+)-dependent neutral sphingomyelinase, which hydrolyses sphingomyelin to phosphocholine and ceramide, and acts as an extracellular hemolysin . The triplet residues, His151-Asp195-His296, of the enzyme are highly conserved among bacterial and mammalian Mg(2+)-dependent neutral sphingomyelinases . The triplet residues converge on the active-site pocket of the 3D model of the enzyme . To investigate the function of these residues in the acid-base catalysis, we introduced several mutations for each residue by site-directed mutagenesis . Hemolytic and hydrolytic activities of the enzyme, abolished by the mutations at Asp195 and His296, revealed that these residues are critical for the catalytic function . The effect of the divalent metal cations on the pH dependency of the hydrolytic activities indicates that His296 corresponds to the most acidic ionizable group as a general base . The mutagenesis at His151 was also deleterious; however, the H151A and H151Q mutant enzymes partially retained their activities . The H151A mutation affected the most basic ionizable group, suggesting that His151 may act as a general acid in catalysis . By the structural basis of the 3D model, Asp195 must maintain not only the appropriate spatial arrangement but also pK(a)s of His151 and His296 . Taking into consideration all of these, we proposed the acid-base catalytic mechanism of B . cereus sphingomyelinase. J Clin Microbiol, 2003 Jul, 41(7), 3441 - 4 Bacillus cereus bacteremia in a preterm neonate; Hilliard NJ et al.; Bacillus cereus is an uncommon but potentially serious bacterial pathogen causing infections of the bloodstream, lungs, and central nervous system of preterm neonates . A case of bacteremia caused by B . cereus in a 19-day-old preterm neonate who was successfully treated with vancomycin, tobramycin, meropenem, and clindamycin is described . Implications for the diagnostic laboratory and clinicians when Bacillus species are detected in normally sterile sites are discussed, and the small numbers of infant infections proven to be due to this organism that have been described previously are reviewed. J Clin Microbiol, 2003 Jul, 41(7), 3070 - 7 Mycobacterium bovis subsp . caprae caused one-third of human M . bovis-associated tuberculosis cases reported in Germany between 1999 and 2001; Kubica T et al.; The prevalence of the Mycobacterium bovis subsp . caprae and M . bovis subsp . bovis among German tuberculosis cases caused by the bovine tubercle bacillus from 1999 to 2001 was determined . Isolates from 166 patients living in Germany and 10 animals were analyzed by conventional laboratory procedures, spoligotyping, and partly by PCR-restriction fragment length polymorphism analysis of the gyrB gene . By spoligotyping, 55 of 176 isolates (31%) could be identified as M . bovis subsp . caprae, and 121 (69%) were confirmed as M . bovis subsp . bovis . In general, a low variability of spoligotypes with 59 distinct patterns and a cluster rate of 77% (136 isolates/19 clusters) was determined . About half of all isolates were grouped in the three main clusters with 29, 30, and 35 isolates, respectively . Differences in age and gender between the patient groups infected with M . bovis subsp . bovis and M . bovis subsp . caprae did not reach statistical significance . However, marked differences in the geographical prevalence of M . bovis subsp . caprae were observed, ranging from fewer than 10% of all M . bovis isolates in the north up to more than 80% of isolates in the south of Germany . In conclusion, M . bovis subsp . caprae accounts for a high ratio of human M . bovis-associated tuberculosis cases in Germany and was more frequently found in the southern part. J Mol Biol, 2003 Jul 11, 330(3), 607 - 20 Crystal structure of a natural circularly permuted jellyroll protein: 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes; Tsai LC et al.; The 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes (Fsbeta-glucanase) is classified as one of the family 16 glycosyl hydrolases . It hydrolyzes the glycosidic bond in the mixed-linked glucans containing beta-1,3- and beta-1,4-glycosidic linkages . We constructed a truncated form of recombinant Fsbeta-glucanase containing the catalytic domain from amino acid residues 1-258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme . The crystal structure of the truncated Fsbeta-glucanase was solved at a resolution of 1.7A by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein . The overall topology of the truncated Fsbeta-glucanase consists mainly of two eight-stranded anti-parallel beta-sheets arranged in a jellyroll beta-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules . Sequence comparison with other bacterial glucanases showed that Fsbeta-glucanase is the only naturally occurring circularly permuted beta-glucanase with reversed sequences . Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share approximately 70% sequence identity, than to the naturally occurring Fsbeta-glucanase of similar topology with 30% identity . This result suggests that protein structure relies more on sequence identity than topology . The high-resolution structure of Fsbeta-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability. J Tongji Med Univ, 1999, 19(3), 161 - 5, 169 The construction of Schistosoma japonicum vaccine BCG-Sj26GST and its identification; Huangfu Y et al.; The expression of foreign gene, Schistosoma Japonicum 26 ku antigen (Sj26GST), in Bacillus Calmette-Guerin (BCG), Mycobacterium (M . smegmatis) and Escherichia coli (E . coli) were studied . The cDNA fragment encoding Sj26GST was amplified by PCR using plasmid pGEX, which could express Sj26GST in E . coli as template . The Sj26GST cDNA was cloned into the down-stream of human M . tuberculosis heat shock protein (hsp) 70 promoter with correct reading frame, and then the DNA fragment containing hsp70 promoter and Sj26GST gene were subcloned together into E . coli-Mycobacteria shuttle plasmid pBCG-2000 to construct the expression shuttle plasmid pBCG-Sj26 . The recombinant BCG and M . smegmatis mc(2)155, which were electroplated with pBCG-Sj26, could express Sj26GST and the recombinant Schistosoma Japonicum vaccine BCG-Sj26GST was made . The recombinant Sj26GST (rSj26GST) were soluble and could be observed on SDS-PAGE at molecular weight of 26 ku . The content of rSj26GST accounted for 15% and 10% of total bacterial protein in BCG and M . smegmatis respectively . The results of Western blot showed the combination of rSj26GST with antibody of GST. Appl Environ Microbiol, 2003 Jul, 69(7), 4111 - 5 Activity of free and clay-bound insecticidal proteins from Bacillus thuringiensis subsp . israelensis against the mosquito Culex pipiens; Lee L et al.; Bacillus thuringiensis subsp . israelensis produces parasporal insecticidal crystal proteins (ICPs) that have larvicidal activity against some members of the order Diptera, such as blackflies and mosquitoes . Hydrolysis of the ICPs in the larval gut results in four major proteins with a molecular mass of 27, 65, 128, and 135 kDa . Toxicity is caused by synergistic interaction between the 25-kDa protein (proteolytic product of the 27-kDa protein) and one or more of the higher-molecular-mass proteins . Equilibrium adsorption of the proteins on the clay minerals montmorillonite and kaolinite, which are homoionic to various cations, was rapid (<30 min for maximal adsorption), increased with protein concentration and then reached a plateau (68 to 96% of the proteins was adsorbed), was significantly lower on kaolinite than on montmorillonite, and was not significantly affected by the valence of the cation to which the clays were homoionic . Binding of the toxins decreased as the pH was increased from 6 to 11, and there was 35 to 66% more binding in phosphate buffer at pH 6 than in distilled water at pH 6 or 7.2 . Only 2 to 12% of the adsorbed proteins was desorbed by two washes with water; additional washings desorbed no more toxins, indicating that they were tightly bound . Formation of clay-toxin complexes did not alter the structure of the proteins, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the equilibrium supernatants and desorption washes and by dot blot enzyme-linked immunosorbent assay of the complexes, which was confirmed by enhanced chemiluminescence Western blot analysis . Free and clay-bound toxins resulted in 85 to 100% mortality of the mosquito Culex pipiens . Persistence of the bound toxins in nonsterile water after 45 days was significantly greater (mortality of 63% +/- 12.7%) than that of the free toxins (mortality of 25% +/- 12.5%). Appl Environ Microbiol, 2003 Jul, 69(7), 3986 - 95 Role of the Bacillus methanolicus citrate synthase II gene, citY, in regulating the secretion of glutamate in L-lysine-secreting mutants; Brautaset T et al.; The thermotolerant, restrictive methylotroph Bacillus methanolicus MGA3 (ATCC 53907) can secrete 55 g of glutamate per liter (maximum yield, 0.36 g/g) at 50 degrees C with methanol as a carbon source and a source of ammonia in fed-batch bioreactors . A homoserine dehydrogenase mutant, 13A52-8A66, secreting up to 35 g of L-lysine per liter in fed-batch fermentations had minimal 2-oxoglutarate dehydrogenase activity {7.3 nmol min(-1) (mg of protein)(-1)}, threefold-increased pyruvate carboxylase activity {535 nmol min(-1) (mg of protein)(-1)}, and elevated citrate synthase (CS) activity {292 nmol min(-1) (mg of protein)(-1)} and simultaneously secreted glutamate (20 to 30 g per liter) and L-lysine . The flow of carbon from oxaloacetate is split between transamination to aspartate and formation of citrate . To investigate the regulation of this branch point, the B . methanolicus gene citY encoding a CSII protein with activity at 50 degrees C was cloned from 13A52-8A66 into a CS-deficient Escherichia coli K2-1-4 strain . A citY-deficient B . methanolicus mutant, NCS-L-7, was also isolated from the parent strain of 13A52-8A66 by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis, followed by selection with monofluoroacetate disks on glutamate plates . Characterization of these strains confirmed that citY in strain 13A52-8A66 was not altered and that B . methanolicus possessed several forms of CS . Analysis of citY cloned from NCS-L-7 showed that the reduced CS activity resulted from a frameshift mutation . The level of glutamate secreted by NCS-L-7 was reduced sevenfold and the ratio of L-lysine to glutamate secreted was increased 4.5-fold compared to the wild type in fed-batch cultures with glutamate feeding . This indicates that glutamate secretion in L-lysine-overproducing mutants can be altered in favor of increased L-lysine secretion by regulating in vivo CS activity. Appl Environ Microbiol, 2003 Jul, 69(7), 3777 - 83 Construction of an expression system for site-directed mutagenesis of the lantibiotic mersacidin; Szekat C et al.; The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp . strain HIL Y-85,54728 . Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II . The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster . For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection . In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed . To test the expression system, three mutants were constructed . In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine . S16I mersacidin was produced only in small amounts . The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin . Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine . E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin . Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity. Scand J Infect Dis, 2003, 35(4), 240 - 3 Diagnosis of tuberculous lymphadenitis in Butajira, rural Ethiopia; Yassin MA et al.; Tuberculous lymphadenitis (TBLN) is a diagnostic challenge in sub-Saharan Africa, where there is a high rate of human immunodeficiency virus (HIV) infection . This study aimed to find ways to improve the diagnosis in Butajira, rural Ethiopia, where TBLN constitutes 40% of the total tuberculosis (TB) diagnosis . Among 147 clinically suspected cases, 107 (72.8%) were confirmed as TBLN by fine-needle aspiration (FNA) cytology and acid-fast bacillus (AFB) smear examination . Of the remaining 40 cases, denoted non-tuberculous lymphadenitis (NTBLN) after this smear examination, 37 (92.5%) showed a cytological pattern with neutrophil aggregates . The clinical manifestations were similar and cervical lymph nodes were the most affected in these 2 groups . 24 of the 107 TBLN cases (22.4%) and 9 (22.5%) of the other cases were seropositive for HIV infection (p > 0.5) . FNA cytology combined with AFB smear examination is a good alternative to histology in rural Ethiopia where the expertise in taking biopsies is very limited . Polymerase chain reaction for Mycobacterium tuberculosis complex DNA was positive in 15 of 23 cases tested with NTBLN cytology, showing that an additional independent criterion for the presence of M . tuberculosis is needed for diagnosis in lymphadenitis cases of this kind . These findings could help to strengthen the diagnostic algorithm suggested by the National TB Control Program. J Bacteriol, 2003 Jul, 185(14), 4256 - 67 Mycobacterium tuberculosis chaperonin 10 is secreted in the macrophage phagosome: is secretion due to dissociation and adoption of a partially helical structure at the membrane? Fossati G, Izzo G, Rizzi E, Gancia E, Modena D, Moras ML, Niccolai N, Giannozzi E, Spiga O, Bono L, Marone P, Leone E, Mangili F, Harding S, Errington N, Walters C, Henderson B, Roberts MM, Coates AR, Casetta B, Mascagni P. To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy . This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages . To understand the structural implications underlying this secretion, a structural study of M . tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment . It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a beta-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature . A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state . Remarkably, zwitterionic lipids had no effect on the protein structure . By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles . When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures . In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately beta-sheet in both dilute and concentrated solutions . Interestingly, human Cpn10 also crosses biological membranes, whereas the E . coli homologue is strictly cytosolic . These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M . tuberculosis Cpn10 to the external environment. J Dairy Sci, 2003 Jun, 86(6), 1947 - 52 Evaluation of screening test for detection of antimicrobial residues in ewe milk; Molina MP et al.; The effects of preservatives (potassium dichromate and sodium azide), heat treatment (untreated and 82 degrees C/10 min), and lactation stage upon the response of the microbial tests (BRT AiM and Delvotest) utilized for the detection of residues of antimicrobial substances in ewe milk were examined . Milk samples were collected from the morning milking of 50 Manchega ewes every 2 wk, from 15 d postpartum until the end of lactation . A total of 2322 samples were analyzed by BRT AiM with prediffusion and Delvotest microbial tests . The specificity of preservative-free milk samples without heat treatment was high (96.3% for BRT and 97.7% for Delvotest), with results improving for those samples thermally treated at 82 degrees C/10 min (99.0% for BRT and 98.7% for Delvotest) . Potassium dichromate produced a total inhibition of growth of Bacillus stearothermophilus with both methods . When acidiol was utilized, the specificity of the samples not treated thermally was lower compared with preservative-free milk samples for the BRT AiM (90.2%) and Delvotest (91.0%) methods, improving when the samples were thermally treated, both for BRT AiM (94.8%) and Delvotest (95.3%), given that the presence of the preservative increased the frequency of doubtful results . The lactation stage significantly affected the results of the methods, with a greater frequency of false-positive and doubtful cases toward the end of the cycle, especially in those samples preserved with acidiol . The greater selectivity in both methods was therefore obtained for preservative-free ewe milk samples with prior heat treatment taken at the beginning or in midlactation period. J Struct Funct Genomics, 2002, 2(3), 145 - 54 The X-ray crystal structure of pyrrolidone-carboxylate peptidase from hyperthermophilic archaea Pyrococcus horikoshii; Sokabe M et al.; The crystal structure of pyrrolidone-carboxylate peptidase (PCP) from hyperthermophilic archaea Pyrococcus horikoshii (PhoPCP) has been determined at 1.6-A resolution by X-ray crystallography . PCP belongs to the C15 family of cysteine protease, and specifically removes the amino terminal pyroglutamate residue from a wide range of N-terminal-blocking peptides . The crystal structure is very similar to that of other hyperthermophiles, Pyrococcus furiosus and Thermococcus litoralis, and even that from the mesophile, Bacillus amyloliquefaciens . The inter-subunit disulfide bonds, which have been proposed as one of the thermostabilizing factors of the PCP from such hyperthermophiles, was not present in PhoPCP . The result suggests that the thermostability of PhoPCP may be obtained by the accumulation of many weak factors. Biochemistry, 2003 Jul 8, 42(26), 8054 - 65 Family 39 alpha-l-iduronidases and beta-D-xylosidases react through similar glycosyl-enzyme intermediates: identification of the human iduronidase nucleophile; Nieman CE et al.; The inclusion of both beta-D-xylosidases and alpha-L-iduronidases within the same sequence-related family (family 39), despite the considerable difference in substrate structures and poor sequence conservation around the putative nucleophile, raises concerns about whether a common mechanism is followed by the two enzymes . A novel anchimeric assistance mechanism for iduronidases involving a lactone intermediate is one possibility . NMR analysis of the methanolysis reaction catalyzed by human alpha-L-iduronidase reveals that, as with the beta-D-xylosidases, alpha-L-iduronidase is a retaining glycosidase . Using two different mechanism-based inactivators, 5-fluoro-alpha-L-iduronyl fluoride and 2-deoxy-2-fluoro-alpha-L-iduronyl fluoride, the active site nucleophile in the human alpha-L-iduronidase was identified as Glu299 within the (295)IYNDEAD(301) sequence . The equivalent, though loosely predicted, glutamic acid was identified as the nucleophile in the family 39 beta-D-xylosidase from Bacillus sp . {Vocadlo, D., et al . (1998) Biochem . J . 335, 449-455}; thus, a common mechanism involving a covalent glycosyl-enzyme intermediate that adopts the rather uncommon (2,5)B conformation is predicted. Biosci Biotechnol Biochem, 2003 May, 67(5), 1094 - 100 Transglycosylation of glycosyl residues to cyclic tetrasaccharide by Bacillus stearothermophilus cyclomaltodextrin glucanotransferase using cyclomaltodextrin as the glycosyl donor; Shibuya T et al.; Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo{-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->}, CTS) . Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase . The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides . Saccharide A was identified as 4-mono-O-alpha-glucosyl-CTS, {-->6)-{alpha-D-Glcp-(1-->4)}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->}, and sachharide D was 4,4'-di-O-alpha-glucosyl-CTS, {-->6)-{alpha-D-Glcp-(1-->4)}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-{alpha-D-Glcp-(1-->4)}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->} . These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS. J Gen Appl Microbiol, 2003 Apr, 49(2), 101 - 9 Real-time quantitative PCR assay on bacterial DNA: In a model soil system and environmental samples; Kabir S et al.; Real-time quantitative PCR (RTQ-PCR) was used to quantify the bacterial target DNA extracted by three commonly used DNA extraction protocols (bead mill homogenization, grinding in presence of liquid nitrogen and hot detergent SDS based enzymatic lysis) . For the purpose of our study, pure culture of Bacillus cereus (model organism), sterilized soil seeded with a known amount of B . cereus (model soil system) and samples from woodland and grassland (environmental samples) were chosen to extract DNA by three different protocols . The extracted DNA was then quantified by RTQ-PCR using 16S rDNA specific universal bacterial primers . The standard curve used for the quantification by RTQ-PCR was linear and revealed a strong linear relationship (r(2)=0.9968) with a higher amplification efficiency, e5=1.02 . High resolution gel electrophoresis was also carried out to observe the effect of these extraction methods on diversity analysis . For the model soil system, the liquid nitrogen method showed the highest target DNA copy number (1.3 x 10(9) copies/microl) . However, for both the environmental samples, the bead beating method was found to be suitable on the basis of the high target DNA copy numbers (5.38 x 10(9) and 4.01 x 10(8) copies/ml for woodland and grassland respectively), high yield (6.4 microg/g and 1.76 microg/g of soil for woodland and grassland respectively) and different band patterns on high resolution gel electrophoresis suggesting an overall high extraction efficiency . This difference in the extraction efficiency between the model soil system and environmental samples may be attributed to different affinity of seeded and native DNA to soil particles. Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1313 - 6 Epub 2003 Jun 27. Expression, purification, crystallization and preliminary crystallographic analysis of Leishmania mexicana phosphoglycerate mutase; Poonperm B et al.; Bacterially expressed 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) from Leishmania mexicana with a six-His tag fused at its C-terminus was expressed from plasmid pET28a after IPTG induction in Escherichia coli cells and gave a yield of 20 mg of highly purified iPGAM per litre of cell culture . Crystals of the protein complexed with 3-phosphoglycerate were obtained by the hanging-drop method of vapour diffusion with PEG 4000 as the precipitating agent in the presence of cobalt chloride and diffracted synchrotron radiation to beyond 1.90 A . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 72.27, c = 129.68 A . A model of Bacillus stearothermophilus iPGAM (33% identity) was used to provide an initial molecular-replacement solution . X-ray data to 2.05 A for the structure of L . mexicana iPGAM complexed with 2-phosphoglycerate have also been collected. Biochim Biophys Acta, 2003 Jun 27, 1613(1-2), 15 - 27 Water-miscible organic cosolvents enhance phosphatidylinositol-specific phospholipase C phosphotransferase as well as phosphodiesterase activity; Wehbi H et al.; Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in a Ca(2+)-independent two-step mechanism: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I-1-P) . Moderate amounts of water-miscible organic solvents have previously been shown to dramatically enhance the cyclic phosphodiesterase activity, that is, hydrolysis of cIP . Cosolvents {isopropanol (iPrOH), dimethylsufoxide (DMSO), and dimethylformamide (DMF)} also enhance the phosphotransferase activity of PI-PLC toward PI initially presented in vesicles, monomers, or micelles . Although these water-miscible organic cosolvents caused large changes in PI particle size and distribution (monitored with pyrene-labeled PI fluorescence, 31P NMR spectroscopy, gel filtration, and electron microscopy) that differed with the activating solvent, the change in PI substrate structure in different cosolvents was not correlated with the enhanced catalytic efficiency of PI-PLC toward its substrates . PI-PLC stability was decreased in water/organic cosolvent mixtures (e.g., the T(m) for PI-PLC thermal denaturation decreased linearly with added iPrOH) . However, the addition of myo-inositol, a water-soluble inhibitor of PI-PLC, helped stabilize the protein . At 30% iPrOH and 4 degrees C (well below the T(m) for PI-PLC in the presence of iPrOH), cosolvent-induced changes in protein secondary structure were minimal . iPrOH and diheptanoylphosphatidylcholine, each of which activates PI-PLC for cIP hydrolysis, exhibited a synergistic effect for cIP hydrolysis that was not observed with PI as substrate . This behavior is consistent with a mechanism for cosolvent activation that involves changes in active site polarity along with small conformational changes involving the barrel rim tryptophan side chains that have little effect on protein secondary structure. J Biol Chem, 2003 Sep 19, 278(38), 36637 - 51 Epub 2003 Jun 26. Lipomannan and lipoarabinomannan from a clinical isolate of Mycobacterium kansasii: novel structural features and apoptosis-inducing properties; Guerardel Y et al.; Although Mycobacterium kansasii has emerged as an important pathogen frequently encountered in immunocompromised patients, little is known about the mechanisms of M . kansasii pathogenicity . Lipoarabinomannan (LAM), a major mycobacterial cell wall lipoglycan, is an important virulence factor for many mycobacteria, as it modulates the host immune response . Therefore, the detailed structures of the of M . kansasii LAM (KanLAM), as well as of its biosynthetic precursor lipomannan (KanLM), were determined in a clinical strain isolated from a human immunodeficiency virus-positive patient . Structural analyses revealed that these lipoglycans possess important differences as compared with those from other mycobacterial species . KanLAM carries a mannooligosaccharide cap but is devoid of the inositol phosphate cap present in Mycobacterium smegmatis . Characterization of the mannan core of KanLM and KanLAM demonstrated the following occurrences: 1) alpha1,2-oligo-mannopyranosyl side chains, contrasting with the single mannopyranosyl residues substituting the mannan core in all the other structures reported so far; and 2) 5-methylthiopentose residues that were described to substitute the arabinan moiety from Mycobacterium tuberculosis LAM . With respect to the arabinan domain of KanLAM, succinyl groups were found to substitute the C-3 position on 5-arabinofuranosyl residues, reported to be linked to the C-2 of the 3,5-arabinofuranose in Mycobacterium bovis bacillus calmette-guerin LAM . Because M . kansasii has been reported to induce apoptosis, we examined the possibility of the M . kansasii lipoglycans to induce apoptosis of THP-1 cells . Our results indicate that, in contrast to KanLAM, KanLM was a potent apoptosis-inducing factor . This work underlines the diversity of LAM structures among various pathogenic mycobacterial species and also provides evidence of LM being a potential virulence factor in M . kansasii infections by inducing apoptosis. Biochim Biophys Acta, 2003 Jun 20, 1622(1), 29 - 35 The cytotoxicity of Bacillus thuringiensis subsp . coreanensis A1519 strain against the human leukemic T cell; Namba A et al.; A novel cytotoxic protein was isolated from the crystal produced by Bacillus thuringiensis subsp . coreanensis A1519 strain . Upon treatment of the crystal proteins by proteinase K, the significant cytotoxicity toward the leukemic T cell, MOLT-4, was exhibited . The microscopic observation indicated that the cell death was accompanied by no extensive rupture of the cell membrane . It was, therefore, suggested that the cell death of MOLT-4 was induced through a mechanism other than the colloid-osmotic swelling and cell lysis as caused by hitherto known B . thuringiensis crystal proteins . The 29-kDa polypeptide proved to be an active component of the proteinase K-digested A1519 crystal proteins . EC(50) of the purified 29-kDa polypeptide was 0.078 microg/ml . The N-terminal amino acid sequence of the 29-kDa polypeptide shared no significant homology with all the known proteins, suggesting that this polypeptide belong to a new family of B . thuringiensis crystal proteins . In the ligand blotting analysis, specific binding proteins for the 29-kDa polypeptide were detected from the cell membrane of MOLT-4. Hum Pathol, 2003 Jun, 34(6), 589 - 96 Whipple's disease: immunospecific and quantitative immunohistochemical study of intestinal biopsy specimens; Lepidi H et al.; Whipple's disease may be diagnosed by periodic acid-Schiff (PAS) staining, electron microscopy, or polymerase chain reaction of intestinal biopsy specimens . The aim of this study was to evaluate the diagnostic value of immunohistochemistry and the quantification of infected cells in intestinal Whipple's disease . A total of 29 duodenal biopsy specimens from 15 patients with untreated and treated Whipple's disease were examined and compared with biopsy specimens from control patients with normal intestinal mucosa or various pathologic processes . Percentages of staining surfaces with PAS stain and antibodies directed against CD68, a macrophage marker, or the Whipple bacillus, Tropheryma whipplei, were studied quantitatively using a computerized system of image analysis . Positive detection of T . whipplei was obtained using immunohistochemistry in all 15 patients with Whipple's disease . No bacteria were detected in any of the negative controls . The use of quantitative image analysis showed a massive intestinal macrophagic infiltration before (20.3%) and after (13.4%) antibiotic therapy completion as compared with controls (2.1%) . The 2 detection methods for T . whipplei, PAS stain and immunohistochemistry, were quantitatively similar before therapy (19.9% versus 17.5%), but the immunodetection-based surface area was significantly lower than the PAS staining surface area after therapy (2.8% versus 7.9%) . Our findings indicate that immunohistochemistry is highly specific and sensitive and is applicable as a diagnostic method on intestinal tissue specimens to detect T . whipplei during active infection or in retrospective studies. Int Microbiol, 2003 Jun, 6(2), 127 - 9 Epub 2003 Jun 24. Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in Bacillus megaterium; Vazquez GJ et al.; Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb) . Enzyme activity was measured throughout the different growth phases of B . megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase . Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine . Overexpression of Act(Bm), a sigma(54) regulator from B . megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B . megaterium. Environ Health Perspect, 2003 Jun, 111(8), 1114 - 21 Clinical and laboratory investigation of allergy to genetically modified foods; Bernstein JA et al.; Technology has improved the food supply since the first cultivation of crops . Genetic engineering facilitates the transfer of genes among organisms . Generally, only minute amounts of a specific protein need to be expressed to obtain the desired trait . Food allergy affects only individuals with an abnormal immunologic response to food--6% of children and 1.5-2% of adults in the United States . Not all diseases caused by food allergy are mediated by IgE . A number of expert committees have advised the U.S . government and international organizations on risk assessment for allergenicity of food proteins . These committees have created decision trees largely based on assessment of IgE-mediated food allergenicity . Difficulties include the limited availability of allergen-specific IgE antisera from allergic persons as validated source material, the utility of specific IgE assays, limited characterization of food proteins, cross-reactivity between food and other allergens, and modifications of food proteins by processing . StarLink was a corn variety modified to produce a (Italic)Bacillus thuringiensis(/Italic) (Bt) endotoxin, Cry9C . The Centers for Disease Control and Prevention investigated 51 reports of possible adverse reactions to corn that occurred after the announcement that StarLink, allowed for animal feed, was found in the human food supply . Allergic reactions were not confirmed, but tools for postmarket assessment were limited . Workers in agricultural and food preparation facilities have potential inhalation exposure to plant dusts and flours . In 1999, researchers found that migrant health workers can become sensitized to certain Bt spore extracts after exposure to Bt spraying. J Am Mosq Control Assoc, 2003 Jun, 19(2), 125 - 9 Biological fitness of a Culex quinquefasciatus population and its resistance to Bacillus sphaericus; de Oliveira CM et al.; Biological fitness components of a field-collected colony of Culex quinquefasciatus Say that was highly resistant to Bacillus sphaericus strain 2362 (resistance ratio greater than 163,000) after 46 generations of selection were compared to those of a susceptible colony (CqSF) that had originated from the same parental cohort but that had not been exposed to B . sphaericus . The effect of B . sphaericus on the fitness of Cx . quinquefasciatus was determined in terms of fecundity, fertility, and development time . The resistant colony (CqRL) showed significantly lower fecundity and fertility, and slower development than the susceptible colony . Development time from egg to egg showed a 20% increase in CqRL compared to CqSF . The generation time increased from 21.6 days to 26 days for highly resistant generations of CqRL. J Comput Aided Mol Des, 2002 Dec, 16(12), 935 - 53 A structure-based design approach for the identification of novel inhibitors: application to an alanine racemase; Mustata GI et al.; We report a new structure-based strategy for the identification of novel inhibitors . This approach has been applied to Bacillus stearothermophilus alanine racemase (AlaR), an enzyme implicated in the biosynthesis of the bacterial cell wall . The enzyme catalyzes the racemization of L- and D-alanine using pyridoxal 5'-phosphate (PLP) as a cofactor . The restriction of AlaR to bacteria and some fungi and the absolute requirement for D-alanine in peptidoglycan biosynthesis make alanine racemase a suitable target for drug design . Unfortunately, known inhibitors of alanine racemase are not specific and inhibit the activity of other PLP-dependent enzymes, leading to neurological and other side effects . This article describes the development of a receptor-based pharmacophore model for AllaR, taking into account receptor flexibility (i.e . a 'dynamic' pharmacophore model) . In order to accomplish this, molecular dynamics (MD) simulations were performed on the full AlaR dimer from Bacillus stearothermophilus (PDB entry, 1 sft) with a D-alanine molecule in one active site and the non-covalent inhibitor, propionate, in the second active site of this homodimer . The basic strategy followed in this study was to utilize conformations of the protein obtained during MD simulations to generate a dynamic pharmacophore model using the property mapping capability of the LigBuilder program . Compounds from the Available Chemicals Directory that fit the pharmacophore model were identified and have been submitted for experimental testing . The approach described here can be used as a valuable tool for the design of novel inhibitors of other biomolecular targets. Protein Sci, 2003 Jul, 12(7), 1538 - 46 Grafting a new metal ligand in the cocatalytic site of B . cereus metallo-beta-lactamase: structural flexibility without loss of activity; Rasia RM et al.; Metallo-beta-lactamases are zinc enzymes able to hydrolyze the four-membered ring of beta-lactam antibiotics, representing one of the latest generations of beta-lactamases . These enzymes belong to the zinc metallo-hydrolase family of the beta-lactamase fold . Enzymes belonging to this family have a bimetallic active site whose structure varies among different members by point substitutions of the metal ligands . In this work, we have grafted new metal ligands into the metal binding site of BcII from Bacillus cereus that mimic the ligands present in other members of this superfamily . We have characterized spectroscopically and modeled the structure of the redesigned sites, which differ substantially from the wild-type enzyme . Despite the changes introduced in the active site, the mutant enzymes retain almost full activity . These results shed some light on the possible evolutionary origin of these metalloenzymes. Trends Microbiol, 2003 Jun, 11(6), 259 - 63 How is the phagocyte lectin keyboard played? Master class lesson by Mycobacterium tuberculosis; Tailleux L et al.; Mammals have evolved surface pattern recognition receptors, such as the Toll-like receptors, to initiate defenses against pathogens, including mycobacterium . In turn, microbes have developed strategies to circumvent defenses of their host and establish persistent infections . Mycobacterium tuberculosis, one of the most successful pathogens worldwide, has the ability to parasitize and manipulate phagocytic cells of its human host . A set of recent reports has shed light on exploitation of phagocyte surface lectins by the tubercle bacillus . These findings could lead the way to innovative therapeutic approaches. Clin Exp Immunol, 2003 Jul, 133(1), 30 - 7 A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes; Lopez B et al.; In the last decade, an unprecedented genetic diversity has been disclosed among Mycobacterium tuberculosis strains found worldwide . However, well-conserved genotypes seem to prevail in areas with high incidence of tuberculosis . As this may be related to selective advantages, such as advanced mechanisms to circumvent {M . bovis Bacille Calmette-Guerin (BCG)-induced} host defence mechanisms, we investigated the influence of strain diversity on the course of experimental disease . Twelve M . tuberculosis strains, representing four major genotype families found worldwide today, and the laboratory strain H37Rv were each used to infect BALB/c mice by direct intratracheal injection . Compared with H37Rv, infections with Beijng strains were characterized by extensive pneumonia, early but ephemeral tumour necrosis factor-alpha (TNF-alpha) and inducible isoform of nitric oxide synthetase (iNOS) expression, and significantly higher earlier mortality . Conversely, Canetti strains induced limited pneumonia, sustained TNF-alpha and iNOS expression in lungs, and almost 100% survival . Strains of the Somali and the Haarlem genotype families displayed less homogeneous, intermediate rates of survival . Previous BCG vaccination protected less effectively against infection with Beijing strains than against the H37Rv strain . In conclusion, genetically different M . tuberculosis strains evoked markedly different immunopathological events . Bacteria with the Beijing genotype, highly prevalent in Asia and the former USSR, elicited a non-protective immune response in mice and were the most virulent . Future immunological research, particularly on candidate vaccines, should include a broad spectrum of M . tuberculosis genotypes rather than a few laboratory strains. Environ Microbiol, 2003 Jul, 5(7), 618 - 27 Molecular characterization of endolithic cyanobacteria inhabiting exposed dolomite in central Switzerland; Sigler WV et al.; The phototrophic microbial community inhabiting exposed dolomite in the alpine Piora Valley (Switzerland) forms a distinct endolithic bilayer that features adjacent red dolomite (exterior) and green dolomite (interior) layers that are c . 0.5-1 mm below the rock surface . Characterization of the community, with an emphasis on cyanobacteria, was conducted with culture-dependent and -independent approaches . Direct microscopy of green dolomite revealed four distinct morphotypes consistent with Chlorophyta genera Chlorella and Stichococcus and the Cyanobacterial genera Nostoc and Calothrix, whereas only Stichococcus and Nostoc were observed in the red dolomite . Enrichment in BG-11 media resulted in the growth of Chlorella and Stichococcus . Denaturing gradient gel electrophoresis (DGGE) analysis of DNA extracted from the enrichment revealed two dominant phylotypes with sequence similarity to Chlorella osrokiniana chloroplast and the cyanobacteria genus Leptolyngbya . 16S rRNA gene-based DGGE analysis of DNA extracted directly from both layers indicated that although both layers harboured phylotypes most similar to the Cyanobacterial genera Nostoc, Chroococcidiopsis, and Microcoleus, and the Chlorophyte Stichococcus bacillaris, the two layers also harboured unique genera such as Scytonema, and Symploca (red, external layer of dolomite) and Chlorella (green, internal layer of dolomite) . The unique community structure of each layer suggests a selection process directed by the pressures of the endolithic environment . We conclude that the overall composition of the phototrophic community closely resembles that of endolithic communities located in extreme habitats, suggesting that a cosmopolitan community inhabits this defined niche. Appl Microbiol Biotechnol, 2003 Nov, 63(1), 51 - 6 Epub 2003 Jun 18. Biochemical studies on cloned Bacillus sp . BP-7 phenolic acid decarboxylase PadA; Prim N et al.; Sequence analysis of a Bacillus sp . BP-7 recombinant clone coding for a previously described carboxylesterase revealed the presence of an additional ORF with homology to bacterial hydroxycinnamic acid decarboxylases . Analysis of the amino acid sequence of the encoded enzyme revealed the presence of a single, highly conserved domain of 161 amino acids, with a predicted molecular mass of 19,143 Da and a pI of 5.5 . Crude cell extracts from the recombinant clone displayed activity on ferulic, p-coumaric and caffeic acids, with no need for added cofactors . The cloned enzyme, named PadA, displayed maximum activity at 40 degrees C and pH 5.5, being stable over a broad range of pH and up to 45 degrees C . HPLC analysis of the products of catalysis revealed the conversion of phenolic acids to their aromatic 4-vinyl derivatives, with no accumulation of other by-products . PadA was found as a homodimer in the parental Bacillus sp . BP-7 strain and its expression was induced by both hydroxycinnamic acids and their corresponding derivative products . The results obtained suggest that the enzyme could be involved in a stress response for conversion of toxic hydroxycinnamic acids released after plant cell wall degradation. Biotechnol Appl Biochem, 2003 Oct, 38(Pt 2), 175 - 81 Similarities between the thermal inactivation kinetics of Bacillus amyloliquefaciens alpha-amylase in an aqueous solution of sodium dodecyl sulphate and the kinetics in the solution of anionic-phospholipid vesicles; Tanaka A et al.; An anionic surfactant, SDS, is commonly used to model compounds of negatively charged phospholipids . The effect of SDS on the thermal inactivation of BAA (alpha-amylase from Bacillus amyloliquefaciens ) was compared with the effect of negatively charged phospholipid vesicles of DOPA (dioleoylphosphatidic acid) at 40-55 degrees C . The inactivation kinetics revealed that both SDS below its c.m.c . (critical micellar concentration) and DOPA vesicles accelerated the inactivation and the unfolding of the enzyme structure . Both SDS and DOPA vesicles lowered the activation enthalpy and entropy for the inactivation . The lowered activation parameters might be due to the relatively small energy requirement needed to reach the transition state from the ground state of BAA in the presence of the negatively charged hydrophobic environments . This study suggests an accelerated unfolding of BAA in the presence of SDS to be energetically similar to the accelerated unfolding in the presence of DOPA vesicles that involve a molten-globule-like state. Izv Akad Nauk Ser Biol, 2003 May-Jun, (3), 311 - 4 {Biological role of lectins from Bacillus polymyxa during interaction with surface carbohydrates of wheat roots }; Karpunina LV et al.; The data obtained demonstrate the capacity of lectins LI and LII from the soil nitrogen-fixing bacteria Bacillus polymyxa 1460 to change their proteolytic activity after interaction with surface carbohydrates of wheat seedling roots. J Biotechnol, 2003 Jun 26, 103(2), 129 - 35 Kinetic parameters of continuous cultures of Bacillus thermoleovorans sp . A2 degrading phenol at 65 degrees C; Feitkenhauer H et al.; In this paper, we report on the kinetics of phenol degradation and cell growth in continuous cultures of suspended cells of Bacillus thermoleovorans sp . A2 at 65 degrees C . A high yield coefficient of Y(x/s)=0.84 g cell dry weight g(-1) phenol was measured at a dilution rate of 0.5 h(-1) . At the same dilution rate the coefficient for maintenance metabolism (m(s)) was determined to be 0.045 g phenol g(-1) cell dry weight h(-1) . The maximal growth rate (wash-out) determined at a phenol inlet concentration of 188 mg l(-1) was 0.9 h(-1) . Up to 7 g phenol l(-1) per day were degraded in a continuously operated 2-l stirred tank reactor with suspended cells (feed concentration 660 mg l(-1)) . Additionally, yield coefficients for oxygen and ammonium are reported. Yi Chuan Xue Bao, 2003 Apr, 30(4), 364 - 9 {Cloning and sequencing of chitinase gene from Bacillus thuringiensis subsp israelensis}; Zhong WF et al.; Chitin, a linear homopolymer of N-acetyl-D-glucosamine, is a common constituent of fungal cell walls, exoskeletons of insects, and shells of invertebrates . Thus, chitinase, a chitin hydrolytic enzyme, has become of interest for potential use as biopesticides for controlling insect pests . Using a pair of specific primers, chitinase gene (ichi) was amplified by touchdown PCR from Bacillus thuringiensis subsp israelensis chromosomal DNA, and then subcloned into pGEM-T easy vector . ichi sequence (GenBank Accession Number: AF526379) with a length of 2570 bp included an open reading frame (ORF) of 2067 bp, which contained 688 amino acids with Mr = 75.79 kDa and pI = 5.90 . The amino acid sequence of ichi gene shows 97.24%, 97.18%, 97.63% and 63.07% identity to that of Bacillus cereus strain 28-9 chitinase CW, Bacillus cereus CH chitinase B, Bt subsp Mexican chitinase, and Bt subsp pakistani chitinase A71, respectively . It was demonstrated that Ichi contains a signal peptide (46 amino acid residues) and three functional domains: an N-terminal catalytic domain (105 amino acid residues), a fibronectin type III like domain (74 amino acid residues), and a C-terminal chitin-binding domain (40 amino acid residues). Int J Food Microbiol, 2003 Aug 15, 85(1-2), 73 - 81 Antibacterial activity of 11 essential oils against Bacillus cereus in tyndallized carrot broth; Valero M et al.; The antibacterial activity of 11 essential oils from aromatic plants against the strain INRA L2104 of the foodborne pathogen Bacillus cereus grown in carrot broth at 16 degrees C was studied . The quantity needed by the essential oils of nutmeg, mint, clove, oregano, cinnamon, sassafras, sage, thyme or rosemary to produce 14-1110% relative extension of the lag phase was determined . Total growth inhibition of bacterial spores was observed for some of the antimicrobial agents assayed . The addition of 5 microl cinnamon essential oil per 100 ml of broth in combination with refrigeration temperatures of <or=8 degrees C produced the conditions necessary to inhibit the growth of B . cereus for at least 60 days in a model, refrigerated minimally processed food product, made with carrots and tyndallized . This is especially important considering that the psychrotrophic enterotoxigenic strain of B . cereus INRA TZ415 was able to grow in this substrate at low temperatures in the absence of any essential oil . Furthermore, the study of the sensory characteristics of the final product suggests that the use of cinnamon essential oil can be considered as an alternative to "traditional food preservatives". Enferm Infecc Microbiol Clin, 2003 Jun-Jul, 21(6), 299 - 307; quiz 308, 326 {Drugs with activity against Mycobacterium tuberculosis}; Coll P; Treatment for Mycobacterium tuberculosis has to be lengthy, since populations of this bacillus differ in metabolic activity, and it has to consist of various associated drugs, since spontaneous chromosome mutations can give rise to drug resistance . The multiresistant phenotype emerges with sequential acquisition of mutations in several loci of separate genes . Knowledge of the mechanisms of resistance permits the development of molecular techniques for the early detection of resistant strains, thereby making proper control possible . Tuberculosis treatment includes isoniazid, rifampicin and pyrazinamide during the first two months and isoniazid and rifampicin to complete six months of treatment . In specific situations, a fourth drug is added, ethambutol for adults and streptomycin for children in whom visual acuity cannot be monitored . This review describes the characteristics, activity, resistance mechanisms and side effects associated with the various antituberculosis drugs. Biochemistry, 2003 Jun 24, 42(24), 7518 - 26 Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: the role of alanine 230 in acceptor subsite +1; Leemhuis H et al.; Cyclodextrin glycosyltransferase (CGTase) preferably catalyzes transglycosylation reactions, whereas many other alpha-amylase family enzymes are hydrolases . Despite the availability of three-dimensional structures of several transglycosylases and hydrolases of this family, the factors that determine the hydrolysis and transglycosylation specificity are far from understood . To identify the amino acid residues that are critical for the transglycosylation reaction specificity, we carried out error-prone PCR mutagenesis and screened for Bacillus circulans strain 251 CGTase mutants with increased hydrolytic activity . After three rounds of mutagenesis the hydrolytic activity had increased 90-fold, reaching the highest hydrolytic activity ever reported for a CGTase . The single mutation with the largest effect (A230V) occurred in a residue not studied before . The structure of this A230V mutant suggests that the larger valine side chain hinders substrate binding at acceptor subsite +1, although not to the extent that catalysis is impossible . The much higher hydrolytic than transglycosylation activity of this mutant indicates that the use of sugar acceptors is hindered especially . This observation is in favor of a proposed induced-fit mechanism, in which sugar acceptor binding at acceptor subsite +1 activates the enzyme in transglycosylation {Uitdehaag et al . (2000) Biochemistry 39, 7772-7780} . As the A230V mutation introduces steric hindrance at subsite +1, this mutation is expected to negatively affect the use of sugar acceptors . Thus, the characteristics of mutant A230V strongly support the existence of the proposed induced-fit mechanism in which sugar acceptor binding activates CGTase in a transglycosylation reaction. Biochemistry, 2003 Jun 24, 42(24), 7390 - 9 HU protein employs similar mechanisms of minor-groove recognition in binding to different B-DNA sites: demonstration by Raman spectroscopy; Serban D et al.; The sequence isomers d(CGCAAATTTGCG) and d(TCAAGGCCTTGA) form self-complementary duplexes that present distinct targets for binding of the homodimeric architectural protein HU of Bacillus stearothermophilus (HUBst) . Raman spectroscopy shows that although each duplex structure is of the B-DNA type, there are subtle conformational dissimilarities between them, involving torsion angles of the phosphodiester backbone and the arrangements of stacked bases . Each DNA duplex forms a stable stoichiometric (1:1) complex with HUBst, in which the structure of the HUBst dimer is largely conserved . However, the Raman signature of each DNA duplex is perturbed significantly and similarly with HUBst binding, as reflected in marker bands assigned to localized vibrations of the phosphodiester moieties and base residues . The spectral perturbations identify a reorganization of the DNA backbone and partial unstacking of bases with HUBst binding, which is consistent with non-sequence-specific minor-groove recognition . Prominent among the HUBst-induced perturbations of B-DNA are a conversion of approximately one-third of the alpha/beta/gamma torsions from the canonical g(-)/t/g(+) conformation to an alternative conformation, an equivalent conversion of deoxyadenosyl moieties from the C2'-endo/anti to the C3'-endo/anti conformation, and appreciable unstacking of purines . The results imply that each solution complex is characterized by structural perturbations extending throughout the 12-bp sequence . Comparison with previously studied protein/DNA complexes suggests that binding of HUBst bends DNA by approximately 70 degrees. Braz J Infect Dis, 2003 Feb, 7(1), 73 - 81 Epub 2003 Dec 02. Clinical and radiological analysis of children and adolescents with tuberculosis in Bahia, Brazil; Franco R et al.; We reviewed the clinical and radiological characteristics of tuberculosis (TB) in children and adolescents at the Hospital Especializado Octavio Mangabeira, (HEOM) in Salvador, Bahia . This study included 275 TB patients aged 1 to 15 years seen between January 1990 and November 2001 . Standardized forms were filled out on the basis of a review of patient records and x-rays . Through a retrospective and descriptive analysis, it was found that 51.6% were male, 35.3% were aged 1 to 5 years, 28% were aged 6 to 10 and 36.7% were aged 11 to 15 . Among all patients, 79.6% lived in the city of Salvador . A history of contact with TB was found in 63.9%, most frequently among children under 5 years old; 77.2% were vaccinated with Bacillus Calmette-Guerin (BCG) . The most frequently observed symptoms were coughing (76%), fever (73.1%) weight loss (53.1%), and 4.7% were asymptomatic . Pulmonary TB was most frequent (57.8%) and extra-pulmonary TB occurred in 24.4%, with a predominance of hilar adenopathy . Both forms occurred simultaneously in 17.8% . In 53.1% of the cases the diagnosis was not determined by bacteriology or pathological anatomy; in these cases diagnosis was reached through clinical and radiological criteria, contact history, a tuberculin test > or = 10mm and a positive response to tuberculostatic drugs. Braz J Infect Dis, 2003 Feb, 7(1), 1 - 6 What do we (not) know about the human bartonelloses? Velho PE, Cintra ML, Uthida-Tanaka AM, de Moraes AM, Mariotto A. The human bartonelloses are a group of diseases with a rapidly increasing clinical spectrum . Well known manifestations such as Carrion's disease, trench fever, cat-scratch disease, and bacillary angiomatosis are examples of Bartonella sp . infection . Along with these diseases, recurrent bacteremia, endocarditis, septicemia, erythema nodosum, erythema multiforme, trombocytopenic purpura and other syndromes have been reported having been caused by bacteria of this genus . The infectious process and the pathogenesis of these microorganisms are poorly understood . The bartonelloses may have a benign and self-limited evolution in a host, or a potentially fatal one . These bacteria can provoke a granulomatous or an angioproliferative histopathologic response . As these diseases are not yet well defined, we have reviewed the four main human bartonelloses and have examined unclear points about these emergent diseases. J Appl Microbiol, 2003, 95(1), 23 - 8 Cloning and study of the expression of a novel cry1Ia-type gene from Bacillus thuringiensis subsp . kurstaki; Tounsi S et al.; AIMS: Cloning and expression of a new cry1Ia-type gene of Bacillus thuringiensis . METHODS AND RESULTS: PCR amplification, using gene cry1I-specific primers revealed the presence of such a gene in the strain BNS3 of Bacillus thuringiensis subsp . kurstaki . The cloning and sequencing from BNS3 of the cry1Ia-type gene, called crybns3-3, showed an open reading frame of 2160-bp, encoding a protein of 719 amino acid residues . Both nucleotide and amino acid sequences similarity analysis revealed that the crybns3-3 is a new cry1Ia-type gene, presenting several differences from the cry1Ia-type genes . The study of the expression of crybns3-3 by Northern blot and RT-PCR showed that it was transcribed . The expression of crybns3-3 under the control of BtI and BtII promoters revealed that Crybns3-3 would co-crystallize with the endogenous delta-endotoxins . CONCLUSIONS: crybns3-3 is a novel cry1Ia gene isolated from B . thuringiensis subsp . kurstaki strain BNS3 . SIGNIFICANCE AND IMPACT OF THE STUDY: The characteristics of crybns3-3 indicate that it is a new cry1Ia-type gene . Amino acid residue substitutions presented in Crybns3-3 could be exploited for both toxicity and specificity studies . Crybns3-3 would interact and co-crystallize at least partially with the endogenous delta-endotoxins of BNS3, and then participate in the formation of the parasporal crystal inclusions. Int J Syst Evol Microbiol, 2003 May, 53(Pt 3), 725 - 30 Bacillus barbaricus sp . nov., isolated from an experimental wall painting; Taubel M et al.; In a project concerning bacterial colonization of experimental wall paintings, a large number of isolates have been acquired with high similarities in their whole-cell protein patterns obtained after SDS-PAGE . Of this group, four strains, designated V2-BIII-A2(T), V2-BI-A9, V2-BI-04 and V2-BII-A8, were chosen for further characterization . Banding patterns obtained after ERIC-PCR were barely distinguishable among these four strains, indicating their affiliation within a single species . The isolates also displayed nearly identical biochemical and physiological features . The chemotaxonomic characteristics, including polar lipids, quinone systems, cell-wall diamino acid composition and fatty acid profiles, were in good agreement with those of numerous previously described Bacillus species . 16S rDNA analysis of strain V2-BIII-A2(T) showed that this bacterium belongs to the genus Bacillus, with highest sequence similarities to Bacillus megaterium (94.6%), Bacillus flexus (94.4%) and the alkaliphilic Bacillus cohnii (94.2%) . Based on almost identical biochemical, physiological and chemotaxonomic traits, ERIC-PCR-generated genomic fingerprints and comparative 16S rDNA sequence analysis, it is demonstrated that the four isolates represent a novel species of the genus Bacillus, for which the name Bacillus barbaricus sp . nov . is proposed . The type strain is V2-BIII-A2(T) (= CCM 4982(T) = DSM 14730(T)). C R Biol, 2003 Mar, 326(3), 317 - 27 Factors influencing the toxicity of xenobiotics against larval mosquitoes; Rey D et al.; In order to examine the factors influencing xenobiotic toxicity against larval mosquitoes, the larvicidal performances of two conventional insecticides (temephos and Bacillus thuringiensis var . israelensis: Bti) and a new potential phyto-insecticide (decomposed leaf litter) were compared under different conditions against three detritivorous larval mosquito types . Bioassays performed under standard conditions indicated differential tolerance levels according to the xenobiotic and the larval type . Bioassays performed under different conditions of xenobiotic dose and geometry of the water column indicated differential effects of those parameters on mortality rates . This allowed us to distinguish the performances of temephos versus those of Bti and leaf litter . These toxicological performances were examined as indicators for analysis of xenobiotic bioavailability for mosquito larvae in environmental water, and also for their comparative interest in field mosquito control. J Biol Chem, 2003 Aug 22, 278(34), 31473 - 8 Epub 2003 Jun 12. A novel double heme substitution produces a functional bo3 variant of the quinol oxidase aa3 of Bacillus cereus . Purification and paratial characterization; Contreras-Zentella M et al.; A novel bo3-type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa3 and caa3 . The purified enzyme contained 12.4 nmol of heme O and 11.5 nmol of heme B mg-1 protein . The enzyme was composed of two subunits with an Mr of 51,000 and 30,000, respectively . Both subunits were immunoreactive to antibodies raised against the B cereus aa3 oxidase . Moreover, amino-terminal sequence analysis of the 30-kDa subunit revealed that the first 19 residues were identical to those from the 30-kDa subunit of the B . cereus aa3 oxidase . The purified bo3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent Km of 498 microM, a Vmax of 21 micromol of O2 min-1mg-1, and a calculated turnover of 55 s-1 . The quinol oxidase activity with tetrachlorohydroquinol was inhibited by potassium cyanide and 2-n-heptyl 4-hydroxyquinoline-N-oxide with an I50 of 24 and 300 microM, respectively . Our results demonstrate that the bo3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa3 apoprotein encoded by the qox operon of the aa3 oxidase of B . cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo3 cytochrome . This is the first reported example of an enzymatically active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites. Vaccine, 2003 Jul 4, 21(23), 3149 - 56 Immunization with recombinant Calmette-Guerin bacillus (BCG)-hepatitis C virus (HCV) elicits HCV-specific cytotoxic T lymphocytes in mice; Uno-Furuta S et al.; Since virus-specific cytotoxic T lymphocytes (CTLs) play a critical role in preventing the spread of hepatitis C virus (HCV), an effective HCV vaccine should be capable of eliciting HCV-specific CTLs . In the present study, we assessed the capability of a novel recombinant vaccine using an attenuated tuberculosis bacillus, Calmette-Guerin bacillus (BCG), as a vaccine vehicle to elicit HCV-specific CTLs . BCG was engineered to express the CTL epitope of HCV-non-structure protein 5a (NS5a) as a chimeric protein with alpha antigen of mycobacteria . Immunization with this recombinant BCG elicited major histocompatibility complex class I-restricted CD8(+) HCV-NS5a-specific CTLs in mice . Immunized mice showed a substantial reduction in the vaccinia virus titer compared with control mice when the immunized mice were challenged with a recombinant vaccinia virus expressing HCV-NS5a genes . These findings provide evidences for the possibility of BCG as a vaccine vector and its continued exploration as a vehicle for eliciting HCV-specific immunity. Can J Urol, 1995 Mar, 2(Supp1), 1 - 6 Optimal treatment of superficial bladder cancer in 1994; Lamm D; Bladder cancer, one of the first cancers associated with industrialization, is not unexpectedly increasing in incidence throughout the world . Despite the increasing incidence of bladder cancer, mortality has actually decreased in recent years . In advanced and metastatic bladder cancer, the platinum/methotrexate-based combination of chemotherapy may be responsible for this improved survival . However, chemotherapy appears not to have influenced survival in patients with superficial disease . Increasing evidence suggests that bacillus calmette-Guerin immunotherapy can reduce both disease progression and mortality. Lett Appl Microbiol, 2003, 37(1), 75 - 80 Enhanced secretion of heterologous cyclodextrin glycosyltransferase by a mutant of Bacillus licheniformis defective in the D-alanylation of teichoic acids; Craynest M et al.; AIMS: To examine whether inactivation of the dlt operon and increased charge density of the wall enhances secretion of heterologous proteins in industrial strains of Bacillus licheniformis . METHODS AND RESULTS: The dltA gene of B . licheniformis was cloned, sequenced and mutated by inserting a chloramphenicol acetyl transferase (cat) gene cassette . The mutation facilitated growth in the late exponential growth phase, increased endogenous autolysis and decreased resistance to a cationic peptide, polylysine . It was observed that dltA mutation increased the production of cyclodextrin glycosyltransferase (CGTase) by 1.5- to sevenfold depending on the growth phase, but decreased the production of penicillinase by twofold . CONCLUSIONS AND SIGNIFICANCE: The results suggest that the d-alanylation of teichoic acids is an element that can be used to improve the production of some secretory proteins in industrial applications based on this important industrial microorganism. Lett Appl Microbiol, 2003, 37(1), 61 - 5 Bacteriocidal effects and inhibition of cell separation of cinnamic aldehyde on Bacillus cereus; Kwon JA et al.; AIMS: In this study, bacteriocidal effects of cinnamic aldehyde on Bacillus cereus were investigated . METHODS: The bacterial culture or cell suspension in 0.85% NaCl was treated with cinnamic aldehyde at a concentration of 0.3 ml l(-1) . Viable cells were counted on a nutrient agar plate . Protein leakage from the cell was determined using a protein dye . Cell morphology was observed using a scanning electron microscope . RESULTS: Bacillus cereus cells were the most sensitive to cinnamic aldehyde among four different food-borne pathogens . When the cells were treated with 0.3 ml l(-1) of cinnamic aldehyde, the viable counts decreased about 6 log cycles after 6 h of incubation . The bacterial cells remained unlysed although they were killed by cinnamic aldehyde . Treatment of cinnamic aldehyde to the exponential phase cells resulted in no significant protein leakage but strong inhibition of cell separation . CONCLUSIONS: The present findings suggest that cinnamic aldehyde exhibits bacteriocidal effects and inhibition of cell separation on B . cereus . SIGNIFICANCE AND IMPACT OF THE STUDY: These data represent an interesting background for a possible mechanism for antibacterial effects of cinnamic aldehyde. Lett Appl Microbiol, 2003, 37(1), 31 - 4 Effect of feed gas composition of gas discharge plasmas on Bacillus pumilus spore mortality; Purevdorj D et al.; AIMS: To investigate the effect of gas composition on the sensitivity of Bacillus pumilus spores to gas plasmas . METHODS AND RESULTS: Inert gas plasmas, oxygen-based plasmas and various moisturized air plasmas were used to inactivate B . pumilus spores in low gas pressure of 50 Pa . Although the treatment temperature did not exceed 55 degrees C when exciting these plasmas, spore survival varied widely depending on the composition of the gas feed . Higher spore mortality was acquired by inert gases of low molecular weight except for helium . The highest spore mortality (4.54log reduction) was obtained when air with a 0.05 molar fraction of water vapour was used as the plasma carrier gas . CONCLUSIONS: Water molecules in the plasma carrier gas play a significant role in inactivation of B . pumilus spores . SIGNIFICANCE AND IMPACT OF THE STUDY: This strong inactivation may occur through hydroxyl free radicals generated from the moisturized air plasma. Clin Exp Allergy, 2003 Jun, 33(6), 731 - 6 Absence of association between delayed type hypersensitivity to tuberculin and atopy in children in The Gambia; Ota MO et al.; BACKGROUND: An inverse association between delayed type hypersensitivity to tuberculin and atopy has been observed in children, suggesting that exposure to mycobacteria may influence the immune response to allergens . OBJECTIVE: To investigate the relationship between tuberculin responses and atopy in children living in three different environments in The Gambia . METHODS: In this cross-sectional study a total of 507 school-aged children were recruited from rural, urban poor or urban affluent communities . They were assessed for skin responses to five common allergens and tuberculin, presence of bacille Calmette-Guerin (BCG) scar, presence of intestinal parasites, and total serum IgE . Atopy was defined as the presence of a skin prick test response > or = 3 x 3 mm to at least one allergen . RESULTS: The overall prevalence of atopy was 33% but there was a significant variation among the three study groups . The prevalence of atopy was 22% in urban poor, 36% in urban affluent, and 43% in rural children . Controlling for potential confounding factors, children in the rural community had a significantly higher odds ratio, 3.3 (95% confidence interval 1.8-6.0) of being atopic than children from the urban poor community . No association between atopy and tuberculin response or BCG scar was observed in any of the three groups . Serum IgE levels were higher among children of the urban poor group but were not associated with tuberculin response or BCG scar in any of the groups . CONCLUSION: Environmental factors have an important influence on the development of atopy in children in The Gambia but delayed type hypersensitivity to tuberculin is not a protective factor. J Food Prot, 2003 Jun, 66(6), 1047 - 54 Potential of Bacillus cereus for producing an emetic toxin, cereulide, in bakery products: quantitative analysis by chemical and biological methods; Jaaskelainen EL et al.; A method for the direct quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, in bakery products was developed . The analysis was based on robotized extraction followed by quantitation of cereulide by liquid chromatography-mass spectrometry and an assay of toxicity by the boar sperm motility inhibition test . The bioassay and the chemical assay gave comparable results, demonstrating that the extracted cereulide was in a biologically active form . Cereulide was formed when cereulide-producing B . cereus strains were present at > or = 10(6) CFU/g in products with water activity values of > 0.953 and pHs of > 5.6 . Rice-containing pastries accumulated high contents of cereulide (0.3 to 5.5 microg/g {wet weight}) when stored at nonrefrigeration temperatures (21 to 23 degrees C) . Cereulide was not formed in products stored at refrigeration temperatures (4 to 8 degrees C) . Cereulide is not inactivated by heating during food processing . Therefore, direct analysis of this toxin in food is preferable to cultivating methods for assessing the risk of food poisoning by emetic B . cereus. J Food Prot, 2003 Jun, 66(6), 978 - 84 Improved model based on the Weibull distribution to describe the combined effect of pH and temperature on the heat resistance of Bacillus cereus in carrot juice; Collado J et al.; The effect of pH and temperature on the thermal inactivation of different strains of Bacillus cereus was modeled . Inactivation tests were carried out in carrot broth, following a full factorial design at four levels for temperature (from 90 to 105 degrees C, depending on the strain) and pH (6.2, 5.8, 5.2, and 4.7) . Individual inactivation curves were analyzed by applying the Weibull model function (with percent discrepancy close to 20% for most cases), and the effects of pH and temperature on the scale parameter (designated D(beta)) and the shape parameter (beta) were also studied . Temperature and pH did not have a significant effect on the shape parameter (beta) . The effect of temperature on the scale parameter was modeled by the zeta concept . The scale parameter decreased with pH, although the behavior of the strains was not homogeneous . Two global models with a small number of parameters were developed, providing a satisfactory description of the thermal inactivation of B . cereus, with percent discrepancy ranging from 18 to 25%. Folia Microbiol (Praha), 2003, 48(2), 157 - 61 Bacteriocin diversity in Bacillus sphaericus; Cetinkaya S et al.; Nondenaturing polyacrylamide gel electrophoresis revealed the presence of diversity among bacteriocins produced by strains of Bacillus sphaericus . Bacteriocin bands of six strains (pathogenic and non pathogenic) were found to be located just below the stacking gel . However, in two other strains (1 pathogenic and 1 collection strain) more than one protein band with bacteriocin activity were seen in the middle of resolving gel . In bacteriocin-treated cultures, electron-microscopy studies revealed the growth of lysedswollen ghost cells, and loss of viability among sensitive strains. Vestn Ross Akad Med Nauk, 2003, (5), 23 - 8 {The state-of-the art and prospects for the pharmacological therapy of infectious and allergic eye diseases}; Maichuk IuF; The inflammatory, infectious and allergic diseases of the eye still remain an acute problem in practical ophthalmology due to their high prevalence, relapsing clinical course, a growing frequency rate of mycotic and acanthoamebic keratitis and a growing number of resistant causative agents of bacterial keratitis . 55.3% of corneal pathologies are related with the herpes simplex virus, 37.3%--with secondary bacterial infection, 33.2%--with primary bacterial infection, 6.7%--with mycotic infection and 5.6%--with acathoamebic infection . Algorithms of pharmacological therapy are suggested for a majority of inflammatory lesions of the eye, i.e . for herpetic keratitis, ulcerous keratitis caused by blue pus bacillus, gonococcus, staphylococcus, fungi, acanthomebas, as well as for adenoviral and Chlamidia conjunctivitis and allergy of the eye . The author points out that the treatment must be complex, including specific therapy--antiviral one (zovirax ointment, interferon locally, and zovirax or valtrex perorally), antibacterial one (okacin, tobrex, vitabact, fucithamic locally, quinine antibiotics perorally, aminoglycosides or cephalosporins subconjunctivally or intramuscularly) and antimycotic one (locally and perorally) . Another part of the complex therapy is equally important--pathogenetic one (carnosine, taufon, vitacic, corneregel ointment), antiallergic one (spersallerg, allergophtal, alomide, lecrolin), antinflammatory one (naclof, maxidex, cyclolip) tears replacing one (natural tears, ophthagel) and immunomodeling one (affinoleicin, lycopid, tactivin). Proc Natl Acad Sci U S A, 2003 Jun 24, 100(13), 7497 - 502 Epub 2003 Jun 10. High-resolution structure of RNase P protein from Thermotoga maritima; Kazantsev AV et al.; The structure of RNase P protein from the hyperthermophilic bacterium Thermotoga maritima was determined at 1.2-A resolution by using x-ray crystallography . This protein structure is from an ancestral-type RNase P and bears remarkable similarity to the recently determined structures of RNase P proteins from bacteria that have the distinct, Bacillus type of RNase P . These two types of protein span the extent of bacterial RNase P diversity, so the results generalize the structure of the bacterial RNase P protein . The broad phylogenetic conservation of structure and distribution of potential RNA-binding elements in the RNase P proteins indicate that all of these homologous proteins bind to their cognate RNAs primarily by interaction with the phylogenetically conserved core of the RNA . The protein is found to dimerize through an extensive, well-ordered interface . This dimerization may reflect a mechanism of thermal stability of the protein before assembly with the RNA moiety of the holoenzyme. FEMS Microbiol Lett, 2003 Jun 6, 223(1), 73 - 82 Diversity of mercury resistance determinants among Bacillus strains isolated from sediment of Minamata Bay; Narita M et al.; Thirty mercury-resistant (Hg R) Bacillus strains were isolated from mercury-polluted sediment of Minamata Bay, Japan . Mercury resistance phenotypes were classified into broad-spectrum (resistant to inorganic Hg(2+) and organomercurials) and narrow-spectrum (resistant to inorganic Hg(2+) and sensitive to organomercurials) groups . Polymerase chain reaction (PCR) product sizes and the restriction nuclease site maps of mer operon regions from all broad-spectrum Hg R Bacillus were identical to that of Bacillus megaterium MB1 . On the other hand, the PCR products of the targeted merP (extracellular mercury-binding protein gene) and merA (intracellular mercury reductase protein gene) regions from the narrow-spectrum Hg R Bacillus were generally smaller than those of the B . megaterium MB1 mer determinant . Diversity of gene structure configurations was also observed by restriction fragment length polymorphism (RFLP) profiles of the merA PCR products from the narrow-spectrum Hg R Bacillus . The genetic diversity of narrow-spectrum mer operons was greater than that of broad-spectrum ones. FEMS Microbiol Lett, 2003 Jun 6, 223(1), 61 - 6 Pulsed field gel electrophoresis of chromosomal DNA reveals a clonal population structure to Bacillus thuringiensis that relates in general to crystal protein gene content; Gaviria Rivera AM et al.; Seventy strains of Bacillus thuringiensis representing 21 serovars were allocated to 38 genomic groups using pulsed field gel electrophoresis (PFGE) of restriction enzyme-digested DNA . There was a broad correlation between PFGE type and serotype for serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, sotto, thuringiensis, and tolworthi, although some serovars included atypical strains . Serovars canadensis and entomocidus were heterogeneous . Detection of crystal protein genes by polymerase chain reaction indicated an approximate correlation between PFGE type and cry gene complement . For example, cry1 products were amplified from DNA from PFGE type 17 strains of serovar aizawai and from PFGE type 23 strains of serovar tolworthi but not from a PFGE 18 strain of aizawai nor from a PFGE type 24 strain of tolworthi . These data suggest a clonal population structure to B . thuringiensis with some consistency of Cry-plasmid composition within PFGE types. Vaccine, 2003 Jun 20, 21(21-22), 2782 - 90 BCG scar and positive tuberculin reaction associated with reduced child mortality in West Africa . A non-specific beneficial effect of BCG? Garly ML, Martins CL, Bale C, Balde MA, Hedegaard KL, Gustafson P, Lisse IM, Whittle HC, Aaby P. Previous studies have suggested that the bacille Calmette-Guerin (BCG) vaccine may have a non-specific beneficial effect on childhood survival in areas with high mortality . We examined whether BCG-vaccinated children with a BCG scar or a positive tuberculin reaction had better survival than children without such reactions . As part of an ongoing two-dose measles vaccine trial for which children were recruited at 6 months of age, we examined 1813 children for BCG scar at 6 months of age and 813 BCG-vaccinated children were skin-tested for delayed hypersensitivity to tuberculin, tetanus and diphtheria . We found that BCG-vaccinated children with a BCG scar had significantly lower mortality compared with BCG scar-negative children, the mortality ratio in the first 12 months of follow-up being 0.41 (0.25-0.67) . BCG-vaccinated children with a positive tuberculin test had a mortality ratio of 0.45 (0.24-0.85) compared with tuberculin negative children . These results were unchanged by control for potential confounders or using different cut-off points for a tuberculin-positive response . Exclusion of dead children who had HIV antibodies did not modify the estimate (mortality rate (MR)=0.46 (0.23-0.94)) . After censoring for tuberculosis (TB) exposure at home, the mortality ratios for having a scar and being tuberculin-positive were 0.46 (0.27-0.79) or 0.42 (0.21-0.84), respectively . Children positive to tetanus or diphtheria in the skin test had the same mortality as children not responding to these vaccine-related antigens . Thus, BCG scar and a positive tuberculin reaction were associated with better survival in early childhood in an area with high mortality . Since nothing similar was found for responders to diphtheria-tetanus-pertussis (DTP) vaccine, and the effect could not be explained by protection against tuberculosis, the effect of BCG vaccination could be due to non-specific immune-stimulation protecting against other infections. Bioresour Technol, 2003 Sep, 89(3), 267 - 74 Biological treatments affect the chemical composition of coffee pulp; Ulloa Rojas JB et al.; Biological treatments were applied to fresh coffee pulp (CoP) to improve its nutritive value for monogastric animals by reducing its content of cellulose and antinutritional factors (ANFs) such as total phenols, tannins and caffeine . Treatments were: (1) ensiling with 0, 50 and 100 gkg(-1) molasses for 2 and 3 months, (2) aerobic decomposition for 0, 7, 14, 21, 28, 35 and 42 days, (3) aerobic bacterial inoculation (Bacillus sp.) for 0, 7, 14, 21 and 28 days . Ensiled CoP (E-CoP) showed higher fat and ash contents than oven-dried-CoP (OD-CoP; P<0.05) . Similarly, true protein values tended to increase . The cellulose and total phenols levels of E-CoP were lower than OD-CoP (P<0.05) . The E-CoP tannins levels tended to be lower than OD-CoP whereas caffeine levels remained unaffected . Improvement in the nutritional quality of E-CoP was associated with higher fat and protein contents and reduction of cellulose, total phenols and tannins . The aerobic decomposition treatment improved the nutritional quality of CoP by increasing true protein and fat contents . In addition, total phenols, tannins, caffeine and cellulose contents were reduced by an increase in treatment time (P<0.05) . Bacterial treatment increased the protein content of CoP after 21 days (from 137 to 392 gkg(-1)) and decreased it after 28 days . Cellulose, total phenols, tannins and caffeine contents reduced with an increase in time of bacterial degradation . Bacterial treatment improved the CoP quality by increasing protein content and reducing cellulose and ANFs, especially after 21 days of treatment . Both the aerobic decomposition (after 21-28 days) and the aerobic bacterial degradation of CoP (after 21 days) appeared more suitable to improve the nutritional quality of CoP than the ensiling. Pediatr Infect Dis J, 2003 May, 22(5), 465 - 7 Arcanobacterium haemolyticum sinusitis and orbital cellulitis; Limjoco-Antonio AD et al.; We present a case of sinusitis and orbital cellulitis in a 9-year-old girl caused by the Gram-positive bacillus Arcanobacterium haemolyticum . In addition to antimicrobial chemotherapy, two surgical procedures were required to drain the ethmoid and maxillary sinus cavities and a subperiosteal abscess. Biochem J, 2003 Aug 15, 374(Pt 1), 255 - 9 Investigation of the pore-forming mechanism of a cytolytic delta-endotoxin from Bacillus thuringiensis; Promdonkoy B et al.; Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp . kyushuensis . Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation . The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose-response curve, indicating that toxin aggregation or oligomerization is required for pore formation . Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore . Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules . When toxin-treated red blood cells at 4 degrees C were transferred to a toxin-free solution at 37 degrees C, no significant increase in haemolysis was observed . This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore . From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer . Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers. Biochemistry, 2003 Jun 17, 42(23), 6996 - 7002 Reaction mechanism of the heterotetrameric (alpha2beta2) E1 component of 2-oxo acid dehydrogenase multienzyme complexes; Fries M et al.; Pyruvate decarboxylase (E1) catalyzes the first two reactions of the four involved in oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase (PDH) multienzyme complex . It requires thiamin diphosphate to bring about the decarboxylation of pyruvate, which is followed by the reductive acetylation of a lipoyl group covalently bound to the N(6) amino group of a lysine residue in the second catalytic component, a dihydrolipoyl acetyltransferase (E2) . Replacement of two histidine residues in the E1alpha and E1beta chains of the heterotetrameric E1 (alpha(2)beta(2)) component of the PDH complex of Bacillus stearothermophilus, considered possible proton donors at the active site, was carried out . Subsequent characterization of the mutants permitted different roles to be assigned to these two particular residues in the reaction catalyzed by E1: E1alpha His271 to stabilize the dianion formed during decarboxylation of the 2-oxo acid and E1beta His128 to provide the proton required to protonate the incoming dithiolane ring in the subsequent reductive acetylation of the lipoyl goup . On the basis of these and other results from a separate investigation into the roles of individual residues in a loop region in the E1alpha chain close to the active site of E1 {Fries, M., Chauhan, H . J., Domingo, G . J., Jung, H., and Perham, R . N . (2002) Eur . J . Biochem . 270, 861-870} together with work from other laboratories, a detailed mechanism for the E1 reaction can be formulated. J Infect Dis, 2003 Jun 15, 187(12), 1959 - 61 Epub 2003 May 29. Field trial of a vaccine against new world cutaneous leishmaniasis in an at-risk child population: how long does protection last? Armijos RX, Weigel MM, Romero L, Garcia V, Salazar J. During 12 months of follow-up in a randomized double-blind controlled field study, a killed whole-promastigote vaccine cocktail plus bacille Calmette-Guerin (BCG) adjuvant significantly reduced the incidence of cutaneous leishmaniasis (CL) in Ecuadorian children, compared with BCG alone . To determine how much longer protection might continue, the study was reblinded to permit 48 additional months of follow-up . During months 13-18, CL incidence remained lower in the vaccine group, compared with that in the control group (5.9% vs . 13.8%; chi2=8.8; P=.003), with vaccine efficacy calculated at 56.5% (95% confidence interval, 18.7%-76.7%); however, during months 24-60, no significant between-group differences were detected . Periodic administration of boosters may be necessary to maintain whole-parasite-vaccine protection against New World CL. Mol Microbiol, 2003 Jun, 48(6), 1553 - 64 The kinetic properties of the carboxy terminal domain of the Bacillus licheniformis 749/I BlaR penicillin-receptor shed a new light on the derepression of beta-lactamase synthesis; Duval V et al.; To study the properties of the BlaR penicillin-receptor involved in the induction of the Bacillus licheniformisbeta-lactamase, the water-soluble carboxy terminal domain of the protein (BlaR-CTD) was overproduced in the periplasm of Escherichia coli JM105 and purified to protein homogeneity . Its interactions with various beta-lactam antibiotics were studied . The second-order acylation rate constants k2/K' ranged from 0.0017 to more than 1 micro M-1s-1 and the deacylation rate constants were lower than 4 x 10-5 s-1 . These values imply a rapid to very rapid formation of a stable acylated adduct . BlaR-CTD is thus one of the most sensitive penicillin-binding proteins presently described . In the light of these results, the kinetics of beta-lactamase induction in Bacillus licheniformis were re-examined . When starting with a rather high cell density, a good beta-lactamase substrate such as benzylpenicillin is too sensitive to beta-lactamase-mediated hydrolysis to allow full induction . By contrast, a poor beta-lactamase substrate (7-aminocephalosporanic acid) can fully derepress beta-lactamase expression under conditions where interference of the antibiotic with cell growth is observed . These results suggest that acylation of the penicillin receptor is a necessary, but not sufficient, condition for full induction. Endocr Relat Cancer, 2003 Jun, 10(2), 301 - 8 Innate immunity in breast carcinoma; Sfondrini L et al.; The innate immune response, which depends on so-called pattern-recognition receptors (PRRs) is an evolutionarily old immune response able to elicit a defensive response against a vast array of pathogens . The purpose of this review is to revisit the role of innate immunity in breast carcinoma from the oldest therapeutic approach using bacillus Calmette-Guerin to the recent findings on the manipulation of the PRR pathways with unmethylated cytosine-guanosine dinucleotides (CpG motifs) . Encouraging results have been obtained in prevention and local treatment of murine mammary tumors using tumor cells engineered to express stably mycobacterial antigens or directly using CpG-containing oligonucleotides . The experimental findings raise the possibility of successful anti-tumor management through stimulation of innate immunity in women at high risk of developing breast cancer and in breast cancer patients with reasonable immunological performance and low tumor load. Acta Cytol, 2003 May-Jun, 47(3), 368 - 72 Fine needle aspiration cytology of leprous neuritis; Singh N et al.; OBJECTIVE: To document the cytomorphologic features of leprous neuritis and their correlation with bacterial density . STUDY DESIGN: A partly retrospective, partly prospective study of the fine needle aspiration cytology of enlarged nerves in leprosy . Cytomorphologic features of nerve aspirates from 28 patients were studied . May-Grunwald-Geimsa and Ziehl-Neelsen staining methods were employed . RESULTS: Five cytomorphologic patterns were observed in smears of nerve aspirates in 19 group I patients with concurrent skin and nerve lesions: (1) inflammation composed of epithelioid cell granulomas (5), bacillary index (BI) = 0; (2) epithelioid cell granulomas with necrosis (5), BI = 0-1+; (3) acellular necrosis (5), BI = 0-4+; (4) macrophage granuloma (3), BI = 5-6+; and (5) granulation tissue (1), BI = 1+ . In 9 group II patients with pure neuritic leprosy, 3 patterns were seen: (1) epithelioid cell granulomas (5), BI 0-6+; (2) epithelioid granulomas with necrosis (1), BI = 0; and (3) acellular necrosis (3), BI = 0-6+ . CONCLUSION: The entire spectrum of leprosy is seen in nerve aspirates . Necrosis is often a prominent feature . Recognition of the range of cytomorphologic patterns and their correlation with BI contribute to accurate calibration of the disease in nerves, resulting in appropriate choice of treatment. Plant Cell Rep, 2003 Apr, 21(8), 814 - 20 Epub 2003 Feb 22. Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures; Isenegger DA et al.; An aberrant random amplified polymorphic DNA (RAPD) marker in genomic DNA of tissue culture plantlets was frequently observed during a comparison of DNA fingerprints derived from potato germplasm grown in tissue culture and the field . The RAPD marker was cloned, sequenced and determined to be of bacterial origin . A bacterial contaminant was isolated from the tissue culture plants and identified as a Bacillus pumilus . A set of sequence characterised amplified region (SCAR) primers were designed from the sequence of the cloned fragment and tested for the specific detection of B . pumilus . Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) were also used to generate B . pumilus profiles specific to our isolate in order to test and confirm the sequence homology of amplified markers generated from a range of DNA samples isolated from tissue culture plants and pure isolates of B . pumilus-like bacteria. Plant Cell Rep, 2003 Apr, 21(8), 789 - 96 Epub 2003 Mar 15. Transgene expression in broccoli (Brassica oleracea var . italica) clones propagated in vitro via leaf explants; Cao J et al.; We have developed an efficient protocol for the in vitro propagation of transgenic broccoli plants using leaf explants as starting material . A high frequency of shoot formation from leaf explants was obtained on Murashige and Skoog medium containing benzyladenine (BA, 5 mg/l) and naphthaleneacetic acid (0.5 mg/l) . Frequent subcultures of existing shoots and shoot clusters to medium containing only BA (2 mg/l) promoted rapid shoot multiplication . The use of a 1:1 mixture of Agargel and Gelrite in the rooting medium increased the number of healthy roots per rooted plant . Applying this protocol, we obtained thousands of clonal rooted plantlets within 6 months from a transgenic broccoli plant carrying the cry1Ac and cry1C genes from Bacillus thuringiensis associated with kanamycin and hygromycin selectable markers, respectively . Thirty randomly selected clones that had been propagated for 1 year on medium containing kanamycin (50 mg/l) all showed resistance to both kanamycin and hygromycin . Genomic DNA and total soluble proteins were isolated from 16 of these clones . Polymerase chain reaction analysis indicated that the cry1Ac and cry1C genes were both maintained . ELISA assays showed that all of the clones produced a high level of Cry1Ac protein similar to the original transgenic plant; however, most clones had significantly lower levels of Cry1C protein than the original plant . This variation indicates that it is important to evaluate transgene expression in transgenic clones propagated long-term in vitro . In vitro propagation starting from leaf explants was also successful with other transgenic and non-transgenic Brassica oleracea materials, including broccoli, cauliflower, and collard. Infection, 2003 Jun, 31(3), 192 - 3 Intestinal perforations in a premature infant caused by Bacillus cereus; Girisch M et al.; Although Bacillus cereus is a ubiquitous bacterium, the incidence of neonatal infections is very low with only a few cases of B . cereus infections in neonates reported in the literature . We report the case of a premature infant with multiple intestinal perforations and an abdominal B . cereus infection . The initial course was characterized by severe cardiovascular shock, anemia, thrombocytopenia and disseminated intravascular coagulation, leading to periventricular leukomalacia, alopecia capitis and toxic epidermal necrolysis . The possible role of B . cereus-associated enterotoxins for the clinical manifestations are discussed . Our case confirms previous reports of severe clinical symptoms in B . cereus infection in premature neonates . We speculate that the systemic complications of B . cereus infection are at least partly related to the effect of B . cereus-associated enterotoxins. J Biochem Mol Biol, 2003 May 31, 36(3), 294 - 8 Ex vivo cytotoxicity of the Bacillus thuringiensis Cry4B delta-endotoxin to isolated midguts of Aedes aegypti larvae; Barusrux S et al.; The pathological effect of the Bacillus thuringiensis Cry delta- endotoxins on susceptible insect larvae had extensive damage on the midgut epithelial cells . In this study, an ex vivo assay was devised for assessing the insecticidal potency of the cloned Cry4B mosquito-larvicidal protein that is expressed in Escherichia coli . Determination of toxicity was carried out by using a cell viability assay on the midguts that were dissected from 5-day old Aedes aegypti mosquito larvae . After incubation with the toxin proteins, the number of viable epithelial cells was determined photometrically by monitoring the quantity of the bioreduced formazan product at 490 nm . The results showed that the 65-kDa trypsin-activated Cry4B toxin exhibited toxic potency ca . 3.5 times higher than the 130-kDa Cry4B protoxin . However, the trypsin-treated products of the non-bioactive Cry4B mutant (R158A) and the lepidopteran-specific Cry1Aa toxin displayed relatively no ex vivo activity on the mosquito-larval midguts . The ex vivo cytotoxicity studies presented here confirms data that was obtained in bioassays. Environ Sci Technol, 2003 May 15, 37(10), 2296 - 301 Photocatalytic activity, antibacterial effect, and photoinduced hydrophilicity of TiO2 films coated on a stainless steel substrate; Yu JC et al.; Transparent TiO2 films on stainless steel prepared by dip coating in a nonionic microemulsions solution have been shown to have much higher photocatalytic activity than those coatings on glass . Fe3+ and Fe2+ ions, diffusing from stainless steel substrate into TiO2 films during high-temperature calcination, behave as dopants to significantly affect the films' photocatalytic activity . An optimum calcination condition, under which the amount of diffused Fe3+ and the ratio of Fe3+ to Fe2+ ions favor the film's photocatalytic reaction, was obtained . In addition, this TiO2 films also exhibits excellent photoinduced hydrophilicity and antibacterial effect for the sterilization of Bacillus pumilus . As stainless steel is a very common material, practical systems for pollution treatment and disinfection may be designed based on this enhanced coating. Klin Med (Mosk), 2003, 81(4), 63 - 6 {Post-tuberculous fibrous mediastinitis: a report of a case followed-up for many years}; Volkova KI et al.; The paper reports a rare case of fibrosing mediastinitis of posttuberculous genesis caused by infection with Mycobacterium bovis . Such cases are not available in the literature so far . The patient was observed from 1956 to 2001 . Bovine tubercle bacilla affect mediastinal lymph nodes which undergo fibrosis resulting in stenosis of the trachea, major bronchi, esophagus . Narrowing of the chest duct leads to formation of severe recurrent transudate in the left pleural cavity (for 3 years 70 pleural punctures were made with removal of a total of 22 l of fluid). Indian J Pathol Microbiol, 2002 Jul, 45(3), 293 - 8 Spectrum of dermatopathologic lesions associated with HIV/AIDS in India; Lanjewar DN et al.; Histopatholgoical analysis of cutaneous lesions in 195 patients with HIV/AIDS was carried out between 1989 to 1997 at tertiary level public hospital in Mumbai . 104/195 (53%) cases showed infectious diseases which comprised of molluscum contagiosum (28), condyloma accuminata (18), verruca vulgaris (7), varicella zoster (5), syphilis (14), tuberculosis (13), donovanosis (4), leprosy (2), chancroid (2), bacillary angiomatosis (2), lymphogranuloma venercum (1), Norwegian scabies (3), leishmaniasis (2), demodicidosis (1), crytococcosis (1), tinea versicolor (1) . In 12 (6%) cases neoplasms were observed which included squamous cell carcinoma (9), basal cell carcinoma (2) and kaposi's sarcoma (1) case . The miscellaneous conditions were observed in 66(33.5%) cases which comprised of psoriasis (21), papular urticaria (13), Reiter's disease (7) and eosinophilic folliculitis (6) . The prevalence of cutaneous tuberculosis observed in this study is high as compared with western literature while the prevalence of kaposis's sarcoma is quite low as compared with reports from Africa, USA and United Kingdom. Biotechnol Bioeng, 2003 Aug 5, 83(3), 344 - 52 Production of savinase and population viability of Bacillus clausii during high-cell-density fed-batch cultivations; Christiansen T et al.; The growth and product formation of a Savinase-producing Bacillus clausii were investigated in high-cell-density fed-batch cultivations with both linear and exponential feed profiles . The highest specific productivity of Savinase was observed shortly after the end of the initial batch phase for all feed profiles applied and, in addition, there was a time-dependent decrease in specific productivity . The specific glucose uptake rate increased with time for constant specific growth rate indicating that the maintenance requirements increased with time, possibly due to a decreasing K(+) concentration . The physiological state of the cells was monitored during the cultivations using a flow cytometry assay based on the permeability of the cell membrane to propidium iodide . In the latter parts of the fed-batch cultures with a linear feed profile, a large portion of the cell population was found to have a permeable membrane, indicating a large percentage of dead cells . By assuming that only cells with a nonpermeable membrane contributed to growth and product formation, the physiological properties of this subpopulation were calculated . Curr Microbiol, 2003 Jul, 47(1), 51 - 4 Toxicity of hexavalent chromium and its reduction by bacteria isolated from soil contaminated with tannery waste; Megharaj M et al.; An Arthrobacter sp . and a Bacillus sp., isolated from a long-term tannery waste contaminated soil, were examined for their tolerance to hexavalent chromium {Cr(VI)} and their ability to reduce Cr(VI) to Cr(III), a detoxification process in cell suspensions and cell extracts . Both bacteria tolerated Cr(VI) at 100 mg/ml on a minimal salts agar medium supplemented with 0.5% glucose, but only Arthrobacter could grow in liquid medium at this concentration . Arthrobacter sp . could reduce Cr(VI) up to 50 microg/ml, while Bacillus sp . was not able to reduce Cr(VI) beyond 20 microg/ml . Arthrobacter sp . was distinctly superior to the Bacillus sp . in terms of their Cr(VI)-reducing ability and resistance to Cr(VI) . Assays with permeabilized (treated with toluene or Triton X 100) cells and crude extracts demonstrated that the Cr(VI) reduction was mainly associated with the soluble protein fraction of the cell . Arthrobacter sp . has a great potential for bioremediation of Cr(VI)-containing waste. Curr Microbiol, 2003 Jul, 47(1), 40 - 5 Overexpression, purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus; Kuo LY et al.; For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII . The His(6)-tagged enzyme was overexpressed in IPTG-induced E . coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76% . The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE . The temperature and pH optima for the purified protein were 60 degrees C and 8.0, respectively . Under optimal condition, the purified enzyme showed a marked preference for Leu- p-nitroanilide, followed by Arg- and Lys-derivatives . The His(6)-tagged enzyme was stimulated by Co(2+) ions, but was strongly inhibited by Cu(2+) and Hg(2+) and by the chelating agents, DTT and EDTA . The EDTA-treated enzyme could be reactivated with Co(2+) ions, indicating that it is a cobalt-dependent exopeptidase . Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme. Curr Microbiol, 2003 Jul, 47(1), 26 - 31 Characterization of Bacillus thuringiensis ser . jordanica (serotype H71), a novel serovariety isolated in Jordan; Khyami-Horani H et al.; The novel strain of Bacillus thuringiensis J112 isolated from a soil sample in Jordan was classified and characterized in terms of toxicity against dipteran and nematode larvae, crystal protein pattern, plasmid profile, and cry gene content . A new name, Bacillus thuringiensis serovariety jordanica (H serotype 71), is proposed for the reference strain J112 . The parasporal crystal proteins were toxic to 3(rd) instar larvae of Drosophila melanogaster and to 2(nd) stage juveniles of root knot nematodes Meloidogyne javanica and M . incognita, but showed poor mosquitocidal activity towards Culex pipiens molestus and Culiseta longiareolata larvae . Solubilized and trypsin-digested crystal proteins possessed moderate hemolytic activity against sheep erythrocytes . SDS-polyacrylamide gel electrophoresis revealed that crystals are composed of several polypeptides ranging from 24 to 170 kDa, of which the 20-, 42-, 140-, and 170-kDa proteins were the major components . Analysis of the plasmid pattern of J112 revealed the presence of two large plasmidic bands of about 160 and 205 kbp . PCR with total DNA from strain J112 and specific primers for cry1, cry2, cry3, cry4, and cyt2A genes revealed that cry1, cry3A, cry4, cry5 and cyt2a genes are present. Int J Parasitol, 2003 May, 33(5-6), 547 - 54 Recent progress in the development and testing of vaccines against human tuberculosis; McMurray DN; The growing pandemic of human tuberculosis has not been affected significantly by the widespread use of the only currently available vaccine, bacille Calmette Guerin . Bacille Calmette Guerin protects uniformly against serious paediatric forms of tuberculosis and against adult pulmonary tuberculosis in some parts of the world, but there are clearly populations in high-burden countries which do not benefit from the current vaccination regimen . New tuberculosis vaccines will be essential for the ultimate control of this ancient disease . Research over the past 10 years has produced literally hundreds of new tuberculosis vaccine candidates representing all of the major vaccine design strategies; protein/peptide vaccines in adjuvants, DNA vaccines, naturally and rationally attenuated strains of mycobacteria, recombinant mycobacteria and other living vaccine vectors expressing genes coding for immunodominant mycobacterial antigens, and non-peptide vaccines . Many of these vaccines have been tested for immunogenicity and protective efficacy in mouse and guinea pig models of low-dose pulmonary tuberculosis . In addition, alternative routes of tuberculosis vaccine delivery (e.g . oral, respiratory, gene gun) and various combinations of priming or boosting an experimental vaccine with bacille Calmette Guerin have been examined in relevant animal models . One of the most promising of these vaccines is currently in Phase I trials in human subjects, and others are expected to follow in the near future . This review will summarise the most recent progress made toward the development and preclinical evaluation of novel vaccines for human tuberculosis. Comp Biochem Physiol B Biochem Mol Biol, 2003 May, 135(1), 125 - 37 Expression of a midgut-specific cadherin BT-R1 during the development of Manduca sexta larva; Midboe EG et al.; The btr-1 gene of Manduca sexta (GenBank AF319973) encodes a cadherin, BT-R(1) (210-kDa), which contains 12 ectodomain modules in association with a number of motifs potentially involved in interactions with cadherin and integrin . The molecule is a target receptor for Bacillus thuringiensis Cry1A toxins that bind to BT-R(1) with high affinity and specificity . BT-R(1) is localized exclusively in the midgut epithelium . The amount of BT-R(1) protein increases dramatically during larval development, paralleling accumulation of its mRNA . The 5'-UTR of the btr-1 gene contains sequence motifs that most likely recruit specific transcription factors, particularly, those that determine posterior patterning and that control intestinal cell proliferation, differentiation and identity during development . The increase in abundance of BT-R(1) may be required to support not only the differentiation of the epithelial cells but also the establishment of physiological function and structural integrity of the midgut during larval development in M . sexta . We believe that BT-R(1) is essential to larval midgut epithelial organization during rapid cell proliferation and tissue growth in M . sexta because disruption of such organization and functionality occasioned by the binding of the Cry1A toxins of B . thuringiensis to BT-R(1) causes death to the insect. Cell Microbiol, 2003 Jun, 5(6), 405 - 15 Deficiency in mycolipenate- and mycosanoate-derived acyltrehaloses enhances early interactions of Mycobacterium tuberculosis with host cells; Rousseau C et al.; Lipids that are uniquely found in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched long-chain fatty acids, have long been thought to play a role in host-pathogen interactions . The recent construction by Dubey et al . (2002) Mol Microbiol 45: 1451-1459, of a Mycobacterium tuberculosis mutant that is deficient in the synthesis of the di- and tri-methylbranched fatty acids, mycolipenates and mycosanoates, found in some forms of diacyltrehaloses (DAT) and polyacyltrehaloses (PAT) provided the opportunity to assess the contribution of these complex lipids to pathogenesis directly . We provide evidence that DAT/PAT deficiency affects the surface global composition of the mycobacterial cell envelope improving the efficiency with which M . tuberculosis binds to and enters phagocytic and non-phagocytic host cells . Interestingly, this property did not affect the overall replication and persistence of the tubercle bacillus in the lungs, spleen and liver of mice infected via the respiratory or intravenous route. J Chromatogr A, 2003 Apr 25, 994(1-2), 207 - 12 Determination of spore concentration in Bacillus thuringiensis through the analysis of dipicolinate by capillary zone electrophoresis; He J et al.; A new capillary zone electrophoresis (CZE) method for the analysis of dipicolinic acid, a specific component found in spores but not in vegetative cells, was used to determine spore concentration in Bacillus thuringiensis according to the relationship between the spore concentration and the content of dipicolinate . The quantitative relationship was established by using purified spores . Electrolyte conditions that affected the separation efficiency of dipicolinate and the reproducibility were investigated . With 10 mM phosphate, 10 mM ethylenediaminetetraacetic acid and 0.25 mM tetradecyltrimethylammonium bromide at pH 6.2 as the carrier electrolyte, dipicolinate can be determined within 8 min at an applied voltage of -25 kV (anode at detector) and a capillary temperature of 25 degrees C . The method has a high separation efficiency with which the number of theoretical plates is above 300,000 plates m(-1) . The relative standard deviations for migration time and peak area are less than 0.5% and 2.0%, respectively . The detection limit for dipicolinate was 10 ng ml(-1), which corresponds to 7.2 x 10(5) spores ml(-1) . The method was used to determine spores in fermentation broths, and the results obtained agreed well with the values obtained by plate counting. Transgenic Res, 2003 Jun, 12(3), 351 - 61 Tritrophic choice experiments with bt plants, the diamondback moth (Plutella xylostella) and the parasitoid Cotesia plutellae; Schuler TH et al.; Parasitoids are important natural enemies of many pest species and are used extensively in biological and integrated control programmes . Crop plants transformed to express toxin genes derived from Bacillus thuringiensis (Bt) provide high levels of resistance to certain pest species, which is likely to have consequent effects on parasitoids specialising on such pests . A better understanding of the interaction between transgenic plants, pests and parasitoids is important to limit disruption of biological control and to provide background knowledge essential for implementing measures for the conservation of parasitoid populations . It is also essential for investigations into the potential role of parasitoids in delaying the build-up of Bt-resistant pest populations . The diamondback moth (Plutella xylostella), a major pest of brassica crops, is normally highly susceptible to a range of Bt toxins . However, extensive use of microbial Bt sprays has led to the selection of resistance to Bt toxins in P . xylostella . Cotesia plutellae is an important endoparasitoid of P . xylostella larvae . Although unable to survive in Bt-susceptible P . xylostella larvae on highly resistant Bt oilseed rape plants due to premature host mortality, C . plutellae is able to complete its larval development in Bt-resistant P . xylostella larvae . Experiments of parasitoid flight and foraging behaviour presented in this paper showed that adult C . plutellae females do not distinguish between Bt and wildtype oilseed rape plants, and are more attracted to Bt plants damaged by Bt-resistant hosts than by susceptible hosts . This stronger attraction to Bt plants damaged by resistant hosts was due to more extensive feeding damage . Population scale experiments with mixtures of Bt and wildtype plants demonstrated that the parasitoid is as effective in controlling Bt-resistant P . xylostella larvae on Bt plants as on wildtype plants . In these experiments equal or higher numbers of parasitoid adults emerged per transgenic as per wildtype plant . The implications for integrated pest management and the evolution of resistance to Bt in P . xylostella are discussed. Acta Crystallogr D Biol Crystallogr, 2003 May, 59(Pt 5), 946 - 9 Epub 2003 Apr 25. Crystallization and preliminary X-ray analysis of a novel unsaturated glucuronyl hydrolase from Bacillus sp . GL1; Mori S et al.; Unsaturated glucuronyl hydrolase from Bacillus sp . GL1 catalyzes the hydrolytic release of unsaturated glucuronic acids from oligosaccharides produced by the reactions of polysaccharide lyases such as gellan, xanthan, hyaluronate and chondroitin lyases . The enzyme was crystallized at 293 K from a droplet containing 56% MPD, 0.1 M NaCl, 0.1 M glycine-NaOH pH 8.2 and 0.1 M dithiothreitol using the vapour-diffusion method . The crystals were hexagonal and belonged to space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 102.8, c = 223.4 A . Diffraction data to 2.4 A were collected from a single crystal. Acta Crystallogr D Biol Crystallogr, 2003 Jun, 59(Pt 6), 1073 - 5 Epub 2003 May 23. Purification, crystallization and preliminary X-ray crystallographic studies on acetolactate decarboxylase; Najmudin S et al.; Acetolactate decarboxylase has the unique ability to decarboxylate both enantiomers of acetolactate to give a single enantiomer of the decarboxylation product, (R)-acetoin . A gene coding for alpha-acetolactate decarboxylase from Bacillus brevis (ATCC 11031) was cloned and overexpressed in B . subtilis . The enzyme was purified in two steps to homogeneity prior to crystallization . Three different diffraction-quality crystal forms were obtained by the hanging-drop vapour-diffusion method using a number of screening conditions . The best crystal form is suitable for structural studies and was grown from solutions containing 20% PEG 2000 MME, 10 mM cadmium chloride and 0.1 M Tris-HCl pH 7.0 . They grew to a maximum dimension of approximately 0.4 mm and belong to the trigonal space group P3(1,2)21, with unit-cell parameters a = 47.0, c = 198.9 A . A complete data set was collected to 2 A from a single native crystal using synchrotron radiation. Mol Biol Evol, 2003 Aug, 20(8), 1326 - 8 Epub 2003 May 30. The effect of positive selection on a sexual reproduction gene in Thalassiosira weissflogii (Bacillariophyta): results obtained from maximum-likelihood and parsimony-based methods; Sorhannus U; Maximum-Likelihood-based and parsimony-based methods were used to test for potential effects of positive selection on the sexually induced gene 1 (Sig1) in Thalassiosira weissflogii . The Sig proteins are thought to play a role in mediating sperm-egg recognition during the sexual reproduction phase . The results obtained from parsimony-based analyses showed that none of the amino acid sites were influenced by positive selection . Maximum-likelihood analyses indicated that positive selection was affecting a maximum of seven and a minimum of four amino acid sites in the polypeptide derived from Sig1 . It was concluded that the results obtained from the maximum-likelihood-based method are more reliable than those obtained from the parsimony-based approach . This is apparently the first study that has shown that reproductive proteins in unicellular eukaryotes are influenced by positive selection. Yi Chuan Xue Bao, 2003 Feb, 30(2), 97 - 102 Transforming and secreting expression of human egf in mutant strain WYBS2001 of Bacillus and its functions; Wang GL et al.; Mutant strain WYBS2001 of B . subtilis with strong anti-pathogenic activity was obtained by mutagenic ultraviolet rays . The gene fragment of Human Epidermal Growth Factor(hegf) of 175 bp was synthesized by PCR and the restriction sites Pst I and Hind III, original code and the signal sequence CTTAGA of secreting vector pUS186 were induced in the fragment . The DNA sequencing result revealed that the synthesized fragment was identical with that of human egf . Then the biological engineering strain WYBS2001T with human egf was obtained by transforming pUSE which was constructed by cloning egf into the secreting plasmid pUS186, into mutant strain WYBS2001 . The result of RIA showed that hEGF can be found in the supernatant of the cultures and its content was 7.6 ng/ml . And the content can be increased if the proteinase inhibitor was added into the medium . After several generations' culturing, WYBS2001T positive engineering strain can still secrete and express hEGF steadily . The result of experiment showed hEGF had biological activity of proliferation and growth of human cell K562 in vitro . WYBS2001T engineering strain had obvious effect on healing the burned animals' models . This research showed microecological gene-engineering bacteria has good applying foreground. J Biol Chem, 2003 Aug 8, 278(32), 29880 - 9 Epub 2003 May 29. Acylation state of the phosphatidylinositol hexamannosides from Mycobacterium bovis bacillus Calmette Guerin and mycobacterium tuberculosis H37Rv and its implication in Toll-like receptor response; Gilleron M et al.; The dimannoside (PIM2) and hexamannoside (PIM6) phosphatidyl-myo-inositol mannosides are the two most abundant classes of PIM found in Mycobacterium bovis bacillus Calmette Guerin, Mycobacterium tuberculosis H37Rv, and Mycobacterium smegmatis 607 . Recently, these long known molecules received a renewed interest due to the fact that PIM2 constitute the anchor motif of an important constituent of the mycobacterial cell wall, the lipoarabinomannans (LAM), and that both LAM (phosphoinositol-capped LAM) and PIM are agonists of Toll-like receptor 2 (TLR2), a pattern recognition receptor involved in innate immunity . Due to the biological importance of these molecules, the chemical structure of PIM was revisited . The structure of PIM2 was recently published (Gilleron, M., Ronet, C., Mempel, M., Monsarrat, B., Gachelin, G., and Puzo, G . (2001) J . Biol . Chem . 276, 34896-34904) . Here we report the purification and molecular characterization of PIM6 in their native form . For the first time, four acyl forms of this molecule have been purified, using hydrophobic interaction chromatography . Mono- to tetra-acylated molecules were identified in M . bovis bacillus Calmette Guerin, M . tuberculosis H37Rv, and M . smegmatis 607 using a sophisticated combination of analytical tools, including matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and two-dimensional homo- and heteronuclear NMR spectroscopy . These experiments revealed that the major acyl forms are similar to the ones described for PIM2 . Finally, we show that PIM6, like PIM2, activate primary macrophages to secrete TNF-alpha through TLR2, irrespective of their acylation pattern, and that they signal through the adaptor MyD88. J Biol Chem, 2003 Aug 8, 278(32), 29600 - 8 Epub 2003 May 28. Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23 . Sequencing, gene expression, purification, and characterization; Oinuma K et al.; Analysis of the nitrile hydratase gene cluster involved in nitrile metabolism of Pseudomonas chlororaphis B23 revealed that it contains one open reading frame encoding aldoxime dehydratase upstream of the amidase gene . The amino acid sequence deduced from this open reading frame shows similarity (32% identity) with that of Bacillus phenylacetaldoxime dehydratase (Kato, Y., Nakamura, K., Sakiyama, H., Mayhew, S . G., and Asano, Y . (2000) Biochemistry 39, 800-809) . The gene product expressed in Escherichia coli catalyzed the dehydration of aldoxime into nitrile . The Pseudomonas aldoxime dehydratase (OxdA) was purified from the E . coli transformant and characterized . OxdA shows an absorption spectrum with a Soret peak that is characteristic of heme, demonstrating that it is a hemoprotein . For its activity, this enzyme required a reducing reagent, Na2S2O4, but did not require FMN, which is crucial for the Bacillus enzyme . The enzymatic reaction was found to be catalyzed when the heme iron of the enzyme was in the ferrous state . Calcium as well as iron was included in the enzyme . OxdA reduced by Na2S2O4 had a molecular mass of 76.2 kDa and consisted of two identical subunits . The kinetic parameters of OxdA indicated that aliphatic aldoximes are more effective substrates than aromatic aldoximes . A variety of spectral shifts in the absorption spectra of OxdA were observed upon the addition of each of various compounds (i.e . redox reagents and heme ligands) . Moreover, the addition of the substrate to OxdA gave a peak that would be derived from the intermediate in the nitrile synthetic reaction . P . chlororaphis B23 grew and showed the OxdA activity when cultured in a medium containing aldoxime as the sole carbon and nitrogen source . Together with these findings, Western blotting analysis of the extracts using anti-OxdA antiserum revealed that OxdA is responsible for the metabolism of aldoxime in vivo in this strain. Am J Respir Crit Care Med, 2003 Aug 15, 168(4), 448 - 55 Epub 2003 May 28. Risk factors for tuberculosis infection in sub-Saharan Africa: a contact study in The Gambia; Lienhardt C et al.; Few studies have investigated the risk factors for tuberculosis (TB) infection in highly endemic countries . We conducted a household study in The Gambia, in which a tuberculin skin test (TST) was performed in members of the households of 315 smear-positive pulmonary TB cases and 305 community control subjects . The risk of being TST positive (10 mm or more) was higher in contacts of cases than in contacts of control subjects . It increased with age, male sex, and duration of stay in the household but was not associated with the presence of a bacille de Calmette-Guerin scar . Within the households of the TB cases, the risk of TST positivity was higher in males and was increased with age, social proximity to the case, and the radiologic extent of the disease in the case's chest X-ray . Adjusting on these, the risk of TST positivity was higher in first-degree relatives compared with more distant relatives and nongenetically related household members, but the effect was not statistically significant . In highly endemic areas, the risk of TB infection in contacts of TB infectious cases is associated with age, sex, intensity of exposure to the case, and severity of disease in the case, but it is possible that genetic factors contribute to the susceptibility to Mycobacterium tuberculosis infection. Can J Urol, 2003 Apr, 10(2), 1790 - 5 Management of bacillus Calmette-Guerin (BCG) refractory superficial bladder cancer: results with intravesical BCG and Interferon combination therapy; Punnen SP et al.; INTRODUCTION AND OBJECTIVE: BCG is the most efficacious intravesical treatment for superficial bladder cancer . However, 30%-40% of tumors are refractory . BCG failure is an indication for cystectomy but several salvage intravesical (IVe) strategies have been proposed . Early results with reduced dose BCG in combination with IFN-a in patients are currently the most promising . We have adopted this approach and now report our preliminary results . This is the first report of this salvage therapy from Canada, the birthplace of IVe BCG therapy for superficial bladder cancer . METHODS: The "O'Donnell protocol" of reduced dose IVe BCG plus IFN-a was followed in 12 patients with BCG refractory superficial transitional cell carcinoma . A retrospective review of the efficacy and toxicity of the treatment was conducted . RESULTS: One year from induction therapy with salvage BCG/IFN-a, 6 of the 12 (50%) of patients were tumor free . Of the six recurrences, 3(50%) did not respond to the IVe therapy and had residual/recurrent tumor at the first follow-up visit . Risk factors for treatment failure were identified . The combinative therapy was well tolerated with minimal toxicity compared to previous full dose BCG . CONCLUSION: Our 12 month data with reduced dose IVe BCG plus IFN-a salvage therapy for BCG refractory superficial TCC confirm previous reports of >50% complete response rates . We need longer follow up in a larger patient population to determine the durability of this promising therapy in patients who would otherwise undergo radical cystectomy. Zhonghua Er Bi Yan Hou Ke Za Zhi, 2002 Aug, 37(4), 267 - 70 {Immunoregulatory effect of Bacillus Calmette Guerin aerosol to allergic airway diseases}; She C et al.; OBJECTIVE: To investigate whether bacillus calmette guerin (BCG) is effective used transairway and its preventive mechanisms to allergic airway diseases . METHODS: Animal experiment was finished . Adult rats were devided into four groups: control group, ovalbumin-sensitized group, BCG used transairway group, BCG used transairway + ovalbumin-sensitized group . Then these animals symptoms were studied and the pathology change were studied under microscope about nasal and bronchi mucosa and bronchoalveolar lavege fluid cells . IL-4 mRNA and IFN-gamma mRNA in lung tissue were detected through RT-PCR, the protein production of IL-4 and IFN-gamma about bronchoalveolar lavege fluid and serum were detected through ELISA . RESULTS: In ovalbumin-sensitized group, allergic animal model were made successfully . In only BCG used transairway group, the symptoms of animals were normal, few inflammation cells infiltrated into the mucosa of nasal and bronchi, the numbers of macrophage were greatly increased in smear of bronchoalveolar lavege fluid, IFN-gamma mRNA and protein production were greatly increased . In BCG used transairway + ovalbumin-sensitized group, the allergic symptoms and inflammation were greatly reduced, not only IFN-gamma mRNA and protein production were increased but also IL-4 mRNA and protein production were greatly decreased . CONCLUSION: It is a good pathway that BCG used transairway . The immunoloregulation mechanisms of BCG to allergic airway diseases are to enhance Th1 response, in the meantime, to suppress Th2 response. J Urol, 2003 Jun, 169(6), 2110 - 2 Intravesical bacillus Calmette-Guerin therapy for stage T1 grade 3 transitional cell carcinoma of the bladder: recurrence, progression and survival in a study of 57 patients; Peyromaure M et al.; PURPOSE: Stage T1 grade 3 transitional cell carcinoma of the bladder is associated with a high risk of tumor recurrence and progression . We report our experience with stage T1 grade 3 bladder tumors treated with bacillus Calmette-Guerin (BCG) therapy in the last 10 years . MATERIALS AND METHODS: We analyzed the outcome in 57 consecutive patients treated with intravesical BCG for stage T1 grade 3 bladder cancer between 1991 and 2001 . After initial transurethral resection all patients received a 6-week course of BCG therapy consisting of 1 instillation weekly . All patients underwent systematic biopsies at the end of the first BCG course . Patients with negative biopsies received maintenance BCG therapy, consisting of intravesical instillations each week for 3 weeks given 3, 6, 12, 18, 24, 30 and 36 months after the first course . Patients with residual tumor received a second course of 6 weekly instillations . Time to tumor recurrence and progression, and the rate of patient survival were retrospectively analyzed . RESULTS: Median followup was 53 months (range 9 to 110) . Minimum followup was 2 years in 36 cases (63.2%) and 5 years in 28 (49.1%) . After the first BCG course 50 patients (87.7%) had no residual disease, while 7 (12.3%) had residual tumor . The recurrence and progression rates were 42.1% and 22.8%, respectively . The rate of delayed cystectomy was 14% . The rate of disease specific survival was 87.7% . CONCLUSIONS: Our study confirms that BCG therapy is effective conservative treatment for patients with stage T1 grade 3 bladder tumors. J Insect Physiol, 1997 Sep, 43(9), 823 - 831 Comparison of the response of midgut epithelial cells and cell lines from lepidopteran larvae to CryIA toxins from Bacillus thuringiensis; Pang A et al.; The cytotoxic responses of midgut epithelial cells (MEC) from spruce budworm (SBW), gypsy moth (GM) and silkworm (SW) larvae were compared with the cytotoxic response of lepidopteran cell lines (SF-9, SE-1a, and CF-1) to CryIA toxins from Bacillus thuringiensis . The MEC from SBW, SW and GM had binding proteins for CryIA(a,b,c) toxins, whereas the lepidopteran cell lines had binding proteins for CryIA(c) . Single MEC exposed to CryIA(a,b,c) toxins in a qualitative lawn assay were equally susceptible to the toxins with a threshold response at about 1ng . The cell lines were not susceptible to CryIA(a,b) toxins in the dose range tested, but had threshold responses for CryIA(c) of 3.4ng for SF-9, 50.2ng for SE-1a and 5.9ng for CF-1 . In the quantitative Live/Dead assay, MEC were equally susceptible to CryIA(a,b,c) toxins with a threshold effect at about 1ng and a maximum effect at about 10ng . CF-1 was most sensitive to CryIA(c) with a threshold effect at 0.39ng and a maximal effect at about 1ng . In contrast, a 25-50 times greater dose of CryIA(a) or CryIA(b) was required to elicit a similar response as CryIA(c) for CF-1 . SF-9 and SE-1a were most susceptible to CryIA(c) with a threshold effect observed at about 0.5ng and maximal effects at about 2ng . SF-9 cells have a threshold and maximum response to CryIA(a,b) of about 10ng and 20ng, respectively . SE-1a cells have a threshold and maximal response to CryIA(a,b) of 5ng and 10ng, respectively . Intact midgut epithelium exposed to CryIA(a,b,c) toxins had a threshold dose of 2ng for CryIA(b), 10-30ng for CryIA(a) and 2-30ng for CryIA(c) . This study has shown that MEC are affected by a broader spectrum of toxins compared to the lepidopteran larvae and insect cell lines. J Insect Physiol, 1997 Apr, 43(4), 345 - 353 Characterization of a Monoclonal Antibody Against a 180 kDa Hemocyte Polypeptide Involved in Cellular Defence Reactions of the Stick Insect Bacillus rossius; MAZZINI M et al.; Defence properties of hemoctyes were investigated using the anti-hemocyte monoclonal antibody BrH1 obtained by immunizing mice with 2% paraformaldehyde-fixed hemoctyes of the stick insect Bacillus rossius . In Western blot analysis, the antibody recognized a 180 kDa antigen in hemocyte cell lysates, whereas fat body lysates and cell-free hemolymph were negative . In immunofluorescence analysis of cultured or freshly collected hemoctyes, BrH1 stained intracellular antigen(s) in detergent-treated cells . Transverse cryosections of adult stick insects probed by immunofluorescence with BrH1 showed in situ the scattered distribution of hemoctyes inside the haemocoel . The antigen(s) recognized by BrH1 appears to be involved in cell defence hemocyte-mediated mechanisms, as evidenced by the fact that cryosections of insects challenged in vivo with yeast cells, bacteria, or polystyrene latex particles and probed with BrH1 showed an accumulation of antigen surrounding the injected stimuli . J Mol Biol, 2003 Jun 6, 329(3), 467 - 77 The multi-layered structure of Dps with a novel di-nuclear ferroxidase center; Ren B et al.; The crystallization of cellular components represents a unique survival strategy for bacterial cells under stressed conditions . A highly ordered, layered structure is often formed in such a process, which may involve one or more than one type of bio-macromolecules . The main advantage of biocrystallization has been attributed to the fact that it is a physical process and thus is independent of energy consumption . Dps is a protein that crystallizes to form a multi-layered structure in starved cells in order to protect DNA against oxidative damage and other detrimental factors . The multi-layered crystal structure of a Dps protein from Bacillus brevis has been revealed for the first time at atomic resolution in the absence of DNA . Inspection of the structure provides the first direct evidence for the existence of a di-nuclear ferroxidase center, which possesses unique features among all the di-iron proteins identified so far . It constitutes the structural basis for the ferroxidase activity of Dps in the crystalline state as well as in solution . This finding proves that the enzymatic process of detoxification of metal ions, which may cause severe oxidative damage to DNA, is the other important aspect of the defense mechanism performed by Dps . In the multi-layered structure, Dps dodecamers are organized in a highly ordered manner . They adopt the classic form of hexagonal packing in each layer of the structure . Such arrangement results in reinforced structural features that would facilitate the attraction and absorption of metal ions from the environment . The highly ordered layered structure may provide an ideal basis for the accommodation of DNA between the layers so that it can be isolated and protected from harmful factors under stress conditions. J Biomol NMR, 2003 May, 26(1), 1 - 11 Biosynthesis of isotopically labeled gramicidins and tyrocidins by Bacillus brevis; Vogt TC et al.; The three-dimensional structure of bilayer-associated gramicidin A is available from a structural data base . This and related peptides are, therefore, ideal model compounds to use during the implementation and development of new NMR techniques for the structural investigations of membrane proteins . As these methods rely on the isotopic labelling of single, selected or all sites, we have, investigated and optimised biochemical protocols using different strains of the Gram-positive bacterium Bacillus brevis . With newly developed schemes for isotopic labelling large amounts of gramicidin and tyrocidin enriched with stable isotopes such as (15)N or (15)N/(13)C have been obtained at low cost . A variety of analytical and spectroscopic techniques, including HPLC, mass spectrometry and NMR spectroscopy are used to characterise the resulting products. J Biochem (Tokyo), 2003 Apr, 133(4), 467 - 74 Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents; Oyama T et al.; The crystal structures of beta-amylase from Bacillus cereus var . mycoides in complexes with five inhibitors were solved . The inhibitors used were three substrate analogs, i.e . glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose . For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor . In addition, another molecule of maltose or GGX was bound about 30 A away from the active site . A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5 . Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172) . A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft. J Biochem (Tokyo), 2003 Mar, 133(3), 317 - 24 Biochemical and genetic analyses of a novel gamma-cyclodextrin glucanotransferase from an alkalophilic Bacillus clarkii 7364; Takada M et al.; On screening for microorganisms in soil obtained in Japan that produce large amounts of gamma-cyclodextrin (gamma-CD), we identified a novel alkalophilic bacterium, Bacillus clarkii 7364 . The cyclodextrin glucanotransferase (CGTase) secreted into the culture medium by this bacterium was purified by affinity chromatography on a gamma-CD-immobilized column, followed by chromatography on a gel filtration column . The enzyme converted 13.7% of pre-gelatinized potato starch (10% w/w per reaction mixture) into CDs, and the majority (79%) of the product CDs was of the gamma form . This property is quite unique among known CGTases and thus we named this enzyme gamma-CGTase . The N-terminal and internal amino acid sequences of gamma-CGTase were determined and used to design PCR primers for amplification of the nucleotide sequence that encodes the gamma-CGTase gene . The entire gene sequence amplified by PCR was determined and then cloned into E . coli . The recombinant enzyme synthesized by E . coli retained biochemical properties quite similar to those of the original one . Comparison of the deduced amino acid sequence of gamma-CGTase with those of other known CGTases that have different product specificities revealed the importance of subsites -3 and -7 for the preferential gamma-cyclization activity. J Biochem (Tokyo), 2003 Mar, 133(3), 279 - 86 Glu-53 of Bacillus cereus sphingomyelinase acts as an indispensable ligand of Mg2+ essential for catalytic activity; Obama T et al.; Bacillus cereus sphingomyelinase (SMase) is an extracellular hemolysin classified into a group of Mg(2+)-dependent neutral SMases (nSMase) . Sequence comparison of bacterial and eukaryotic Mg(2+)-dependent nSMases has shown that several amino acid residues, including Glu-53 of B . cereus SMase, are conserved, suggesting a catalytic mechanism common to these enzymes . Mutational analysis has revealed that hemolytic and SM-hydrolyzing activities are abolished by E53A and E53Q mutations . Only the E53D mutant enzyme partially retains these activities, however, a significant decrease in the apparent k(cat)/K(m) for SM hydrolysis is observed by this mutation . Mg(2+) activates the wild-type enzyme in a two-step manner, i.e., at least two binding sites for Mg(2+), high- and low-affinity, are present on the enzyme . The binding affinity of essential Mg(2+) for the high-affinity site is decreased by the mutation . In addition, the binding affinities of Mn(2+) and Co(2+) (substitutes for Mg(2+)) are also decreased . On the contrary, the inhibitory effects of Ca(2+), Cu(2+), and Zn(2+) on SM-hydrolyzing activity are not influenced by the mutation . The results indicate that Glu-53 of B . cereus SMase acts as a ligand for Mg(2+) and is involved in the high-affinity Mg(2+)-binding site, which is independent of the binding site for inhibitory metals. Infect Immun, 2003 Jun, 71(6), 3116 - 24 Relationship of plcR-regulated factors to Bacillus endophthalmitis virulence; Callegan MC et al.; The explosive, destructive course of Bacillus endophthalmitis has been attributed to the production of toxins during infection . In this study we analyzed the contribution of toxins controlled by the global regulator plcR to the pathogenesis of experimental Bacillus endophthalmitis . Isogenic plcR-deficient mutants of Bacillus cereus and Bacillus thuringiensis were constructed by insertional inactivation of plcR by the kanamycin resistance cassette, aphA3 . Rabbit eyes were injected intravitreally with approximately 100 CFU of wild-type B . cereus or B . thuringiensis or a plcR-deficient mutant . The evolution of endophthalmitis resulting from each plcR-deficient mutant was considerably slower than that caused by each wild-type strain . Retinal function was not eliminated until 42 h postinfection in rabbits with endophthalmitis caused by the plcR-deficient mutants, whereas wild-type infections resulted in a complete loss of retinal function within 18 h . The intraocular inflammatory cell influx and retinal destruction in plcR-deficient endophthalmitis approached the severity observed in wild-ype infections, but not until 36 h postinfection . Gross and histological examinations of eyes infected with plcR mutants demonstrated that the anterior and posterior segment changes were muted compared to the changes observed in eyes infected with the wild types . The loss of plcR-regulated factors significantly attenuated the severity of Bacillus endophthalmitis . The results therefore suggest that plcR may represent a target for which adjunct therapies could be designed for the prevention of blindness during Bacillus endophthalmitis. Curr Infect Dis Rep, 2003 Jun, 5(3), 213 - 219 Progress in the Diagnosis, Prevention, and Treatment of Pertussis; Munoz FM et al.; Pertussis ("whooping cough"), caused by the gram- negative pleomorphic bacillus Bordetella pertussis, is a highly contagious, potentially life-threatening respiratory tract illness that has re-emerged worldwide as a cause of substantial morbidity and mortality in infants, children, and adolescents, even in countries with high vaccination rates . Waning immunity after immunization during childhood has been associated with a growing pool of susceptible adolescents and adults who are capable of transmitting pertussis to vulnerable unvaccinated or incompletely vaccinated infants . The use of acellular pertussis vaccine boosters in adolescents has been proposed and is likely to be recommended . Active immunization and improved methods for early diagnosis are key in the management of pertussis, and represent the most rapidly evolving aspects of this disease. Infez Med, 1999, 7(2), 129 - 132 {Fl gge's droplets}; Pistacchio E; After a review of the main theories by various authors on the aetiology and transmission modes of tubercular infection, the paper deals with the discovery of the tubercular bacillus made by Koch and the experiments conducted by his co-worker, Cornet, who concluded that the tubercular bacillus was more concentrated around the subject suffering from open pulmonary TBC . However, it was Flugge who eventually clarified beyond any doubt, after many experiments, the real, direct transmission mode of tuberculosis through droplets of saliva emitted by the sick and which are still known today as Flugge's droplets. J Immunol, 2003 Jun 1, 170(11), 5345 - 8 Cutting edge: major CD8 T cell response to live bacillus Calmette-Guérin is mediated by CD1 molecules; Kawashima T et al.; MHC class I-restricted CD8(+) T cells are a crucial component of the host defense against mycobacterial infection in mice, but it has often proved very difficult to identify the CD8 T cell response in humans . Human group 1 CD1 molecules (CD1a, -b, -c) mediate MHC-independent presentation of mycobacteria-derived lipid and glycolipid Ags to CD8(+) T cells, and their intracellular localization to the endocytic system may favor efficient monitoring of phagosome-resident mycobacteria . Here, we show that bacillus Calmette-Guerin (BCG)-immunized subjects contain a significant circulating pool of CD8(+) T cells that recognize BCG-infected DCs in a CD1-dependent, but MHC-independent, manner . These CD1-restricted T cells efficiently detected live, rather than dead, BCG and produced IFN-gamma, an important cytokine for protection against mycobacterial infection . These results emphasize that lipid-reactive CD8(+) T cells may contribute to host defense against mycobacterial infection. Tuberculosis (Edinb), 2003, 83(1-3), 201 - 7 Progress in TB drug development and what is still needed; Duncan K; Highly effective drugs for treating TB were introduced over 30 years ago, yet deaths from the disease continue to increase . New tools are needed, including drugs with activity against multi-drug resistant strains of Mycobacterium tuberculosis . Agents that reduce the duration and complexity of the current therapy would have a major impact on compliance and overall cure rate . In recent years, our understanding of the tubercle bacillus and its interaction with the human host has improved dramatically, particularly with the publication in 1998 of the complete genome sequence of M . tuberculosis H37Rv . New genetic tools have been developed and we can now ascertain the function of individual genes . Thus, many potential drug targets have been identified and a number demonstrated to be essential . Several lead compounds have been found, as well as a potential drug candidate, the nitroimidazopyran PA-824 . A far greater effort is needed to translate basic research into drug discovery programmes . High throughput screening and rational design must be employed to find lead compounds acting against well-validated targets and a substantial increase in resources devoted to medicinal chemistry is required to take these leads and turn them into drugs . Models of mycobacterial persistence, in which compounds with potent sterilizing activity can be rapidly analysed, must be characterized . Finally, surrogate markers that give an early indication of treatment outcome would facilitate clinical trials. Tuberculosis (Edinb), 2003, 83(1-3), 131 - 4 Hematogenous reseeding of the lung in low-dose, aerosol-infected guinea pigs: unique features of the host-pathogen interface in secondary tubercles; McMurray DN; The ability to study the early events in the pathogenesis of pulmonary tuberculosis in guinea pigs following very low dose (3-5 cfu) infection by the respiratory route has revealed that early (10-14 days) extrapulmonary dissemination results in reseeding of previously uninfected lobes of the lung by the hematogenous route . Thus, in every guinea pig, the lung is challenged twice, once by the airway and 2-3 weeks later by the circulatory system . The so called "secondary" pulmonary lesions which result from the bacillemia differ fundamentally from the primary lesions, in part, because the host has already developed a strong T cell mediated immunity when the hematogenous reseeding occurs . Secondary lung lesions in non-vaccinated guinea pigs behave similarly to primary lung lesions in previously vaccinated guinea pigs . Since the secondary, blood-borne lesions are thought to be the "reactivatable foci" which result in reactivation tuberculosis following prolonged persistent infection, it is important to understand the nature of the host-pathogen interaction in secondary lesions . The guinea pig model provides a unique opportunity to examine both the microbial and host factors which constitute that interface. Immunology, 2003 Jun, 109(2), 308 - 14 The combination of plasmid interleukin-12 with a single DNA vaccine is more effective than Mycobacterium bovis (bacille Calmette-Guèrin) in protecting against systemic Mycobacterim avium infection; Martin E et al.; Sub-unit vaccines utilizing purified mycobacterial proteins or DNA vaccines induce partial protection against mycobacterial infections . For example, immunization with DNA vaccines expressing the gene for the immunodominant 35000 MW protein, common to Mycobacterium avium and Mycobacterium leprae but absent from the Mycobacterium tuberculosis complex, conferred significant protection against infection with either virulent M . avium or M . leprae in mice . However, the level of protection was equivalent to that obtained with the viable, attenuated vaccine, Mycobacterium bovis, bacille Calmette-Guerin (BCG) . The cytokine, interleukin (IL)-12, is essential for priming naive CD4+ T lymphocytes to differentiate into interferon-gamma (IFN-gamma)-secreting T cells . We have used a novel self-splicing vector expressing both chains of murine IL-12 to determine if plasmid IL-12 would increase the efficacy of a vaccine expressing the M . avium 35000 MW protein (DNA-Av35) . Co-immunization with p2AIL-12 and DNA-Av35 led to a significant increase in the number of antigen-specific IFN-gamma secreting cells and total amount of IFN-gamma released, but a concomitant fall in the antibody response to the 35000 MW protein . This pattern of response was associated with enhanced clearance of M . avium from the liver and spleen of coimmunized mice, and was significantly more effective than BCG or DNA-Av35 . alone . Following M . avium challenge there was significant increase in the expansion of the 35000 MW antigen-reactive T cells in the coimmunized mice . Therefore, plasmid-delivered IL-12 acts as an effective adjuvant to increase the protective efficacy of a single DNA vaccine against M . avium infection above that achieved by BCG, and this strategy may improve the efficacy of subunit vaccines against M . leprae and M . tuberculosis. Mol Genet Genomics, 2003 May, 269(2), 252 - 60 Epub 2003 Mar 19. Cloning and expression of a novel lipase gene from Bacillus sphaericus 205y; Rahman RN et al.; A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia . The gene for the lipase was recovered from a genomic library and sequenced . Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database . The analysis suggested that the B . sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far . Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG . The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene. Mol Ecol, 2003 Apr, 12(4), 1077 - 86 Effects of transgenic Bt corn litter on the earthworm Lumbricus terrestris; Zwahlen C et al.; A 200-day study was carried out to investigate the impact of transgenic Bacillus thuringiensis (Bt) corn on immature and adult Lumbricus terrestris in the field and in the laboratory . Another objective of this study was to develop test methods that could be used for standard testing of the impact of transgenic plants on different earthworm species in the field and in the laboratory . For this purpose two different experiments were involved, a laboratory experiment with adult L . terrestris and a field experiment with immature L . terrestris . No lethal effects of transgenic Bt corn on immature and adult earthworms were observed . Immature L . terrestris in the field had a very similar growth pattern when fed either (Bt+) or (Bt-) corn litter . No significant differences in relative weights of (Bt+) and (Bt-) corn-fed adult L . terrestris were observed during the first 160 days of the laboratory trial, but after 200 days adult L . terrestris had a significant weight loss of 18% of their initial weight when fed (Bt+) corn litter compared to a weight gain of 4% of the initial weight of (Bt-) corn-fed earthworms . Further studies are necessary to see whether or not this difference in relative weight was due to the Bt toxin or other factors discussed in the study . Degradation of Cry1Ab toxin in corn residues was significantly slower in the field than at 10 degrees C in the laboratory . Enzyme-linked immunosorbent assay results indicated that earthworms in both experiments were exposed to the Bt toxin throughout the whole experimental time. Mikrobiologiia, 2003 Mar-Apr, 72(2), 268 - 74 {Seed bacterization and rhizosphere of wheat seedlings colonization by Bacillus Cohn}; Kus'mina LIu et al.; The dynamics of introduced antagonistic bacteria in the spring wheat rhizosphere was studied in small-plot field experiments during several growing seasons . The population density of introduced bacteria was found to considerably depend on the inoculum dose . At sufficiently high inoculum doses, the introduced bacteria remained in the wheat rhizosphere over the entire vegetative period (88-109 days) . The maximum population density of introduced bacteria was observed in the early terms of plant development . No correlation was found between the population density of introduced bacteria and the degree of suppression of root rot or the structural crop yield parameters . The beneficial effect of preplanting seed bacterization on wheat plants was, as a rule, profound only during unfavorable growing seasons. Plasmid, 2003 May, 49(3), 205 - 32 The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes; Andrup L et al.; Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb . Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host . In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B . thuringiensis strains . The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared . Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed . Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3) . On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified . Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets) . These genes were termed bcol for Bacillus-collagen-like genes. J Epidemiol, 2003 May, 13(3), 127 - 35 BCG vaccination and tuberculosis in Japan; Rahman M et al.; This paper summarizes Bacillus Calmette-Guerin (BCG) vaccination and revaccination policies in Japan, its cost-effectiveness, side effects, proposed selective vaccination strategy, and present tuberculosis situation in Japanese perspectives based on Medline database and other published reports . Universal BCG vaccination in infants and revaccination among children were not found economically justifiable . Overall tuberculosis incidence in Japan is higher than that of other developed countries . Trend of decline in tuberculosis incidence is similar to that of the countries where universal BCG vaccination has never been implemented . In the recent years, the number of tuberculosis group infection has been escalating . Since BCG revaccination program has already been discontinued, a consensus on universal BCG vaccination is also essential based on social, political, and economical factors . Side by side, more pragmatic strategies such as well-defined tuberculin test, selective vaccination policy based on tuberculosis incidence in each administrative zone, and early vaccination of high risk groups, should be formulated. Proteomics, 2003 May, 3(5), 798 - 802 Identification of novel proteins in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin in the isoelectric point range 6-11; Florio W et al.; Two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins in the isoelectric point range 6-11 in culture filtrates of Mycobacterium bovis bacillus Calmette-Guerin (BCG) . Twelve proteins were identified, three of which had not been described previously . The expression of the identified proteins was comparatively analyzed in culture filtrates of BCG in different growth phases and culture conditions . For some of these proteins, the relative protein abundance in the different culture filtrate preparations was significantly different . The differential expression of the identified proteins is discussed in relation to their putative localization and/or biological function. Theor Appl Genet, 2003 May, 106(7), 1225 - 33 Epub 2002 Dec 10. Frequency of alleles conferring resistance to Bt maize in French and US corn belt populations of the European corn borer, Ostrinia nubilalis; Bourguet D et al.; Farmers, industry, governments and environmental groups agree that it would be useful to manage transgenic crops producing insecticidal proteins to delay the evolution of resistance in target pests . The main strategy proposed for delaying resistance to Bacillus thuringiensis ( Bt) toxins in transgenic crops is the high-dose/refuge strategy . This strategy is based on the unverified assumption that resistance alleles are initially rare (<10(-3)) . We used an F(2) screen on >1,200 isofemale lines of Ostrinia nubilalis Hubner (Lepidoptera: Crambidae) collected in France and the US corn belt during 1999-2001 . In none of the isofemale lines did we detect alleles conferring resistance to Bt maize producing the Cry1Ab toxin . A Bayesian analysis of the data indicates that the frequency of resistance alleles in France was <9.20 x 10(-4) with 95% probability, and a detection probability of >80% . In the northern US corn belt, the frequency of resistance to Bt maize was <4.23 x 10(-4) with 95% probability, and a detection probability of >90% . Only 95 lines have been screened from the southern US corn belt, so these data are still inconclusive . These results suggest that resistance is probably rare enough in France and the northern US corn belt for the high-dose plus refuge strategy to delay resistance to Bt maize. Infez Med, 1999, 7(4), 260 - 263 {Plaut-Vincent's angina}; Pistacchio E; The clinical appearance of fuso-spirillar infection in patients with a serious impairment of the immunitary system was the starting-point for a historical study . At the end of nineteenth century two european researchers clarified the cause of many infections . Association of a spindle-shaped bacillus with a spirillar one was shown to cause several pathologies: necrotic ulcerous stomatitis, pseudomembranous angina, hepatic abscess, otitis, pneumonia, etc . The identification of such bacteria contributed to improving further differential diagnosis with diphteria Infez Med, 1999, 7(4), 257 - 259 {A case of sepsis caused by BCG (Bacillus Calmette Guerin) after its bladder instillation in a 71-year-old patient affected by bladder carcinoma}; Sica S et al.; Sometimes the Calmette-Guerin bacillus can be used with success as local immunotherapy for superficial bladder carcinoma . BCG is a living attenuated strain of Mycobacterium bovis and therefore its biadder instillation may give rise, in some particular cases, to serious systemic effects, caused either by hypersensitivity reaction or by systemic dissemination in case with low efficiency of cellular immunity . We describe a case of sepsis in a 71-year-old patient after bladder instillation of BCG with a rare complication of granulomatous hepatitis . The etiological diagnosis of granulomatous hepatitis with PCR on liver biopsy is very important for the specific therapy before the patient can undergo a steroid regime. Am J Respir Cell Mol Biol, 2003 Nov, 29(5), 545 - 51 Epub 2003 May 14. Apoptosis genes in human alveolar macrophages infected with virulent or attenuated Mycobacterium tuberculosis: a pivotal role for tumor necrosis factor; Spira A et al.; Tumor necrosis factor (TNF)-alpha-dependent apoptosis of alveolar macrophages (AM) after infection with avirulent Mycobacterium tuberculosis (Mtb) results in bacillary death and the destruction of a growth niche for the pathogen . This response is minimized after infection with virulent strains of Mtb . To study the genetic control of Mtb-induced apoptosis, we used microarrays to interrogate the expression profile of infected human AM . Although we found variation in gene expression between different donors of AM, a set of genes were constant for each condition . A group of proapoptotic genes were downregulated after infection by virulent Mtb strain H37Rv, whereas infection with avirulent Mtb H37Ra led to a gene expression profile that would favor macrophage apoptosis . Neutralizing TNF in macrophage cultures infected with H37Ra changed the gene expression profile to one that resembled the profile of macrophages infected with H37Rv . These data reveal that apoptosis-related genes are regulated differently by virulent or attenuated Mtb strains, and are consistent with the hypothesis that virulent Mtb interfere with TNF death signaling . Given the importance of TNF in host defense against tuberculosis, the ability to repress the expression of genes activated by TNF may constitute a bacillary virulence mechanism. Biomed Environ Sci, 2003 Mar, 16(1), 17 - 28 Genetically engineered corn rootworm resistance: potential for reduction of human health effects from pesticides; Oehme FW et al.; OBJECTIVE AND METHODS: Insecticide use, grower preferences regarding genetically engineered (GE) corn resistant to corn rootworm (CRW), and the health effects of using various CRW insecticides (organophosphates, pyrethroids, fipronil and carbamates) are reviewed for current and future farm practices . RESULTS: Pest damage to corn has been reduced only one-third by insecticide applications . Health costs from insecticide use appear significant, but costs attributable to CRW control are not quantifiable from available data . Methods reducing health-related costs of insecticide-based CRW control should be evaluated . As a first step, organophosphate insecticide use has been reduced as they have high acute toxicity and risk of long-term neurological consequences . A second step is to use agents which more specifically target the CRW . CONCLUSION: Whereas current insecticides may be poisonous to many species of insects, birds, mammals and humans, a protein derived from Bacillus thurigiensis and produced in plants via genetic modification can target the specific insect of CRW (Coleoptra), sparing other insect and non-insect species from injury. J Eukaryot Microbiol, 2003 Mar-Apr, 50(2), 140 - 3 Molecular cloning and sequencing of the merozoite surface antigen 2 gene from Plasmodium falciparum strain FCC-1/HN and expression of the gene in mycobacteria; Zheng C et al.; Strain bacillus Calmette-Guerin (BCG) of Mycobacterium bovis has been used as a live bacterial vaccine to immunize more than 3 billion people against tuberculosis . In an attempt to use this vaccine strain as a vehicle for protective antigens, the gene encoding merozoite surface antigen 2 (MSA2) was amplified from strain FCC-1/HN Plasmodium falciparum genome, sequenced, and expressed in M . bovis BCG under the control of an expression cassette carrying the promoter of heat shock protein 70 (HSP70) from Mycobacterium tuberculosis . The recombinant shuttle plasmid pBCG/MSA2 was introduced into mycobacteria by electroporation, and the recombinant mycobacteria harboring pBCG/MSA2 could be induced by heating to express MSA2; the molecular mass of recombinant MSA2 was about 31 kDa . This first report of expression of the full-length P . falciparum MSA2 gene in BCG provides evidence for use of the HSP70 promoter in expressing a foreign gene in BCG and in development of BCG as a multivalent vectoral vaccine for malaria. Biochemistry, 2003 May 20, 42(19), 5775 - 83 A side reaction of alanine racemase: transamination of cycloserine; Fenn TD et al.; Alanine racemase (EC 5.1.1.1) catalyzes the interconversion of alanine enantiomers, and thus represents the first committed step involved in bacterial cell wall biosynthesis . Cycloserine acts as a suicide inhibitor of alanine racemase and as such, serves as an antimicrobial agent . The chemical means by which cycloserine inhibits alanine racemase is unknown . Through spectroscopic assays, we show here evidence of a pyridoxal derivative (arising from either isomer of cycloserine) saturated at the C4' carbon position . We additionally report the L- and D-cycloserine inactivated crystal structures of Bacillus stearothermophilus alanine racemase, which corroborates the spectroscopy via evidence of a 3-hydroxyisoxazole pyridoxamine derivative . Upon the basis of the kinetic and structural properties of both the L- and D-isomers of the inhibitor, we propose a mechanism of alanine racemase inactivation by cycloserine . This pathway involves an initial transamination step followed by tautomerization to form a stable aromatic adduct, a scheme similar to that seen in cycloserine inactivation of aminotransferases. Biochemistry, 2003 May 20, 42(19), 5574 - 81 Crystal structure of a catalytic site mutant of beta-amylase from Bacillus cereus var . mycoides cocrystallized with maltopentaose; Miyake H et al.; The X-ray crystal structure of a catalytic site mutant of beta-amylase, E172A (Glu172 --> Ala), from Bacillus cereus var . mycoides complexed with a substrate, maltopentaose (G5), and the wild-type enzyme complexed with maltose were determined at 2.1 and 2.0 A resolution, respectively . Clear and continuous density corresponding to G5 was observed in the active site of E172A, and thus, the substrate, G5, was not hydrolyzed . All glucose residues adopted a relaxed (4)C(1) conformation, and the conformation of the maltose unit for Glc2 and Glc3 was much different from those of other maltose units, where each glucose residue of G5 is named Glc1-Glc5 (Glc1 is at the nonreducing end) . A water molecule was observed 3.3 A from the C1 atom of Glc2, and 3.0 A apart from the OE1 atom of Glu367 which acts as a general base . In the wild-type enzyme-maltose complex, two maltose molecules bind at subsites -2 and -1 and at subsites +1 and +2 in tandem . The conformation of the maltose molecules was similar to that of the condensation product of soybean beta-amylase, but differed from that of G5 in E172A . When the substrate flips between Glc2 and Glc3, the conformational energy of the maltose unit was calculated to be 20 kcal/mol higher than that of the cis conformation by MM3 . We suggest that beta-amylase destabilizes the bond that is to be broken in the ES complex, decreasing the activation energy, DeltaG(++), which is the difference in free energy between this state and the transition state. J Egypt Soc Parasitol, 2003 Apr, 33(1), 219 - 28 Vaccination trial against experimental trichrinellosis using autoclaved Trichinella spiralis larvae vaccine (ATSLV); Eissa MM et al.; The autoclaved Trichinella spiralis larvae vaccine (ATSLV) was tested and showed a surprising and somewhat unpredictable effect on the immune system of mice experimentally infected with T . spiralis . The vaccine was given with Bacille Calmette Guerin (BCG) as an adjuvant at different durations and by different routes of administration . The best result was achieved by given the vaccine twice intradermally with two weeks interval, as evidenced by a significant reduction in adult and larval count, as well as reproductive capacity index . Histopathologically, there was significant reduction in number of the encysted larvae which showed degeneration and hyalinization of the cyst wall accompanied by early pericystic fibrosis. Microb Ecol, 2003 Jul, 46(1), 55 - 61 Epub 2003 May 13. Sediment microbes of deep-sea bioherms on the northwest shelf of Australia; Johnson JE et al.; The northwest shelf of Australia is a region with known petroleum reserves . Recent geological investigations have revealed carbonate knolls postulated to be of biological origin . Sediment microbial populations at three sites on the northwest shelf, Pee Shoal, Mermaid Reef, and Scott Reef, were investigated for the presence of hydrocarbon-degrading bacteria . From two sampling trips, 246 bacterial strains were purified, and 182 of these strains displayed preferential growth on hydrocarbon-selective media . Strains were tested for nutrient specificity using single hydrocarbon fermentations . Metabolic profiles were constructed using biochemical testing, fatty acid analysis, and metabolic rate experiments . 16S rRNA gene sequence analysis of four strains identified these strains as members of the genera Sphingomonas, Bacillus, and Microbacterium, and one strain as a member of the alpha-Proteobacteria . The potential of these strains as bioindicators of hydrocarbon pollutants or for bioremediation in marine environments is discussed. Clin Diagn Lab Immunol, 2003 May, 10(3), 376 - 82 NF-kappaB is involved in regulation of CD40 ligand expression on Mycobacterium bovis bacillus Calmette-Guérin-activated human T cells; Mendez-Samperio P et al.; Interaction between CD40L (CD154) on activated T cells and its receptor CD40 on antigen-presenting cells has been reported to be important in the resolution of infection by mycobacteria . However, the mechanism(s) by which Mycobacterium bovis bacillus Calmette-Guerin (BCG) up-regulates membrane expression of CD40L molecules is poorly understood . This study was done to investigate the role of the nuclear factor kappaB (NF-kappaB) signaling pathway in the regulation of CD40L expression in human CD4(+) T cells stimulated with BCG . Specific pharmacologic inhibition of the NF-kappaB pathway revealed that this signaling cascade was required in the regulation of CD40L expression on the surface of BCG-activated CD4(+) T cells . These results were further supported by the fact that treatment of BCG-activated CD4(+) T cells with these pharmacological inhibitors significantly down-regulated CD40L mRNA . In this study, inhibitor kappaBalpha (IkappaBalpha) and IkappaBbeta protein production was not affected by the chemical protease inhibitors and, more importantly, BCG led to the rapid but transient induction of NF-kappaB activity . Our results also indicated that CD40L expression on BCG-activated CD4(+) T cells resulted from transcriptional up-regulation of the CD40L gene by a mechanism which is independent of de novo protein synthesis . Interestingly, BCG-induced activation of NF-kappaB and the increased CD40L cell surface expression were blocked by the protein kinase C (PKC) inhibitors 1-{5-isoquinolinesulfonyl}-2-methylpiperazine and salicylate, both of which block phosphorylation of IkappaB . Moreover, rottlerin a Ca(2+)-independent PKC isoform inhibitor, significantly down-regulated CD40L mRNA in BCG-activated CD4(+) T cells . These data strongly suggest that CD40L expression by BCG-activated CD4(+) T cells is regulated via the PKC pathway and by NF-kappaB DNA binding activity. Proc R Soc Lond B Biol Sci, 2003 Apr 22, 270(1517), 791 - 7 Initial frequency of alleles conferring resistance to Bacillus thuringiensis poplar in a field population of Chrysomela tremulae; Genissel A et al.; Globally, the estimated total area planted with transgenic plants producing Bacillus thuringiensis (Bt) toxins was 12 million hectares in 2001 . The risk of target pests becoming resistant to these toxins has led to the implementation of resistance-management strategies . The efficiency and sustainability of these strategies, including the high-dose plus refuge strategy currently recommended for North American maize, depend on the initial frequency of resistance alleles . In this study, we estimated the initial frequencies of alleles conferring resistance to transgenic Bt poplars producing Cry3A in a natural population of the poplar pest Chrysomela tremulae (Coleoptera: Chrysomelidae) . We used the F(2) screen method developed for detecting resistance alleles in natural pest populations . At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele . We estimated mean resistance-allele frequency for the period 1999-2001 at 0.0037 (95% confidence interval = 0.00045-0.0080) with a detection probability of 90% . These results demonstrate that (i) the F(2) screen method can be used to detect major alleles conferring resistance to Bt-producing plants in insects and (ii) the initial frequency of alleles conferring resistance to Bt toxin can be close to the highest theoretical values that are expected prior to the use of Bt plants if considering fitness costs and typical mutation rates. Salud Publica Mex, 2003 Mar-Apr, 45(2), 78 - 83 {Epidemiology of Tuberculosis in Mexico, 1981-1998 . Inconsistencies between reports of the WHO and the Ministry of health.}; Baez-Saldana AR et al.; OBJECTIVE: To describe the tuberculosis morbidity and mortality trends in Mexico, by comparing the data reported by the Ministry of Health (MH) and the World Health Organization (WHO) between 1981 and 1998 . MATERIAL AND METHODS: The number of cases notified in the past few years, their rates, and the trends of the disease in Mexico were analyzed . The incidence of smear-positive pulmonary tuberculosis was estimated for 1997 and 1998 with the annual tuberculosis infection risk (ATIR), to estimate the percentage of bacilliferous cases in 1997-1998 . RESULTS: WHO reported more tuberculosis cases for Mexico than the MH . However, this difference has decreased throughout the years . The notification of smear-positive cases remained stable during 1993-1998 . The estimated percentages of detection were 66% for 1997 and 26% for 1998 (based on ATIR of 0.5%) . Tuberculosis mortality decreased gradually (6.7% per year) between 1990 and 1998, whereas the number of new cases increased, suggesting the persistence of disease transmission in the population . CONCLUSIONS: Inconsistencies between case notifications from national data and WHO were considerable, but decreased progressively during the study period . According to ATIR estimations, a considerable number of infectious tuberculosis cases are not detected . The English version of this paper is available at: http://www.insp.mx/salud/index.html. Breast Cancer, 2003, 10(2), 175 - 8 Tuberculosis of axillary lymph nodes with primary breast cancer; Fujii T et al.; A rare case of tuberculosis of axillary lymph nodes occurring with primary breast cancer is presented . A 78-year-old woman with no history of pulmonary tuberculosis was admitted to our hospital to undergo examination for a lump in her right breast . The tumor was in the upper outer quadrant of the right breast . On palpation, the tumor was 1.2 cm in diameter and axillary lymph node swelling was noted . Mammography disclosed a spiculated mass and swelling and calcification of the axillary lymph nodes . Sonography showed an irregular hypoechoic mass in the right breast and lymph node swelling in the right axilla, indicating breast cancer with axillary lymph nodes metastases . Chest X-ray showed clustered calcifications in the right axilla and a granular shadow in the right upper lobe . Breast conserving therapy was carried out . Invasive papillotubular carcinoma of the right breast and granulomas with calcification of lymph nodes, compatible with tuberculosis, was diagnosed . Tubercle bacillis were detected by culture of lymph nodes . This case suggests that X-ray is useful for diagnosing lymph node tuberculosis . Lymph node tuberculosis should be suspected when lymph node swelling is noted and X-ray shows clustered calcifications in axillary lymph nodes. Protein Eng, 2003 Apr, 16(4), 287 - 93 Hyperthermostabilization of Bacillus licheniformis alpha-amylase and modulation of its stability over a 50 degrees C temperature range; Declerck N et al.; Bacillus licheniformis alpha-amylase (BLA) is a highly thermostable starch-degrading enzyme that has been extensively studied in both academic and industrial laboratories . For over a decade, we have investigated BLA thermal properties and identified amino acid substitutions that significantly increase or decrease the thermostability . This paper describes the cumulative effect of some of the most beneficial point mutations identified in BLA . Remarkably, the Q264S-N265Y double mutation led to a rather limited gain in stability but significantly improved the amylolytic function . The most hyperthermostable variants combined seven amino acid substitutions and inactivated over 100 times more slowly and at temperatures up to 23 degrees C higher than the wild-type enzyme . In addition, two highly destabilizing mutations were introduced in the metal binding site and resulted in a decrease of 25 degrees C in the half-inactivation temperature of the double mutant enzyme compared with wild-type . These mutational effects were analysed by protein modelling based on the recently determined crystal structure of a hyperthermostable BLA variant . Our engineering work on BLA shows that the thermostability of an already naturally highly thermostable enzyme can be substantially improved and modulated over a temperature range of 50 degrees C through a few point mutations. Am J Infect Control, 2003 May, 31(3), 144 - 50 Do we practice what we preach? Health care worker screening and vaccination; Brotherton JM et al.; OBJECTIVE: To describe the current screening and immunization practices in New South Wales (NSW) hospitals and the experience of NSW nurses in relation to screening and immunization and to identify areas that can be targeted for improvement . DESIGN: This was a cross-sectional survey . SETTING: The study was performed in NSW, Australia . METHODS: We used a written questionnaire to survey the infection control/occupational health coordinators of all of the 85 private hospitals and 204 eligible public hospitals in NSW and 800 randomly sampled registered nurses . RESULTS: Response rates were high (hospitals {90%}, nurses {70%}) . Hospitals almost universally offered hepatitis B vaccination to nurses (251/261, 96%), but more than one quarter (132/473, 28%) of nurses reported incomplete vaccination . Provision to physicians was relatively poor (142/261, 54%) . The majority of nurses (> 80%) had been vaccinated with bacille Calmette-Guerin vaccine, but hospitals reported variable tuberculosis screening practices . Both hospitals and nurses reported low rates (< 30%) of screening and vaccination provision for varicella and measles-mumps-rubella . Two thirds of NSW hospitals (174/261, 67%) provided annual influenza vaccination . CONCLUSIONS: Even though hepatitis B immunization programs were widespread, their effectiveness could be improved by ensuring that vaccination schedules are completed and by targeting physicians . Varicella and measles-mumps-rubella screening and immunization programs are currently lacking . Better strategies are needed to improve the implementation of health care worker protection guidelines in hospitals. J Clin Microbiol, 2003 May, 41(5), 2247 - 8 Bacteremia due to three Bacillus species in a case of Munchausen's syndrome; Galanos J et al.; We report on a case of recurrent bacteremia due to three Bacillus spp . in an immunocompetent patient with no history of intravenous drug use . The source of the organisms was postulated to be a self-injected compound containing Bacillus spores, given the patient's past history of psychiatric illness and self-destructive behavior. Biochem J, 2003 Aug 1, 373(Pt 3), 875 - 83 Structural and thermal stability analysis of Escherichia coli and Alicyclobacillus acidocaldarius thioredoxin revealed a molten globule-like state in thermal denaturation pathway of the proteins: an infrared spectroscopic study; Pedone E et al.; The structure of thioredoxin from Alicyclobacillus acidocaldarius (previously named Bacillus acidocaldarius ) (BacTrx) and from Escherichia coli ( E . coli Trx) was studied by Fourier-transform IR spectroscopy . Two mutants of BacTrx {Lys(18)-->Gly (K18G) and Arg(82)-->Glu (R82E)} were also analysed . The data revealed similar secondary structures in all proteins, but BacTrx and its mutants showed a more compact structure than E . coli Trx . In BacTrx and its mutants, the compactness was p(2)H-dependent . All proteins revealed the existence of a molten globule-like state . At p(2)H 5.8, the temperature at which this state was detected was higher in BacTrx and decreased in the different proteins in the following order: BacTrx>R82E>K18G> E . coli Trx . At neutral or basic p(2)H, the molten globule-like state was detected at the same temperature in both BacTrx and R82E, whereas it was found at the same temperature in all p(2)Hs tested for E . coli Trx . The thermal stability of the proteins was in the following order at all p(2)Hs tested: BacTrx>R82E>K18G> E . coli Trx, and was lower for each protein at p(2)H 8.4 than at neutral or acidic p(2)Hs . The formation of protein aggregates, brought about by thermal denaturation, were observed for BacTrx and K18G at all p(2)Hs tested, whereas they were present in R82E and E . coli Trx samples only at p(2)H 5.8 . The results indicated that a single mutation might affect the structural properties of a protein, including its propensity to aggregate at high temperatures . The data also indicated a possible application of Fourier-transform IR spectroscopy for assessing molten globule-like states in small proteins. Bioresour Technol, 2003 Mar, 87(1), 57 - 61 Production of alpha amylase by Bacillus licheniformis using an economical medium; Ikram-ul-Haq et al.; The present study is concerned with the selection of new medium for the production of alpha amylase by Bacillus licheniformis . Different agricultural by-products such as wheat bran, sunflower meal, cotton seed meal, soybean meal, rice husk or rice bran were tested for the production of alpha amylase . Among different agricultural by-products evaluated, wheat bran was found to be the best basal and standardized medium for optimal production of alpha amylase . The production was increased 2-folds when soluble starch was replaced with pearl millet starch at 1% level and nutrient broth concentrations was reduced from 1% level to 0.5% . The newly selected fermentation medium containing (% w/v) wheat bran 1.25, nutrient broth 0.5, pearl millet starch 1.0, lactose 0.5, NaCl 0.5, CaCl2 0.2 in 100 ml of phosphate buffer . The kinetic values of Y(p/x), Y(p/s), and Q(p) indicated that the production of enzyme was greater in newly selected medium than the conventional more expensive medium. Curr Microbiol, 2003 Apr, 46(4), 256 - 60 Molecular typing by pulsed-field gel electrophoresis of Bacillus thuringiensis from root voles; Swiecicka I; The root voles intestinal strains of Bacillus thuringiensis, were characterised by pulsed-field gel electrophoresis (PFGE) . For 14 isolates, three pulsotypes were found, with the use of SmaI or NotI as restriction enzymes . Strains in each pulsotypes presented identical DNA patterns, indicating that the population structure of B . thuringiensis from root voles is clonal . The similarities in banding patterns were estimated at 56% and 33% for SmaI and NotI digests, respectively . The strains under study differed significantly in the size of their entire genome, which varied between 2.4 and 4.2 Mb . No significant differences were detected among the isolates subjected to biochemical properties determined by API tests . Present study showed that genomic diversity is a common feature of B . thuringiensis originating from one ecological niche . PFGE appears to be a useful technique for use in studies on the spread of B . thuringiensis in the environment. Curr Microbiol, 2003 Jun, 46(6), 432 - 4 Involvement of glycine and aspartate residues in the binding capacity of FAD in the NADH dehydrogenase from an alkaliphilic Bacillus; Shiraki M et al.; There is a region exhibiting a similarity of amino acid sequence near the carboxyl-terminal segment of each FAD-containing oxidoreductase . In this region, four amino acid residues-Thr, Ala, Gly, and Asp-are highly conserved . To determine the involvement of the four amino acid residues (Thr-469, Ala-476, Gly-478, and Asp-479) in the activity of NADH dehydrogenase of an alkaliphilic Bacillus, mutations of these amino acid residues were conducted . In spite of high conservation, mutations of Thr-469 and Ala-476 to Ala and Ser, respectively, did not lead to a critical loss of enzyme activity . However, mutations of Gly-478 and Asp-479 to Ala caused a complete loss of the activity, which appears to result from the loss of binding capacity of FAD. J Biol Chem, 2003 Jul 11, 278(28), 25766 - 72 Epub 2003 May 05. Purification, characterization, and molecular cloning of a novel keratan sulfate hydrolase, endo-beta-N-acetylglucosaminidase, from Bacillus circulans; Yamagishi K et al.; Keratan sulfate (KS) is degraded by various enzymes including endo-beta-galactosidase, keratanase, and keratanase II, which are used for the structural analysis of KS . We purified a novel KS hydrolase, endo-beta-N-acetylglucosaminidase, from the cell pellet and conditioned medium of Bacillus circulans, by sequential chromatography using DE52 and phenyl-Sepharose columns with approximately 63- and 180-fold purity and 58 and 12.5% recovery, respectively . Like keratanase II of Bacillus sp . Ks36, the enzyme, designated Bc keratanase II, hydrolyzed KS between the 4GlcNAcbeta1-3Gal1 structure (endo-beta-N-acetylglucosaminidase), but not hyaluronan, heparan sulfate, heparin, and chondroitin sulfate C, demonstrating a strict specificity to KS . The enzyme digested shark cartilage KS to disaccharides and tetrasaccharides and bovine cornea KS to hexasaccharide, indicating that it prefers highly sulfated KS . Distinct from keratanase II of strain Ks36, the enzyme digested shark cartilage KS at an optimal temperature of 55 degrees C . Based on partial peptide sequencing of the enzyme, we molecularly cloned the gene, which encodes a protein with a predicted molecular mass of approximately 200 kDa . From the deduced protein sequence, Bc keratanase II contained a domain at the C terminus, homologous to the S-layer-like domain of pullulanase from Thermoanaerobacterium thermosulfurigenes and endoxylanase from Thermoanaerobacterium saccharolyticum, and a carbohydrate-binding domain, which may serve to specifically recognize KS chains . A full-length recombinant enzyme showed keratanase II activity . These results may prove useful for the structural analysis of KS toward achieving an understanding of its function. Appl Environ Microbiol, 2003 May, 69(5), 2684 - 91 A census of rRNA genes and linked genomic sequences within a soil metagenomic library; Liles MR et al.; We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil . A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes . The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil . The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, alpha-Proteobacteria, and CFB groups; greater representation among the beta- and gamma-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea . In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives . The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation . This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division. J Mol Biol, 2003 May 9, 328(4), 909 - 20 Insights into the catalytic mechanism of cofactor-independent phosphoglycerate mutase from X-ray crystallography, simulated dynamics and molecular modeling; Rigden DJ et al.; Phosphoglycerate mutases catalyze the isomerization of 2 and 3-phosphoglycerates, and are essential for glucose metabolism in most organisms . Here, we further characterize the 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGM) from Bacillus stearothermophilus by determination of a high-resolution (1.4A) crystal structure of the wild-type enzyme and the crystal structure of its S62A mutant . The mutant structure surprisingly showed the replacement of one of the two catalytically essential manganese ions with a water molecule, offering an additional possible explanation for its lack of catalytic activity . Crystal structures invariably show substrate phosphoglycerate to be entirely buried in a deep cleft between the two iPGM domains . Flexibility analyses were therefore employed to reveal the likely route of substrate access to the catalytic site through an aperture created in the enzyme's surface during certain stages of the catalytic process . Several conserved residues lining this aperture may contribute to orientation of the substrate as it enters . Factors responsible for the retention of glycerate within the phosphoenzyme structure in the proposed mechanism are identified by molecular modeling of the glycerate complex of the phosphoenzyme . Taken together, these results allow for a better understanding of the mechanism of action of iPGMs . Many of the results are relevant to a series of evolutionarily related enzymes . These studies will facilitate the development of iPGM inhibitors which, due to the demonstrated importance of this enzyme in many bacteria, would be of great potential clinical significance. Int J Tuberc Lung Dis, 2003 Apr, 7(4), 399 - 402 Rates of adverse reactions to first and second doses of BCG vaccination: results of a large community trial in Brazilian schoolchildren; Dourado I et al.; OBJECTIVE: To evaluate the incidence of adverse reactions to first and second bacille Calmette-Guerin (BCG) vaccination in schoolchildren . SETTING AND DESIGN: Enhanced surveillance in a Brazilian trial . Suspected reactions were reported to a nurse who visited cases and completed a standard form . RESULTS: Among 71341 schoolchildren studied, 33 reactions were reported . Of these, 25 fulfilled the criteria, resulting in a rate of one per 2854 vaccinations, with no deaths or BCG-osis . Reactions to second doses were more common than to first BCG vaccinations, but this difference was not statistically significant . CONCLUSIONS: Adverse reactions to a second dose of BCG may be more frequent than reactions to a first dose, but they are still rare events. Int J Tuberc Lung Dis, 2003 Apr, 7(4), 320 - 6 Tuberculin reactivity in adult BCG-vaccinated subjects: a cross-sectional study; Bugiani M et al.; INTRODUCTION: Interpretation of the tuberculin skin test (TST) may be complicated by prior bacille Calmette-Guerin (BCG) vaccination . The skin reaction to the vaccination interferes with the management of individuals who may be infected with Mycobacterium tuberculosis . OBJECTIVE: To discriminate between TST reactions due to infection and those due to vaccination in subjects with unknown BCG status . METHODS: Among 60200 subjects tested with 5TU PPD for screening purposes, 4987 contacts of infectious TB cases (Group A), 4962 BCG-vaccinated subjects (Group B) and 5000 subjects from the general population (Group C) were sampled . The frequencies of TST cut-off diameters were calculated for the three groups using a logistic regression model . The frequency of positive subjects in each group and the sensitivity, specificity and predictive values were also computed by means of these cut-offs . RESULTS: The risk of being a contact versus BCG-vaccinated increases 2.43-fold with every mm of TST diameter . The 11 mm cut-off point seems to be the best discriminating value . CONCLUSIONS: Using the traditional 10 mm cut-off, we can consider all vaccinated subjects with a positive TST to be infected . The TST remains a valuable tool for the evaluation of household contacts and suspected cases of tuberculosis in BCG-vaccinated subjects and in populations with high vaccination coverage. J Chin Med Assoc, 2003 Jan, 66(1), 72 - 5 Transplantation of related histocompatible marrow and peripheral blood stem cells in a patient with severe combined immunodeficiency and disseminated BCG infection; Hung GY et al.; We report the results of allogeneic bone marrow transplantation (BMT) in a 15-month-old boy with severe combined immunodeficiency (SCID) complicated by disseminated Bacillus Calmette-Guerin (BCG) infection . Slow immunological recovery after BMT is considered to be associated with prolonged BCG infection . After one booster dose of allogeneic peripheral blood stem cell transplantation (PBSCT), there was still little improvement in immune reconstitution . The patient finally expired because of failure to achieve a functioning immune system after BMT and septic shock. Rev Esp Salud Publica, 2003 Mar-Apr, 77(2), 211 - 20 {Epidemiological surveillance of pulmonary tuberculosis treated at the specialized care level based on 2 data sources, Valladolid; Spain}; Tejero Encinas S et al.; BACKGROUND: Pulmonary tuberculosis is still more frequent that it should be in Spain given the degree of Spain's social and healthcare-related development . Apart from some individual studies, such as the Multicenter Tuberculosis Research Project, the incidence of tuberculosis is known by way of the Compulsory Notifiable Disease System, in which some degree of under-notification has been detected . The question has been raised as to whether this data can be improved through the additional use of another registry, specifically the Minimum Basic Data Set (MBDS) . METHODS: This is a retrospective study referring back to the 1996-2000 period conducted on the population of a healthcare district totaling 220,572 inhabitants . The data from the Compulsory Notifiable Disease registry was used to the specialized care level, and that of the MBDS registry for the diagnosis of pulmonary tuberculosis . The incidence rates were calculated for each source by the capture-recapture method . An analysis was made of epidemiological characteristics such as age, gender, place of residence, bacillus in sputum and treatment data on the hospitalized cases, such as average length of stay, type of admission, type of release, clinical department, HIV co-morbility . RESULTS: The mean annual incidence recorded at the specialized care level was 16.6 cases/100,000 inhabitants in the Compulsory Notifiable Disease registry; 20.4 cases/100,000 inhabitants in the MBDS registry, and 23.1 cases/100,000 inhabitants combining both of these two sources . The incidence estimated using the capture-recapture method was that of 24.4 cases/100,000 inhabitants (IC95%: 23.5-25.3) . CONCLUSIONS: Any information system which provides reliable data serves to improve epidemiological surveillance even though it may have been designed for a different purpose . It is all a matter of knowing the limitations and unique aspects thereof . The MBDS provides information of epidemiological interest which is not included in the Compulsory Notifiable Disease reports . Using the capture-recapture method is one alternative for estimating truer pulmonary tuberculosis rates. Pediatrics, 2003 May, 111(5 Pt 1), e608 - 14 Risk factors for tuberculosis infection in children in contact with infectious tuberculosis cases in the Gambia, West Africa; Lienhardt C et al.; OBJECTIVE: Tuberculosis (TB) infection is highly prevalent in developing countries . As infected children represent a large proportion of the pool from which TB cases will arise, knowledge of the factors that influence TB infection in children are of importance to evaluate transmission of infection in the community and adapt TB control activities . There are limited data on the risk of infection in child populations in developing countries . METHODS: We performed a household contact study in The Gambia (West Africa), in which children who were living in contact with individuals who had proven smear-positive pulmonary TB cases were investigated . A questionnaire was addressed to the mother or caregiver of the child to investigate the presence of various risk factors and assess the degree of exposure of the child to the individual with TB within the household . A tuberculin skin test (TST) was performed on each child . TST sizes > or =5 and 10 mm, respectively, were considered positive . RESULTS: Households of 206 TB cases were visited, and 384 children aged <5 years were examined . The median age was 2, and 48% were girls . The distribution of TST responses followed a bimodal pattern, with 135 (35%) children presenting a palpable induration . Random effects logistic regression analysis demonstrated that the risk of positive TST response in the child increased with the geographic proximity of the child to the individual with TB within the household and with the degree of activities shared with the individual with TB . It was also associated with the clinical severity of the disease in the index case . Nutritional status and presence of a bacille Calmette-Guerin (BCG) scar were not independent risk factors for TST positivity in this population . On multivariate analysis, the effect of geographic proximity to the individual with TB, household size, and duration of cough in the index case persisted for TST responses > or =5 mm . CONCLUSIONS: In a highly endemic country with high BCG vaccination coverage in Africa, TB infection in children who were in contact with individual with infectious TB was directly related to the intensity of exposure of the child to the individual with TB . Our data suggest that a positive TST in a child reflects most probably TB infection rather than previous BCG vaccination . Contact tracing can play a major role in the control of TB in developing countries. FEMS Immunol Med Microbiol, 2003 May 15, 36(1-2), 63 - 9 The CD14 receptor does not mediate entry of Mycobacterium tuberculosis into human mononuclear phagocytes; Shams H et al.; Prior reports have suggested that CD14 mediates uptake of Mycobacterium tuberculosis into porcine alveolar macrophages and human fetal microglia, but the contribution of CD14 to cell entry in human macrophages has not been studied . To address this question, we used flow cytometry to quantify uptake by human monocytes and alveolar macrophages of M . tuberculosis expressing green fluorescent protein . Neutralizing anti-CD14 antibodies did not affect bacillary uptake and the efficiency of bacillary entry was similar in THP-1 cells expressing low and high levels of CD14 . However, most internalized bacteria were found in CD14+ but not in CD14- monocytes because M . tuberculosis infection upregulated CD14 expression . We conclude that: (1) CD14 does not mediate cellular entry by M . tuberculosis; (2) M . tuberculosis infection upregulates CD14 expression on mononuclear phagocytes, and this may facilitate the pathogen's capacity to modulate the immune response. Biochem Biophys Res Commun, 2003 May 16, 304(4), 684 - 90 Altering the substrate chain-length specificity of an alpha-glucosidase; Noguchi A et al.; Dextran glucosidases show high sequence identity (50%) to Bacillus sp . SAM1606 alpha-glucosidase, which is more specific for short-chain substrates . Sequence comparison of these enzymes as well as molecular modeling studies predicted that the extension of loop 4 of the (beta/alpha)(8)-barrel fold may be responsible for the narrower specificity of SAM1606 alpha-glucosidase with respect to substrate chain length . Indeed, deletion mutants of SAM1606 alpha-glucosidase that lack this extension showed higher relative activities toward dextran and long-chain isomaltooligosaccharides . Kinetic and thermodynamic analyses of oligosaccharide hydrolysis catalyzed by SAM1606 alpha-glucosidase and its deletion mutants suggested that the loss of such extension(s) in loop 4 should energetically destabilize the Michaelis complexes with long-chain substrates to result in smaller differences between the activation free energies for the enzymatic hydrolyses of isomaltoheptaose and isomaltose than those observed for the wild-type enzyme . This is the reason that dextran glucosidase, whose loop 4 is shorter in length, shows broader substrate chain-length specificity than does SAM1606 alpha-glucosidase. J Biol Chem, 2003 Aug 1, 278(31), 29240 - 51 Epub 2003 Apr 29. The inhibitor thiomandelic acid binds to both metal ions in metallo-beta-lactamase and induces positive cooperativity in metal binding; Damblon C et al.; Thiomandelic acid is a simple, broad spectrum, and reasonably potent inhibitor of metallo-beta-lactamases, enzymes that mediate resistance to beta-lactam antibiotics . We report studies by NMR and perturbed angular correlation (PAC) spectroscopy of the mode of binding of the R and S enantiomers of thiomandelic acid, focusing on their interaction with the two metal ions in cadmium-substituted Bacillus cereus metallo-beta-lactamase . The 113Cd resonances are specifically assigned to the metals in the two individual sites on the protein by using 113Cd-edited 1H NMR spectra . Each enantiomer of thiomandelate produces large downfield shifts of both 113Cd resonances and changes in the PAC spectra, which indicate that they bind such that the thiol of the inhibitor bridges between the two metals . For R-thiomandelate, this is unambiguously confirmed by the observation of scalar coupling between Halpha of the inhibitor and both cadmium ions . The NMR and PAC spectra reveal that the two chiral forms of the inhibitor differ in the details of their coordination geometry . The complex with R-thiomandelate, but not that with the S-enantiomer, shows evidence in the PAC spectra of a dynamic process in the nanosecond time regime, the possible nature of which is discussed . The thiomandelate complex of the mononuclear enzyme can be detected only at low metal to enzyme stoichiometry; the relative populations of mononuclear and binuclear enzyme as a function of cadmium concentration provide clear evidence for positive cooperativity in metal ion binding in the presence of the inhibitor, in contrast to the negative cooperativity observed in the free enzyme. J Biol Chem, 2003 Jul 18, 278(29), 26517 - 25 Epub 2003 Apr 30. Execution of macrophage apoptosis by Mycobacterium avium through apoptosis signal-regulating kinase 1/p38 mitogen-activated protein kinase signaling and caspase 8 activation; Bhattacharyya A et al.; Macrophage apoptosis is an important component of the innate immune defense machinery (against pathogenic mycobacteria) responsible for limiting bacillary viability . However, little is known about the mechanism of how apoptosis is executed in mycobacteria-infected macrophages . Apoptosis signal-regulating kinase 1 (ASK1) was activated in Mycobacterium avium-treated macrophages and in turn activated p38 mitogen-activated protein (MAP) kinase . M . avium-induced macrophage cell death could be blocked in cells transfected with a catalytically inactive mutant of ASK1 or with dominant negative p38 MAP kinase arguing in favor of a central role of ASK1/p38 MAP kinase signaling in apoptosis of macrophages challenged with M . avium . ASK1/p38 MAP kinase signaling was linked to the activation of caspase 8 . At the same time, M . avium triggered caspase 8 activation, and cell death occurred in a Fas-associated death domain (FADD)-dependent manner . The death signal induced upon caspase 8 activation linked to mitochondrial death signaling through the formation of truncated Bid (t-Bid), its translocation to the mitochondria and release of cytochrome c . Caspase 8 inhibitor (z-IETD-FMK) could block the release of cytochrome c as well as the activation of caspases 9 and 3 . The final steps of apoptosis probably involved caspases 9 and 3, since inhibitors of both caspases could block cell death . Of foremost interest in the present study was the finding that ASK1/p38 signaling was essential for caspase 8 activation linked to M . avium-induced death signaling . This work provides the first elucidation of a signaling pathway in which ASK1 plays a central role in innate immunity. An Pediatr (Barc), 2003 May, 58(5), 432 - 7 {Epidemic outbreak of tuberculosis in a primary and secondary school in Granada (Spain)}; Sanchez Marenco A et al.; OBJECTIVE: To describe the diagnosis and early treatment of an epidemic outbreak of tuberculosis and determine the utility of polymerase chain reaction (PCR) compared with routine culture in gastric juice . MATERIAL AND METHODS: A computer studies teacher, with clinical features suggestive of tuberculosis and caverns on X-ray, was diagnosed with bacilliferous tuberculosis . Primary health care services carried out a Mantoux test on the school's 387 students as well as on teachers and other staff . The children with a positive Mantoux test underwent laboratory, radiological, and microbiological investigations for one week in the Pediatric Respiratory Unit of Hospital Clinico in Granada.In the teaching and non-teaching staff, active tuberculosis was ruled out through bacilloscopy of sputum samples, Mantoux test, and chest X-ray . RESULTS: In the first screening, the Mantoux test was positive in 67 children . Of these, 7 children were diagnosed with tuberculosis and 60 were found to be infected . Of the 7 children with tuberculosis, five presented positive gastric juice culture in Lowenstein medium while Roche COBAS PCR was negative . In the second screening, 9 children became tuberculin positive . Of these, 8 were diagnosed with tuberculosis and one was infected . Cultures were positive in 3 and PCR was negative . In 77.6 % of the children (59/76), the Mantoux induration was equal to or higher than 18 mm . All of the 15 children with tuberculosis were aged between 9 and 14 years old, except one who was 5 years old . CONCLUSIONS: The Mantoux test remains a basic screening method in diagnosis and epidemiological research, whereas the results of microbiological investigation remain poor and in our study the results DNA were disappointing . The screening of tuberculosis and of other infectious diseases should be more closely monitored in professional groups, such as teachers, that are in contact with large numbers of children . This would identify infected adults and prevent epidemics such as that described in the present study. Biosci Biotechnol Biochem, 2003 Mar, 67(3), 525 - 31 Maltosyl-erythritol, a major transglycosylation product of erythritol by Bacillus stearothermophilus maltogenic amylase; Yoon JW et al.; This study was done to modify erythritol to change its physicochemical and sensory properties . Erythritol, a four-carbon sugar alcohol, was transglycosylated by Bacillus stearothermophilus maltogenic amylase with maltotriose as a donor molecule . The presence of various transglycosylation products of erythritol was confirmed by TLC and high performance ion exchange chromatography (HPIC) . The major transfer product was purified by gel filtration chromatography on Bio-Gel P-2 . Examination by LC-MS, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), and 13C NMR showed that the major transfer product was maltosyl-erythritol . Results of 13C NMR of maltosyl-erythritol suggested that linkage was formed between the C1 carbon of glucose unit in maltose and either one of the two carbon atoms of the terminal hydroxyl groups of erythritol, so that a mixture of 1-O- and 4-O-alpha-maltosyl-erythritol was produced . The sweetness of maltosyl-erythritol was about 40% that of sucrose, and its negative sensory properties were less than those of erythritol. J Infect Dis, 2003 May 15, 187(10), 1544 - 51 Epub 2003 Apr 30. Failure to control growth of mycobacteria in blood from children infected with human immunodeficiency virus and its relationship to T cell function; Tena GN et al.; The mechanisms of protective immunity to tuberculosis remain poorly understood in humans . A whole-blood infection model that employs a luminescent readout was used to analyze the role of T cells in control of mycobacterial infection . Control of mycobacterial growth in blood from healthy tuberculin-positive individuals was shown to be mediated predominantly by CD4(+) T cells . Comparison of age-matched cohorts of human immunodeficiency virus (HIV)-infected and -uninfected children from South Africa demonstrated an association between low CD4 cell counts, low interferon (IFN)-gamma production, and impaired ability to regulate growth of Mycobacterium bovis bacille Calmette-Guerin in blood from HIV-infected children . Impaired control of infection was not reconstituted by the addition of exogenous IFN-gamma . The whole-blood assay provides an important tool for monitoring and dissecting of human immune responses to mycobacterial infection. Arch Virol, 2003 May, 148(5), 937 - 49 Genomic characterisation of taro bacilliform virus; Yang IC et al.; Taro bacilliform virus (TaBV) has been classified as a putative badnavirus based on its non-enveloped, bacilliform virion morphology and transmission by mealybugs . The complete nucleotide sequence of a Papua New Guinea isolate of TaBV has now been determined and comprises 7458 bp . The genome contains four open reading frames (ORFs) on the plus-strand that potentially encode proteins of 17, 16, 214 and 13 kDa . The size and organisation of TaBV ORFs 1-3 is similar to that of most other badnaviruses, while the location of ORF 4 is similar to that of ORF 4 and ORF X of the atypical badnaviruses Citrus yellow mosaic virus and Cacao swollen shoot virus, respectively . The putative amino acid sequence of TaBV ORF 3 contained motifs that are conserved amongst badnavirus proteins including aspartic protease, reverse transcriptase (RT) and ribonuclease H (RNase H) . The highly conserved putative plant tRNA(met)-binding site was also present in the 935 bp intergenic region of TaBV . Phylogenetic analysis using the amino acid sequence of ORF 3 showed that TaBV branched most closely to Dioscorea bacilliform virus . These results confirm that TaBV is a pararetrovirus of the genus Badnavirus, family Caulimoviridae. Mol Biotechnol, 2003 May, 24(1), 11 - 20 Specific mutations within the alpha4-alpha5 loop of the Bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170; Kanintronkul Y et al.; The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry delta-endotoxins suggests that helices alpha4 and alpha5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation . In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the alpha4-alpha5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis . As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-beta-Dthiogalactopyranoside induction . When E . coli cells expressing each mutant toxin were assayed against Aedes aegypti mosquito larvae, the activity was almost completely abolished for N166A and Y170A mutations, whereas E171A showed only a small reduction in toxicity . Further analysis of these two critical residues by induction of specific mutations revealed that polarity at position 166 and highly conserved aromaticity at position 170 within the alpha4-alpha5 loop play a crucial role in the larvicidal activity of the Cry4B toxin. Appl Biochem Biotechnol, 2003 Spring, 105 -108, 393 - 402 Effect of Bacillus circulans D1 thermostable xylanase on biobleaching of eucalyptus kraft pulp; Bocchini DA et al.; The alkalophilic Bacillus circulans D1 was isolated from decayed wood . It produced high levels of extracellular cellulase-free xylanase . The enzyme was thermally stable up to 60 degrees C, with an optimal hydrolysis temperature of 70 degrees C . It was stable over a wide pH range (5.5-10.5), with an optimum pH at 5.5 and 80% of its activity at pH 9.0 . This cellulase-free xylanase preparation was used to biobleach kraft pulp . Enzymatic treatment of kraft pulp decreased chlorine dioxide use by 23 and 37% to obtain the same kappa number (kappa number) and brightness, respectively . Separation on Sephadex G-50 isolated three fractions with xylanase activity with distinct molecular weights. Appl Biochem Biotechnol, 2003 Spring, 105 -108, 295 - 301 Cassava flour wastewater as a substrate for biosurfactant production; Nitschke M et al.; Five cassava flour wastewater (manipueira) preparations were tested as culture media for biosurfactant production by a wild-type Bacillus sp . isolate . No-solids (F), no-solids diluted (F/2), natural (I), natural diluted (I/2), and decanted (IPS) were the tested manipueira media . The microorganism was able to grow and to produce biosurfactant on all manipueira preparations . The media whose solids were removed (F and F/2) showed better results than preparations with the presence of solids (I, I/2, and IPS) . No-solids medium (F) showed a surface tension of 26,59 mN/m and reciprocal of critical micelle concentration of over 100 and was selected as a potential substrate for biosurfactant production. Appl Biochem Biotechnol, 2003 Spring, 105 -108, 287 - 94 Effect of media on spore yield and thermal resistance of Bacillus stearothermophilus; Penna TC et al.; The interference of eight components in the yield of sporulation and thermal resistance to moist heat (121 degrees C) of Bacillus stearothermophilus spores suspended in 0.02 M calcium acetate solution and inoculated on paper strips previously treated with calcium acetate/calcium hydroxide was studied . The spore yield of 1.0 x 10(8)mL was developed at 62 degrees C in 17 media containing different concentrations of D-glucose, sodium chloride, L-glutamic acid, yeast extract, peptone, manganese sulfate, potassium phosphate, and ammonium phosphate . The combined effects of yeast extract, peptone, and glucose contributed positively to the spore yield and to the stability of the thermal resistance of both spores in suspension and on strips. Appl Biochem Biotechnol, 2003 Spring, 105 -108, 809 - 19 Evaluation of supports and methods for immobilization of enzyme cyclodextringlycosyltransferase; Sobral KA et al.; An experimental design with factorial planning was used for the immobilization of the enzyme cyclodextringlycosyltransferase (CGTase) from Bacillus firmus (strain no . 37) to select the best combination of support, method of immobilization, and conditions that gives primarily higher average values for the specific immobilized enzyme activity, and secondarily, higher average values for the percentage of protein fixation . The experimental design factors were as follows: supports-controlled-pore silica, chitosan, and alumina; immobilization methods-adsorption, and two covalent bonding methods, either with gamma-aminopropyltriethoxysilane or hexamethylenediamine (HEMDA); conditions-7 degrees C without agitation and 26 degrees C with stirring . The best combination of factors that lead to higher average values of the response variables was obtained with immobilization of CGTase in silica with HEMDA at 7 degrees C . However, immobilization in chitosan at 7 degrees C gave the highest immobilized CGTase specific activity, 0.25 micromole of beta-CD/ (min mg protein) . Physical adsorption gave low specific enzyme activities, and, in general, a high load of enzyme leads to lower specific enzyme activity. J Biol Chem, 2003 Jul 4, 278(27), 24818 - 24 Epub 2003 Apr 28. Crystal structure of calcium-free alpha-amylase from Bacillus sp . strain KSM-K38 (AmyK38) and its sodium ion binding sites; Nonaka T et al.; The crystal structure of a calcium-free alpha-amylase (AmyK38) from Bacillus sp . strain KSM-K38, which resists chelating reagents and chemical oxidants, has been determined by the molecular replacement method and refined to a crystallographic R-factor of 19.9% (R-free of 23.2%) at 2.13-A resolution . The main chain folding of AmyK38 is almost homologous to that of Bacillus licheniformis alpha-amylase . However, neither a highly conserved calcium ion, which is located at the interface between domains A and B, nor any other calcium ions appear to exist in the AmyK38 molecule, although three sodium ions were found, one of which is located at the position corresponding to that of a highly conserved calcium ion of other alpha-amylases . The existence of these sodium ions was crystallographically confirmed by the structures of three metal-exchanged and mutated enzymes . This is the first case in which the structure of the calcium-free alpha-amylase has been determined by crystallography, and it was suggested that these sodium ions, instead of calcium ions, are used to retain the structure and function of AmyK38. Biophys J, 2003 May, 84(5), 3264 - 75 Allosteric interactions within subsites of a monomeric enzyme: kinetics of fluorogenic substrates of PI-specific phospholipase C; Birrell GB et al.; Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C . Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration . The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites . Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity . The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme . The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics . An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine . The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported. Biochemistry, 2003 May 6, 42(17), 5099 - 107 Multiple hydrogen kinetic isotope effects for enzymes catalyzing exchange with solvent: application to alanine racemase; Spies MA et al.; Alanine racemase catalyzes the pyridoxal phosphate-dependent interconversion of the D- and L-isomers of alanine . Previous studies have shown that the enzyme employs a two-base mechanism in which Lys39 and Tyr265 are the acid/base catalysts . It is thus possible that stereoisomerization of the external aldimine intermediates occurs through a concerted double proton transfer without the existence of a distinct carbanionic intermediate . This possibility was tested by the application of multiple kinetic isotope effect (KIE) methodology to alanine racemase . The mutual dependence of primary substrate and solvent deuterium KIEs has been measured using equilibrium perturbation-type experiments . The conceptually straightforward measurement of the substrate KIE in H(2)O is complemented with a less intuitive protium washout perturbation-type measurement in D(2)O . The primary substrate KIE in the D --> L direction at 25 degrees C is reduced from 1.297 in H(2)O to 1.176 in D(2)O, while in the L --> D direction it is reduced from 1.877 in H(2)O to 1.824 in D(2)O . Similar reductions are also observed at 65 degrees C, the temperature to which the Bacillus stearothermophilus enzyme is adapted . These data strongly support a stepwise racemization of stereoisomeric aldimine intermediates in which a substrate-based carbanion is an obligatory intermediate . The ionizations observed in k(cat)/K(M) pH profiles have been definitively assigned based on the DeltaH(ion) values of the observed pK(a)'s with alanine and on the pH dependence of k(cat)/K(M) for the alternative substrate serine . The acidic pK(a) in the bell-shaped curve is due to the phenolic hydroxyl of Tyr265, which must be unprotonated for reaction with either isomer of alanine . The basic pK(a) is due to the substrate amino group, which must be protonated to react with Tyr265-unprotonated enzyme . A detailed reaction mechanism incorporating these results is proposed. World J Gastroenterol, 2003 May, 9(5), 1072 - 6 Modulation of GdCl3 and Angelica sinensis polysaccharides on differentially expressed genes in liver of hepatic immunological injury mice by cDNA microarray; Ding H et al.; AIM: To study the modulating effect of GdCl(3) and Angelica Sinensis polysaccharides (ASP) on differentially expressed genes in liver of hepatic immunological mice by cDNA microarray . METHODS: Hepatic immunological injury was induced by lipopolysaccharide (LPS ip, 0.2 mg/kg(-1)) in bacillus calmetteguerin (BCG ip, 1 mg/kg(-1)) primed mice; A single dose of 20 mg/kg(-1) GdCl(3) was simultaneously pretreated and 30 mg/kg(-1) ASP (ig, qdX7 d) was administrated when the BCG+LPS was primed . The mice were sacrificed at the end of the 7(th) day after ip LPS for 6 h and the liver was removed quickly . The PCR products of 512 genes were spotted onto a chemical material-coated glass plate in array . The DNAs were fixed to the glass plate after series of treatments . The total RNAs were isolated from the liver tissue, and were purified to mRNAs by Oligotex . Both mRNAs from the normal liver tissue and the liver tissue from the mice with hepatic immunological injury or that pretreated with GdCl(3) or ASP were reversely transcribed to cDNAs with the incorporation of fluorescent dUTP to prepare the hybridization probes . The mixed probes were hybridized to the cDNA microarray . After high-stringent washing, the cDNA microarray was scanned for fluorescent signals and showed differences between the two tissues . RESULTS: Among the 512 target genes, 18 differed in liver tissue of hepatic immunological injury mice, and 6 differed in those pretreated by ASP, 7 differed in those pretreated by GdCl(3) . CONCLUSION: cDNA microarray technique is effective in screening the differentially expressed genes between two different kinds of tissue . Further analysis of those obtained genes will be helpful to understand the molecular mechanism of hepatic immunological injury and to study the intervention of drug . Both ASP and GdCl(3) can decrease the number of the differentially expressed genes in liver tissue of mice with hepatic immunological injury. J Antibiot (Tokyo), 2003 Feb, 56(2), 129 - 34 YM-266183 and YM-266184, novel thiopeptide antibiotics produced by Bacillus cereus isolated from a marine sponge II . Structure elucidation; Suzumura K et al.; YM-266183 and YM-266184 are new antibacterial substances that have activity against drug-resistant bacteria produced by Bacillus cereus QN03323 . These structures were elucidated by MS and NMR spectral analysis . YM-266183 and YM-266184 are the cyclic thiopeptides containing thiazole and pyridine moieties, and several unusual amino acids. Ann Diagn Pathol, 2003 Apr, 7(2), 78 - 81 Role of fluorescent microscopy in detecting Mycobacterium leprae in tissue sections; Nayak SV et al.; We compared the sensitivity of the fluorescent method with that of he modified Fite-Faraco method in the detection of Mycobacterium leprae in tissue sections . Fifty-six skin biopsies were obtained from patients having leprosy, particularly the paucibacillary type . Minor alterations were made in the deparaffinization and staining technique, as compared with Kuper and May's method, to obtain optimum fluorescence . Of 56 biopsies studied, 39 showed organisms by the fluorescent method and only 25 showed organisms by the modified Fite-Faraco method . The fluorescent method was found to be more advantageous than the modified Fite-Faraco method, particularly in paucibacillary cases . Fluorescent microscopy has the advantage of speed and ease of screening and reduces observer fatigue . Bacillary positivity rates were higher in the fluorescent method than in the modified Fite-Faraco method in each type of leprosy . J Ind Microbiol Biotechnol, 2003 Mar, 30(3), 141 - 5 Epub 2003 Jan 14. Identification of bacteria contaminating pulp and a paper machine in a Canadian paper mill; Desjardins E et al.; Over 100 bacteria from pulp and slime samples in a Canadian paper mill were identified by partial sequencing of their 16S rDNAs . Seventy-one percent of the isolates could be assigned to a bacterial genus with a high level of confidence . Another 12% exhibited at least 95% similarity within their 16S rDNA sequence with unidentified organisms that originate from warm or wet environments . Pseudomonas, Bacillus, and Pseudoxanthomonas isolates were represented at a relatively high proportion in both pulp and slime samples . This is the first time that Pseudoxanthomonas strains have been isolated from pulp and slime samples on a paper machine. J Ind Microbiol Biotechnol, 2003 Mar, 30(3), 135 - 40 Epub 2003 Mar 20. Potential biological indicators for glutaraldehyde and formaldehyde sterilization processes; Serry FM et al.; The present study aimed to isolate, select, and evaluate bacterial isolates with potential for use as biological indicators for sterilization with glutaraldehyde and/or formaldehyde . A total of 340 local Bacillus isolates were screened for glutaraldehyde and/or formaldehyde resistance by determination of minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and extinction time and were compared with B . subtilis (var . niger) ATCC 9372, the biological indicator for ethylene oxide sterilization, as reference . Of these, 85 isolates had glutaraldehyde MICs of 0.5% or higher, while 29 had formaldehyde MICs of 0.04% or higher . Of the 29 resistant isolates, 15 had MBCs of 0.05% or more . Extinction times were used to evaluate the bactericidal/sporicidal activity of glutaraldehyde . Eight had inactivation times of more than 5 h in 2% glutaraldehyde (pH 8), whereas 12 had inactivation times of more than 3 h in l% formaldehyde, with one isolate in common . These 19 isolates were selected and evaluated as potential biological indicators for aldehydes by determination of the decimal reduction times ( D values), compared with the reference strain . Eight glutaraldehyde-resistant isolates exhibited D values 2.0- to 3.5-fold higher than the reference strain (30 min.) . Only five of 12 formaldehyde resistant isolates had D values higher than that of the reference strain . Using six resistant isolates, temperature coefficient values between 2.11 and 3.02 were obtained for 2% formaldehyde . Finally, 14 isolates were tested for potential pathogenicity and were identified to species level . All of the eight glutaraldehyde-resistant isolates, including the isolate with dual resistance, and three formaldehyde-resistant isolates were B . licheniformis, while two other formaldehyde-resistant isolates were B . cereus . Six of the selected B . licheniformis isolates are potential biological indicators for sterilization processes using aldehydes . Three can be suggested for glutaraldehyde only and three for both aldehydes. J Biol Chem, 2003 Jul 4, 278(27), 24651 - 7 Epub 2003 Apr 24. Optimizing the interfacial binding and activity of a bacterial phosphatidylinositol-specific phospholipase C; Feng J et al.; The phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis can be activated by nonsubstrate interfaces such as phosphatidylcholine micelles or bilayers . This activation corresponds with partial insertion into the interface of two tryptophans, Trp-47 in helix B and Trp-242 in a loop, in the rim of the alphabeta-barrel . Both W47A and W242A have much weaker binding to interfaces and considerably lower kinetic interfacial activation . Tryptophan rescue mutagenesis, reinsertion of a tryptophan at a different place in helix B in the W47A mutant or in the loop (residues 232-244) of the W242A mutant, has been used to determine the importance and orientation of a tryptophan in these two structural features . Phosphotransferase and phosphodiesterase assays, and binding to phosphatidylcholine vesicles were used to assess both orientation and position of tryptophans needed for interfacial activity . Of the helix B double mutants, only one mutant, I43W/W47A, has tryptophan in the same orientation as Trp-47 . I43W/W47A shows recovery of phosphatidylinositol-specific phospholipase C (PC) activation of d-myo-inositol 1,2-cyclic phosphate hydrolysis . However, the specific activity toward phosphatidylinositol is still lower than wild type enzyme and high activity with phosphatidylinositol solubilized in 30% isopropyl alcohol (a hallmark of the native enzyme) is lost . Reinserting a tryptophan at several positions in the loop composed of residues 232-244 partially recovers PC activation and affinity of the enzyme for lipid interfaces as well as activation by isopropyl alcohol . G238W/W242A shows an enhanced activation and affinity for PC interfaces above that of wild type . These results provide constraints on how this bacterial phosphatidylinositol-specific phospholipase C binds to activating PC interfaces. Biosens Bioelectron, 2003 Jul, 18(7), 881 - 9 Demonstration of labeless detection of food pathogens using electrochemical redox probe and screen printed gold electrodes; Susmel S et al.; The demonstration of a labeless immunosensor for the detection of pathogenic bacteria using screen printed gold electrodes (SPGEs) and a potassium hexacyanoferrate(II) redox probe is reported . Gold electrodes were produced using screen printing and the gold surfaces were modified by a thiol based self assembled monolayer (SAM) to facilitate antibody immobilisation . SAMs based on the use of thioctic acid (TA), mercaptopropionic acid (MPA) and mercaptoundecanoic acid (MUA) were evaluated . Following antibody immobilisation via the optimum SAM, the redox behaviour and diffusion co-efficient (D) of the potassium hexacyanoferrate(II) probe was monitored in the absence and presence of analyte . In the presence of analyte, a change in the apparent diffusion co-efficient of the redox probe was observed, attributable to impedance of the diffusion of redox electrons to the electrode surface due to the formation of the antibody-bacteria immunocomplex . No change in the diffusion co-efficient was observed when a non-specific antibody (mouse IgG) was immobilised and antigen added . The system has been demonstrated with Listeria monocytogenes and Bacillus cereus. Tokai J Exp Clin Med, 2002 Dec, 27(4), 97 - 100 Pulmonary paragonimiasis misdiagnosed as tuberculosis: with special references on paragonimiasis; Nagakura K et al.; The diagnosis of tuberculosis by X-ray radiogram is often confused with pulmonary carcinoma, bacillary and parasitic infections, and chronic mycosis . A case of pulmonary paragonimiasis misdiagnosed as tuberculosis by X-ray radiogram is reported . With this case, the smears of sputum were rechecked by an inspection technician's discernment, and Paragonimus eggs along with numerous eosinophils and Charcot-Leyden crystals were detected . In suspected cases of tuberculosis, a history of crab-eating plus sputum examinations, image findings, and serodiagnosis are necessary to rule out paragonimiasis. Int J Syst Evol Microbiol, 2003 Mar, 53(Pt 2), 459 - 63 Bacillus decolorationis sp . nov., isolated from biodeteriorated parts of the mural paintings at the Servilia tomb (Roman necropolis of Carmona, Spain) and the Saint-Catherine chapel (Castle Herberstein, Austria); Heyrman J et al.; Microbial growths causing discoloration on the Roman wall paintings of the Servilia tomb at the necropolis of Carmona (Spain) and the medieval wall paintings of the Saint-Catherine chapel at Castle Herberstein (Austria) were investigated and from four different samples, a group of ten strains with similar characteristics was isolated . The isolates were characterized in a polyphasic taxonomic study, including 16S rDNA sequence analysis, (GTG)5-PCR genomic fingerprinting, DNA-DNA hybridization, DNA base ratio, fatty acid analysis, morphological and biochemical characterization . The data obtained attribute the isolates to a novel species of the genus Bacillus, for which the name Bacillus decolorationis sp . nov . is proposed . The type strain is strain LMG 19507T (=DSM 14890T). Poult Sci, 2003 Apr, 82(4), 551 - 9 Evaluation of transgenic event Bt11 hybrid corn in broiler chickens; Brake J et al.; A feeding study evaluated whether standard broiler diets prepared with grain derived from Syngenta Seeds NK Brand Bacillus thuringiensis (Bt) Corn hybrids had any adverse effects on male or female broiler chickens . Four kinds of corn grain were used in this study: (1) grain from the Bt-expressing field corn hybrid N7070Bt, (2) grain from the N7070Bt hybrid that had been sprayed with Liberty brand herbicide (glufosinate) according to manufacturer's instructions (N7070Bt + Liberty), (3) grain from standard N7070 (non-Bt isoline of N707OBt) grain, and (4) a lot of North Carolina grown grain from the 2000 growing season (NC2000) . The amino acid balance for the four lots of corn was similar relative to their crude protein content; however, the NC2000 corn had higher protein content . Diets with the higher protein NC2000 season corn were amended with a combination of sand, ground cardboard (Solka Floc), and poultry fat so that the metabolizable energy and crude protein content of the diluted diets would be similar to that of the isoline and transgenic diets . Growth of broilers was excellent with males being significantly heavier than females (2,497 g vs . 2,103 g) at 42 d of age . BW of live birds at 42 d was within 26 g for the three treatment groups fed corn that was from the same genetic background, i.e., the two Bt transgenic groups (N7070Bt, N7070Bt + Liberty), and the non-Bt N7070 isoline corn group, while BW for the NC2000 group was significantly lower by 93 g . There was no overall corn source effect on feed conversion ratio (FCR) among the isoline and transgenic corn sources to 42 d of age, but FCR was poorer for broilers consuming the commercial NC2000 corn . There was no overall effect of corn source on survivability to 42 d . Carcass analysis at 48 d demonstrated no differences in percentage carcass yield due to corn source among males and females . The transgenic N7070Bt and N7070Bt + Liberty hybrid diets supported excellent broiler chicken growth with mortality and FCR that were similar to that supported by the N7070 isoline control and better than rates from the commercial NC2000 corn without significant differences among treatment groups in carcass yield . It was clear that the transgenic corn had no deleterious or unintended effects on production traits of broiler chickens in this study. Vestn Khir Im I I Grek, 2003, 162(1), 65 - 9 {Effectiveness of sporobacterin in the prevention and treatment (of postoperative wound infections)}; Slepykh NI et al.; Peculiarities of penetration of living bacterial cultures of Bacillus strain into the structures of the abdominal wall, their positive influence upon the reparative regeneration of surgical wounds were studied in experiments in 240 rats . It was found the living bacterial preparation to be expedient to use as an antagonist of pyogenic infection for purulent postoperative wounds and in order to prevent its development . The clinical antiinflammatory and stimulating the reparative process effects of sporobacterin were studied in 50 patients with infectious wound complications after urgent and scheduled operations . A prophylactic effect was noted in 145 patients operated on for complications of cholelithiasis and commissural ileus . Sporobacterin was proved to give better results of treatment as compared with traditional methods of antiinflammatory therapy with antibiotics. J Mol Biol, 2003 May 2, 328(3), 635 - 54 Crystal structure of thermostable aspartase from Bacillus sp . YM55-1: structure-based exploration of functional sites in the aspartase family; Fujii T et al.; The crystal structure of the thermostable aspartase from Bacillus sp . YM55-1 has been solved and refined for 2.5A resolution data with an R-factor of 22.1% . The present enzyme is a homotetramer with subunits composed of three domains . It exhibits no allosteric effects, in contrast to the Escherichia coli aspartase, which is activated by divalent metal cation and L-aspartate, but is four-times more active than the E.coli enzyme . The overall folding of the present enzyme subunit is similar to those of the E.coli aspartase and the E.coli fumarase C, both of which belong to the same superfamily as the present enzyme . A local structural comparison of these three enzymes revealed seven structurally different regions . Five of the regions were located around putative functional sites, suggesting the involvement of these regions into the functions characteristic of the enzymes . Of these regions, the region of Gln96-Gly100 is proposed as a part of the recognition site of the alpha-amino group in L-aspartate for aspartase and the hydroxyl group in L-malate for fumarase . The region of Gln315-Gly323 is a flexible loop with a well-conserved sequence that is suggested to be involved in the catalytic reaction . The region of Lys123-Lys128 corresponds to a part of the putative activator-binding site in the E.coli fumarase C . The region in the Bacillus aspartase, however, adopts a main-chain conformation that prevents the activator binding . The regions of Gly228-Glu241 and Val265-Asp272, which form a part of the active-site wall, are suggested to be involved in the allosteric activation of the E.coli aspartase by the binding of the metal ion and the activator . Moreover, an increase in the numbers of intersubunit hydrogen bonds and salt-bridges is observed in the Bacillus aspartase relative to those of the E.coli enzyme, implying a contribution to the thermostability of the present aspartase. Vaccine, 2003 May 16, 21(17-18), 2152 - 60 BCG (Bacille of Calmette-Guérin) revaccination leads to improved in vitro IFN-gamma response to mycobacterial antigen independent of tuberculin sensitization in Brazilian school-age children; Barbosa T et al.; Tuberculin skin test (TST) response and cytokine production in finger stab-derived whole blood cultures from 136 BCG scar-positive school-age children were evaluated before and after BCG revaccination . Fifty-four percent of the children increased in vitro production of IFN-gamma after revaccination, and this increase was highly significant for previously unresponsive children (P<0.0001) . No correlation was found between TST response and cytokine production . Our data suggest that the in vitro IFN-gamma response to mycobacterial antigens can be boosted by BCG revaccination and may contribute to the search of correlates of protection to be used for the evaluation of new mycobacterial vaccines. Insect Biochem Mol Biol, 2003 May, 33(5), 499 - 508 Cloning of a Heliothis virescens 110 kDa aminopeptidase N and expression in Drosophila S2 cells; Banks DJ et al.; We previously identified a novel Heliothis virescens 110 kDa aminopeptidase N (APN) that binds Bacillus thuringiensis (Bt) Cry1Ac and Cry1Fa delta-endotoxins, and cloned an internal region of the 110 kDa APN gene (Banks et al., 2001) . Here we describe the RACE-PCR cloning and sequence of a cDNA encoding 110 kDa APN . The 110 kDa APN gene was transiently co-expressed with green fluorescent protein (GFP) in Drosophila S2 cells using the pIZT expression vector . Enrichment of total membranes purified from S2 cells transfected with the 110 kDa APN gene had 3.3 fold increased APN enzymatic activity relative to enriched total membranes purified from S2 cells transfected with vector alone . Whereas the majority of S2 cells transfected with the 110 kDa APN gene bound rhodamine-labeled Cry1Ac toxin, no S2 cells transfected with vector alone bound rhodamine-labeled Cry1Ac toxin . This indicates that toxin binding to whole cells is APN mediated . However, flow cytometry and microscopy indicated that 110 kDa APN transfected S2 cells exposed to Cry1Ac or Cry1Fa toxin did not experience an increase in membrane permeability, indicating that APN transfected cells were resistant to toxin . This suggests while the H . virescens 110 kDa APN functions as a Bt toxin binding protein, it does not mediate cytotoxicity when expressed in S2 cells. Cell, 2003 Apr 18, 113(2), 183 - 93 DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis; Boshoff HI et al.; The presence of multiple copies of the major replicative DNA polymerase (DnaE) in some organisms, including important pathogens and symbionts, has remained an unresolved enigma . We postulated that one copy might participate in error-prone DNA repair synthesis . We found that UV irradiation of Mycobacterium tuberculosis results in increased mutation frequency in the surviving fraction . We identified dnaE2 as a gene that is upregulated in vitro by several DNA damaging agents, as well as during infection of mice . Loss of this protein reduces both survival of the bacillus after UV irradiation and the virulence of the organism in mice . Our data suggest that DnaE2, and not a member of the Y family of error-prone DNA polymerases, is the primary mediator of survival through inducible mutagenesis and can contribute directly to the emergence of drug resistance in vivo . These results may indicate a potential new target for therapeutic intervention. Biochemistry, 2003 Apr 29, 42(16), 4631 - 9 Dual coenzyme specificity of photosynthetic glyceraldehyde-3-phosphate dehydrogenase interpreted by the crystal structure of A4 isoform complexed with NAD; Falini G et al.; Photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Spinacia oleracea belongs to a wide group of GAPDHs found in most organisms displaying oxygenic photosynthesis, including cyanobacteria, green and red algae, and higher plants . As a major catalytic difference with respect to glycolytic GAPDH, photosynthetic GAPDH exhibits dual cofactor specificity toward pyridine nucleotides with a preference for NADP(H) . Here we report the crystal structure of NAD-complexed recombinant A(4)-GAPDH (NAD-A(4)-GAPDH) from Spinacia oleracea, expressed in Escherichia coli . Its superimposition onto native A(4)-GAPDH complexed with NADP (NADP-A(4)-GAPDH) pinpoints specific conformational changes resulting from cofactor replacement . In photosynthetic NAD-A(4)-GAPDH, the side chain of Asp32 is oriented toward the coenzyme to interact with the adenine ribose diol, similar to glycolytic GAPDHs (NAD-specific) . On the contrary, in NADP-A(4)-GAPDH Asp32 moves away to accommodate the additional 2'-phosphate group of the coenzyme and to minimize electrostatic repulsion . Asp32 rotation is allowed by the presence of the small residue Ala40, conserved in most photosynthetic GAPDHs, replacing bulky amino acid side chains in glycolytic GAPDHs . While in NADP-A(4)-GAPDH two amino acids, Thr33 and Ser188, are involved in hydrogen bonds with the 2'-phosphate group of NADP, in the NAD-complexed enzyme these interactions are lacking . The crystallographic structure of NAD-A(4)-GAPDH highlights that four residues, Thr33, Ala40, Ser188, and Ala187 (Leu, Leu, Pro, and Leu respectively, in glycolytic Bacillus stearothermophilus GAPDH sequence) are of primary importance for the dual cofactor specificity of photosynthetic GAPDH . These modifications seem to trace the minimum evolutionary route for a primitive NAD-specific GAPDH to be converted into the NADP-preferring enzyme of oxygenic photosynthetic organisms. Anal Chem, 2003 Apr 1, 75(7), 1628 - 37 Detection of cyclic lipopeptide biomarkers from Bacillus species using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry; Madonna AJ et al.; A novel approach to microbial detection using atmospheric pressure matrix-assisted laser desorption/ionization with an ion trap mass spectrometer to analyze whole cell bacteria is introduced . This new approach was tested with lyophilized spores and cultures of Bacillus globigii (BG) grown on agar media for 4 days or longer . At each stage of growth, it was found that biomarkers, identified as cyclic lipopeptides known as fengycin and surfactin, could be detected by pulsed ultraviolet laser irradiation of intact BG cells (approximately 5 mg) cocrystallized with alpha-cyano-4-hydroxycinnamic acid . Furthermore, definitive amino acid sequence information was obtained by performing tandem mass spectrometry on the precursor ions of the cyclic lipopeptides . The investigation was broadened to include the examination of aerosolized BG spores collected from the atmosphere and directly deposited onto double-sided tape . Subsequent analysis of the recovered spores resulted in the production of mass peaks consistent with fengycin . Other Bacillus species were analyzed for comparison and showed mass spectral peaks also identified as originating from various cyclic lipopeptides . Further studies were conducted using a pulsed infrared laser as the excitation source to analyze BG cells (approximately 5 mg) suspended in a matrix of 0.03 M ammonium citrate and glycerol resulting in the production of ions characteristic of fengycin and surfactin. Poult Sci, 2003 Mar, 82(3), 371 - 80 Attempts to detect transgenic and endogenous plant DNA and transgenic protein in muscle from broilers fed YieldGard Corn Borer Corn; Jennings JC et al.; Questions regarding the digestive fate of DNA and protein from transgenic grain have been raised in regard to human consumption and trade of animal products (e.g., meat, milk, and eggs) from farm animals fed transgenic crops . Using highly sensitive, fully characterized analytical methods, fragments of transgenic and endogenous plant DNA, as well as transgenic protein, were not detected in chicken breast muscle samples from animals fed YieldGard Corn Borer Corn event MON 810 (YG) . Total DNA was extracted from breast muscle samples from chickens fed for 42 d with a diet including either 55 to 60% YG grain or 55 to 60% conventional corn grain . DNA preparations were analyzed by PCR followed by Southern blot hybridization for the presence of a 211-bp fragment of the Bacillus thuringiensis (Bt) cry1Ab gene and a 213-bp fragment of the endogenous corn gene sh2 (encoding ADP glucose pyrophosphorylase) . By using 1 microg of input DNA per reaction, none of the extracted samples was positive for cry1Ab or sh2 at the limit of detection for these PCR assays . A 396-bp fragment of the chicken ovalbumin (ov) gene, used as a positive control, was amplified from all samples showing that the DNA preparations were amenable to PCR amplification . By using a competitive immunoassay with a limit of detection of approximately 60 ng of CrylAb protein per gram of chicken muscle, neither the CrylAb protein nor immunoreactive peptide fragments were detectable in the breast muscle homogenates from chickens fed YG grain. Cardiovasc Surg, 2003 Jun, 11(3), 231 - 5 Tuberculous abdominal aortic pseudoaneurysm penetrating the left psoas muscle after BCG therapy for bladder cancer; Wada S et al.; We describe a case of a 75-year-old man with abdominal aortic and right femoral tuberculous pseudoaneurysms 32 months after intravesical bacillus Calmette-Guerin therapy for bladder cancer . These aneurysms were probably brought on by systemic infection by Mycobacterium bovis . The infrarenal aorta and right common femoral artery were successfully replaced with an in situ expanded polytetrafluoroethylene graft.Tuberculous pseudoaneurysm after bacillus Calmette-Guerin therapy for malignancy is very rare, and we review the related literature. Infect Immun, 2003 May, 71(5), 2933 - 7 Immune response induced by recombinant Mycobacterium bovis BCG producing the cholera toxin B subunit; Biet F et al.; The pentameric form of the cholera toxin B subunit (CTB) is known to be a strong mucosal adjuvant and stimulates antigen-specific secretory immunoglobulin A (IgA) and systemic antibody responses to antigens when given by mucosal routes . To deliver CTB for prolonged periods of time to the respiratory mucosa, we constructed a Mycobacterium bovis bacillus Calmette-Guerin (BCG) strain that produces and secretes assembled pentameric CTB . Mice immunized intranasally (i.n.) with recombinant BCG (rBCG) developed a stronger anti-BCG IgA response in bronchoalveolar lavage fluids (BALF) than mice immunized with nonrecombinant BCG . The total IgA response in the BALF of mice immunized with rBCG was also stronger than that in BALF of mice immunized with the nonrecombinant strain . The induction of IgA was well correlated with an increased production of transforming growth factor beta1 . Simultaneous administration of intraperitoneally delivered ovalbumin and of i.n . delivered CTB-producing BCG induced a long-lasting ovalbumin-specific mucosal IgA response as well as a systemic IgG response, both of which were significantly higher than those in mice immunized with nonrecombinant BCG together with ovalbumin . These results suggest that the CTB-producing BCG may be a powerful adjuvant to be considered for future mucosal vaccine development. Opt Lett, 2003 Apr 15, 28(8), 589 - 91 Characterizing and monitoring respiratory aerosols by light scattering; Pan YL et al.; The elastic-scattering intensity pattern from a single particle as a function of spherical coordinate angles theta and phi provides detailed information on the pattern's morphology . By use of an ellipsoidal reflector and a CCD camera, a single-laser-shot intensity pattern from a large angular range (theta from 90 degrees to 168 degrees and phi from 0 degrees to 360 degrees) was detected from a single aerosol (e.g., a Bacillus subtilisspore, a 1-microm-diameter polystyrene latex sphere, or a cluster of either of these) flowing through the reflectors focal volume at 5 m/s . Noticeable difference in the large-angle-range two-dimensional angular optical scattering (LATAOS) suggest that the LATAOS pattern could be useful in differentiating and classifying life-threatening aerosols from normal background aerosols. Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5437 - 42 Epub 2003 Apr 17. Mycobacterium tuberculosis glycosylated phosphatidylinositol causes phagosome maturation arrest; Fratti RA et al.; The tubercle bacillus parasitizes macrophages by inhibiting phagosome maturation into the phagolysosome . This phenomenon underlies the tuberculosis pandemic involving 2 billion people . We report here how Mycobacterium tuberculosis causes phagosome maturation arrest . A glycosylated M . tuberculosis phosphatidylinositol {mannose-capped lipoarabinomannan (ManLAM)} interfered with the phagosomal acquisition of the lysosomal cargo and syntaxin 6 from the trans-Golgi network . ManLAM specifically inhibited the pathway dependent on phosphatidylinositol 3-kinase activity and phosphatidylinositol 3-phosphate-binding effectors . These findings identify ManLAM as the M . tuberculosis product responsible for the inhibition of phagosomal maturation. Am J Respir Crit Care Med, 2003 Jul 15, 168(2), 185 - 91 Epub 2003 Apr 17. Randomized trial of adjunctive interleukin-2 in adults with pulmonary tuberculosis; Johnson JL et al.; Interleukin (IL)-2 has a central role in regulating T cell responses to Mycobacterium tuberculosis . Adjunctive immunotherapy with recombinant human IL-2 was studied in a randomized, placebo-controlled, double-blinded trial in 110 human immunodeficiency virus-seronegative adults in whom smear-positive, drug-susceptible pulmonary tuberculosis was newly diagnosed . Patients were randomly assigned to receive twice-daily injections of 225, 000 IU of IL-2 or placebo for the first 30 days of treatment in addition to standard chemotherapy . Subjects were followed for 1 year . The primary endpoint was the proportion of patients with sputum culture conversion after 1 and 2 months of treatment . After 1 month, the proportion of patients for whom sputum culture converted to negative was 17% for the IL-2 group compared with 30% in the control group (p = 0.14; chi2) . After 2 months, 77% in the IL-2 group were culture negative compared with 85% of those receiving placebo (p = 0.29, chi2) . Results were similar when patients with isoniazid monoresistance were included in the analysis . There were no differences in weight gain and no improvement in fever, cough, and chest pain between groups . One patient in each arm relapsed . IL-2 did not enhance bacillary clearance or improvement in symptoms in human immunodeficiency virus-seronegative adults with drug-susceptible tuberculosis. Int J Tuberc Lung Dis, 2003 Jan, 7(1), 22 - 9 A cost-effectiveness analysis of universal versus selective immunization with Mycobacterium bovis bacille Calmette-Guérin in Finland; Hersh AL et al.; SETTING: Mycobacterium bovis bacille Calmette-Guerin (BCG) is provided to all infants born in Finland . OBJECTIVE: To analyze the cost-effectiveness of universal versus selective BCG immunization . DESIGN: A Markov model was developed to simulate rates of tuberculosis (TB) and non-tuberculous mycobacterial disease (NTM), and to examine the cost-effectiveness in terms of cost per case averted of three different strategies: universal BCG, selective BCG (10% of infants at higher TB risk than other infants) or no BCG immunization . RESULTS: In a cohort of 60,000 infants over 15 years, the model predicts five cases each of TB and NTM disease with universal immunization, 8-21 TB and 31 NTM cases with various strategies of selective immunization, and 25 TB and 34 NTM cases with no BCG immunization . BCG side-effects are predicted in 5, 0.5 and 0 infants, respectively . The cost per case averted for immunization strategies ranges from a cost of 38,311 US dollars to a savings of 323 dollars as selective immunization becomes more efficient at targeting infants at highest risk of TB . CONCLUSIONS: In a country with a low incidence of pediatric tuberculosis, selective BCG immunization is a more cost-effective strategy than universal BCG immunization for the prevention of tuberculosis, but results in an increase in NTM cases. Arch Esp Urol, 2003 Jan-Feb, 56(1), 19 - 22 {Changes in prostate specific antigen levels during intravesical instillations with Calmette-Guerin bacillus: relationship with transurethral resection of the prostate}; Lopez Llaurado H et al.; OBJECTIVES: To evaluate variations on the serum PSA levels during and after administration of intravesical BCG and its relationship with previous transurethral resection of the prostate (TURP) . METHODS: PSA serum concentration variations were studied after each instillation, at 1 month and at 3 months in 24 patients with superficial bladder cancer and/or CIS under weekly BCG instillations treatment . Three patients had undergone TURP . RESULTS: A raise in PSA was observed in 87.5% of the cases during BCG instillations, although the increase was only significative in patients who had undergone TURP . PSA values descended at one and three months . CONCLUSIONS: Endovesical BCG administration produces an increase on serum PSA levels . This variation is higher in patients with history of TURP. J Clin Microbiol, 2003 Apr, 41(4), 1637 - 50 PCR-based method to differentiate the subspecies of the Mycobacterium tuberculosis complex on the basis of genomic deletions; Huard RC et al.; The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M . bovis bacillus Calmette-Guerin {BCG}), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp . caprae and "Mycobacterium tuberculosis subsp . canettii." Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis . However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings . Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci . Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561', Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions . Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA . The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M . tuberculosis (or M . africanum subtype II), M . africanum subtype I, M . bovis, M . bovis BCG, M . caprae, M . microti, or "M . canettii" or as a Mycobacterium other than MtbC (MOTT) . This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species . It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use. J Biol Chem, 2003 Jun 20, 278(25), 22341 - 9 Epub 2003 Apr 07. Sphingosine phosphate lyase expression is essential for normal development in Caenorhabditis elegans; Mendel J et al.; Sphingolipids are ubiquitous membrane constituents whose metabolites function as signaling molecules in eukaryotic cells . Sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, motility, invasiveness, and programmed cell death . These effects of sphingosine 1-phosphate and similar phosphorylated sphingoid bases have been observed in organisms as diverse as yeast and humans . Intracellular levels of sphingosine 1-phosphate are tightly regulated by the actions of sphingosine kinase, which is responsible for its synthesis and sphingosine-1-phosphate phosphatase and sphingosine phosphate lyase, the two enzymes responsible for its catabolism . In this study, we describe the cloning of the Caenorhabditis elegans sphingosine phosphate lyase gene along with its functional expression in Saccharomyces cerevisiae . Promoter analysis indicates tissue-specific and developmental regulation of sphingosine phosphate lyase gene expression . Inhibition of C . elegans sphingosine phosphate lyase expression by RNA interference causes accumulation of phosphorylated and unphosphorylated long-chain bases and leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage similar to the effects seen with exposure to Bacillus thuringiensis toxin . Our results show that sphingosine phosphate lyase is an essential gene in C . elegans and suggest that the sphingolipid degradative pathway plays a conserved role in regulating animal development. Toxicol Sci, 2003 May, 73(1), 66 - 71 Epub 2003 Apr 15. Effect of diesel exhaust particulate on bacillus Calmette-Guerin lung infection in mice and attendant changes in lung interstitial lymphoid subpopulations and IFNgamma response; Saxena RK et al.; The effect of exposure to diesel exhaust particulate (DEP) on bacillus Calmette-Guerin (BCG) lung infection in mice was studied . C57Bl/6J female mice were infected with BCG (2.5 x 104 bacteria/mouse) by intrapulmonary instillation, with or without coadministration of DEP (100 microg/mouse) . Five weeks later, mice exposed to DEP + BCG had about a four-fold higher BCG load in the lungs than mice exposed only to BCG (p < 0.05) . DEP treatment alone had no effect on the total number of lung lymphocytes or numbers of T, B, or NK cells recovered from lungs . In contrast, BCG infection significantly increased (p< 0.05) recovery levels of all types of lymphocytes from lungs . Coexposure to DEP + BCG further increased the recovery of lymphocytes from lungs of BCG-infected mice . The pulmonary lymphocyte subpopulation expressing the greatest levels of mRNA for IFNgamma after BCG infection was CD4+ T cells . Expression levels were similar in mice exposed to BCG or BCG + DEP and were elevated as compared to noninfected mice and mice treated with DEP alone . Recovery of IFNgamma-secreting lymphocytes and IFNgamma-secreting T cells was significantly higher (p < 0.05) from lungs of BCG-infected mice as compared to control or DEP-exposed mice . BCG and BCG + DEP groups of mice did not differ significantly in the numbers of IFNgamma-secreting lymphocytes in lungs . Taken together, these results indicated that coexposure to DEP + BCG did not significantly affect the level of IFNgamma response of mice to BCG infection . However, DEP treatment was found to inhibit IFNgamma-induced nitric oxide (NO) production by mouse alveolar macrophages in vitro . Our results indicate that DEP exposure did not alter the IFNgamma response to BCG infection, but reduced responsiveness of alveolar macrophages to IFNgamma . Reduced sensitivity of DEP-exposed alveolar macrophages to IFNgamma may contribute to a greater load of BCG in the lungs of BCG-infected mice given DEP. J Bacteriol, 2003 May, 185(9), 2820 - 5 The Bacillus thuringiensis PlcR-regulated gene inhA2 is necessary, but not sufficient, for virulence; Fedhila S et al.; We previously reported that Bacillus thuringiensis strain 407 Cry 32(-) secretes a zinc-requiring metalloprotease, InhA2, that is essential for virulence in orally infected insects . Analysis of the inhA2-lacZ transcriptional fusion showed that inhA2 expression is repressed in a PlcR(-) background . Using DNase I footprinting experiments, we demonstrated that PlcR activates inhA2 transcription directly by binding to a DNA sequence showing a one-residue mismatch with the previously reported PlcR box . It was previously reported that PlcR is essential for B . thuringiensis virulence in oral infection by contributing to the synergistic properties of the spores on the insecticidal activity of the Cry1C protein . We used complementation experiments to investigate whether the PlcR(-) phenotype was due to the absence of InhA2 . The results indicated that overexpression of inhA2 in the (Delta)plcR strain did not restore the wild-type phenotype . However, virulence was fully restored in the (Delta)inhA2 complemented mutant . Thus, inhA2 is the first example of a PlcR-regulated gene found to be directly involved in virulence . However, it is not sufficient for pathogenicity when the other members of the PlcR regulon are lacking . This suggests that InhA2 may act in concert with other PlcR-regulated gene products to provide virulence. Bioresour Technol, 2003 Sep, 89(2), 125 - 31 Effects of temperature, inoculum size and starch hydrolyzate concentration on butanediol production by Bacillus licheniformis; Perego P et al.; An optimization study has been performed on 2,3-butanediol production by Bacillus licheniformis NCIMB 8059 from different carbon sources (glucose, sucrose and cornstarch hydrolyzate), alternately varying temperature (34<T<40 degrees C), inoculum size (0.5<X(0)<10 gl(-1)), and starting substrate concentration (20<S(0)<70 gl(-1)) . The results of average volumetric productivity obtained from tests at variable temperature have been worked out according to Arrhenius to estimate the thermodynamic parameters of both 2,3-butanediol formation (Deltah(*)=69.5 kJ mol(-1); Deltas(*)=-0.12 kJ mol(-1)K(-1)) and thermal inactivation equilibrium (Deltah( composite function )(D)=179 kJ mol(-1); Deltas( composite function )(D)=0.73 kJ mol(-1)K(-1)) . The highest butanediol yield on starting glucose (Y=0.87 mol x mol(-1)) and average diol plus acetoin productivity (nu(av)=0.58 gl(-1)h(-1)) were obtained on cornstarch hydrolyzate at T=37 degrees C, pH 6.0, X(0)=10 gl(-1); S(0)=30 gl(-1) which suggests some stimulation in this raw material of the fermentative metabolism of B . licheniformis . Therefore, cornstarch hydrolyzate can be proposed as an alternative carbon source for industrial production of 2,3-butanediol with no need for growth factor addition. Croat Med J, 2003 Apr, 44(2), 187 - 92 Immunoprophylactic intravesical application of bacillus Calmette-Guerin after transurethral resection of superficial bladder cancer; Librenjak D et al.; AIM: To evaluate the effect of intravesical instillation of Bacillus Calmette-Guerin (BCG) in the prevention of recurrence and progression of the superficial bladder cancer . METHODS: Between February 1989 and May 1994, 170 patients with histologically proven superficial transitional cell carcinoma of the bladder stage Ta and T1 were assessed as eligible for 6-week + 6-month protocol of intravesical BCG instillation at the Split University Hospital . All patients underwent complete transurethral resection of the tumor, which established tumor size, histology, stage, and absence of muscle invasion . Out of 170 patients offered to receive intravesical BCG instillations, 80 agreed to undergo the treatment (BCG group), and 90 refused it (control group) . The median duration of follow-up was 64 months (range, 16-128) . RESULTS: The BCG group had lower incidence rates of recurrence (12 vs 26 events per 100 patient-years in controls, p<0.001) and progression (3.0 vs 6.6 events per 100 patient-years in controls, p=0.017, large-sample one-sample binomial test in both cases) than the control group, but similar mean intervals to first recurrence or progression . The 5-year recurrence-free rates were 55% in BCG patients and 31% in controls, and in case of progression, 86% and 70%, respectively . Cox regression showed that the independent predictors of recurrence were tumor size (p<0.001), absence of BCG treatment (p=0.002), and patient age (p=0.05) . The single independent predictor of tumor progression was absence of BCG treatment, but only in case of tumor grade III (roughly doubling the relative risk of the event) . CONCLUSION: Our data suggest that BCG intravesical instillation, using 6 week + 6 month scheme, prevents against recurrence and progression of superficial bladder tumors . This treatment should be especially advocated in patients with advanced grade tumors, but the scheme remains to be evaluated against other BCG treatment schemes. J Mol Evol, 2003 May, 56(5), 587 - 96 Unisexuality and molecular drive: bag320 sequence diversity in bacillus taxa (insecta phasmatodea); Luchetti A et al.; Satellite DNA variability follows a pattern of concerted evolution through homogenization of new variants by genomic turnover mechanisms and variant fixation by chromosome redistribution into new combinations with the sexual process . Bacillus taxa share the same Bag320 satellite family and their reproduction ranges from strict bisexuality (B . grandii) to automictic (B . atticus) and apomictic (B . whitei = rossius/ grandii; B . lynceorum = rossius/grandii/atticus) unisexuality . Thelytokous reproduction clearly allows uncoupling of homogenization from fixation . Both trends and absolute values of satellite variability were analyzed in all Bacillus taxa but B . rossius, on 906 sequenced monomers at all level of comparisons: intraspecimen, intrapopulation, interpopulation, intersubspecies, and interspecies . For unisexuals, allozymic and mitochondrial clones were also taken into account . Different reproductive modes (sexual/parthenogenetic) appear to explain observed variability trends, supporting Dover's hypothesis of sexuality acting as a driving force in the fixation of sequence variants, but the present analyses also highlight current spreading of new variants in B . grandii maretimi specimens and point to a biased sequence inheritance at the time of hybrid onset in the apomictic hybrids B . whitei and B . lynceorum . Evidence of biased gene conversion events suggests that, given enough time, sequence homogenization can take place in a unisexual such as B . lynceorum . On the contrary, the absolute values of sequence diversity in each taxon are linked to the species' range, time of divergence, and repeat copy number and, possibly, to transposon features . Satellite dynamics appears therefore to be the outcome of both general molecular processes and specific organismal traits. Di Yi Jun Yi Da Xue Xue Bao, 2003 Apr, 23(4), 323 - 5 Cloning and sequence analysis of Bacillus thuringiensis gene fragments isolated by restriction digest PCR; Li X et al.; OBJECTIVE: To clone and analyze Bacillus thuringiensis gene fragments isolated by restriction digest PCR (RD-PCR) . METHOD: Specific primers were designed to amplify the genes of Bacillus thuringiensis israelensis (Bti), and the PCR products were classified and re-amplified by RD-PCR to obtain the fragments for subsequent purification and cloning into the pMD18-T vectors, followed by rapid identification . The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced . RESULTS: Sequence analysis showed that all the fragments amplified were Bti genes . CONCLUSION: RD-PCR is reliable in breaking down large gene fragments into confined and shorter gene fragments for preparing microarray probes. J Food Prot, 2003 Apr, 66(4), 599 - 603 Inactivation of Bacillus cereus spores by high hydrostatic pressure at different temperatures; Oh S et al.; The effect of pH on the initiation of germination and on the inactivation of Bacillus cereus (KCTC 1012) spores during high hydrostatic pressure processing (HPP) with pressures of 0.1 to 600 MPa at different temperatures was investigated . Two different high-pressure treatments were adopted to evaluate the effect of pH on the inactivation of B . cereus on sporulation medium and in suspension medium . Inactivation of B . cereus spores with HPP treatment was affected more by sporulation medium pH than by suspension medium pH . B . cereus spores obtained through sporulation at pH 6.0 showed more resistance to inactivation by HPP at 20, 40, and 60 degrees C than did those obtained through sporulation at pHs of 7.0 and 8.0 . Constituents of B . cereus spores obtained through sporulation at pH 6.0 may undergo electrochemical charge changes comparable to those for spores obtained through sporulation at pH 7.0 . The initiation of B . cereus spore germination was more sensitive to pressure around 300 MPa at 20 degrees C . Increasing processing temperatures during HPP enhanced the effect of sporulation medium pH (i.e., environmental pH) on the inactivation of B . cereus spores. Biomedica, 2003 Mar, 23(1), 87 - 102 {Systematics of the Lutzomyia species of the verrucarum Theodor group, 1965 (Diptera: Psychodiadae)}; Bejarano EE et al.; The verrucarum group of phlebotomine sand flies includes vectors of Leishmania spp . and Bartonella bacilliformis, and from the perspective of public health is considered as one of the most important groups of neotropical phlebotomine sand flies . Due to marked morphological similarity among species constituting this group, the identification based on conventional taxonomic characters can be difficult . Consequently, the verrucarum group has been the focus of numerous taxonomic comparisons which have included the following methods: chaetotaxy, morphometry, larval spiracular system, chorionic structure, morphology of the genital atrium, cytogenetics, morphological phylogenetics, isoenzymes, random amplified polymorphic DNA, cuticular hydrocarbons, DNA probes, and nuclear and mitochondrial nucleotide sequences . Based on morphological characters of the male terminalia, the verrucarum group has been divided in four series, i.e., verrucarum, serrana, townsendi and pia . Since the revision of the group made by Young and Duncan in 1994, ten new species, principally of Andean origin, have been assigned to 3 of the series verrucarum (L . maranonensis, L . cajamarcensis, L . antioquiensis, L . falcaorum), serrana (L . robusta, L . guilvardae) and pia (L . suapiensis, L . tihuiliensis, L . tocaniensis, L . limafalcaoae) . The total number of verrucarum group members is now 40 . Explanations for this diversity of species include the isolation of ancestral populations in refugia of humid forest during the quaternary period, the Andean cordilleras as geographical barrier, and the appearance of the Isthmus of Panama . Biology systematics and evolution of the verrucarum group is reviewed with emphasis on the 19 species extant in Colombia. Proc Natl Acad Sci U S A, 2003 Apr 29, 100(9), 5004 - 9 Epub 2003 Apr 14. Three cadherin alleles associated with resistance to Bacillus thuringiensis in pink bollworm; Morin S et al.; Evolution of resistance by pests is the main threat to long-term insect control by transgenic crops that produce Bacillus thuringiensis (Bt) toxins . Because inheritance of resistance to the Bt toxins in transgenic crops is typically recessive, DNA-based screening for resistance alleles in heterozygotes is potentially much more efficient than detection of resistant homozygotes with bioassays . Such screening, however, requires knowledge of the resistance alleles in field populations of pests that are associated with survival on Bt crops . Here we report that field populations of pink bollworm (Pectinophora gossypiella), a major cotton pest, harbored three mutant alleles of a cadherin-encoding gene linked with resistance to Bt toxin Cry1Ac and survival on transgenic Bt cotton . Each of the three resistance alleles has a deletion expected to eliminate at least eight amino acids upstream of the putative toxin-binding region of the cadherin protein . Larvae with two resistance alleles in any combination were resistant, whereas those with one or none were susceptible to Cry1Ac . Together with previous evidence, the results reported here identify the cadherin gene as a leading target for DNA-based screening of resistance to Bt crops in lepidopteran pests. Lab Invest, 2003 Apr, 83(4), 561 - 70 Recombinant adenovirus vector bearing antisense macrophage migration inhibitory factor cDNA prevents acute lipopolysaccharide-induced liver failure in mice; Iwaki T et al.; Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine involved in delayed hypersensitivity and cellular immunity . MIF also acts as a proinflammatory cytokine and counterregulates the anti-inflammatory effects of glucocorticoids . Exogenous gene transfer mediated by adenovirus is useful to study a particular molecular function as well as to develop gene therapy strategies . A recombinant adenovirus containing sense and antisense murine MIF (mMIF) cDNA inserts was constructed using a cosmid-terminal protein complex method . The sense mMIF adenovirus (AxCA-mMIFS) efficiently induced mMIF in COS-7 cells that endogenously lack mMIF in a dose-dependent manner . In contrast, the antisense mMIF adenovirus (AxCA-mMIFAS) inhibited the expression of mMIF in NIH3T3 cells in a dose-dependent manner . To assess the pathophysiologic role of MIF in acute liver failure, we induced acute onset of liver damage in mice (male Jcl:ICR) by a combined treatment of Bacille Calmette-Guerin (BCG) and lipopolysaccharide (LPS) . mMIF level in the liver of mice infected with AxCA-mMIFAS showed a significant reduction in MIF production in response to BCG-LPS compared with mice treated without viral infection and with AxCA-mMIFS . In addition, the immunohistochemical staining demonstrated that F4/80 antigen on macrophage was enhanced in liver infected with AxCA-mMIFS but reduced in liver infected with AxCA-mMIFAS . The staining intensity is correlated with the mMIF antigen level in liver tissue . The survival rate of mice infected with AxCA-mMIFAS was significantly higher than that of mice treated with PBS and infected with AxCA-LacZ in BCG-LPS . These results suggest that inhibition of MIF production, using recombinant adenovirus bearing the antisense MIF gene, reduced the mortality rate in BCG-LPS-induced liver failure in mice . This finding might aid in the further development of gene therapy targeting MIF. Ned Tijdschr Geneeskd, 2003 Mar 22, 147(12), 569 - 72 {Lymphadenitis as a complication following vaccination with Bacillus Calmette-Guérin (BCG)}; Cerda de Palou E et al.; A healthy 14-months-old boy developed suppurative adenitis some weeks after Bacillus Calmette Guerin (BCG) vaccination . The tumour grew rapidly, showed fluctuation and eventually incision was needed . Culture of the abscess was negative . The diagnosis was: regional lymphadenitis after BCG vaccination . This suppurative lymphadenitis is a non-serious complication of BCG vaccination and has been reported in 0.1 to 4% of those vaccinated . As the BCG vaccination is given only to the at-risk population in the Netherlands, BCG complications are rare . Non-suppurative nodal swellings are considered a normal post-vaccination reaction and do not require treatment. Ned Tijdschr Geneeskd, 2003 Mar 22, 147(12), 561 - 5 {Limited tuberculin screening participation amongst travellers to countries with high tuberculosis incidence; reason to consider BCG vaccination for some travellers}; Cobelens FG et al.; OBJECTIVE: To determine the compliance amongst Dutch travellers to high tuberculosis-incidence countries with a screening procedure involving a tuberculin skin test before and after the trip . DESIGN: Prospective study . METHOD: Nine hundred and eighty-eight tuberculin-negative Dutch people who travelled to high tuberculosis-incidence countries for 3 to 12 months were studied for their compliance with an advised screening procedure of repeat tuberculin skin testing 2 to 4 months after return . At 2 of the 4 participating health services, data were also collected on extra calls made and the pertinent time investments . RESULTS: Five hundred and ninety-nine travellers (61%) were compliant with the screening procedure . Of those for whom the data was available (n = 417), 33% (98/300) of the compliant travellers required extra calls . These took an average of 30 min per extra traveller tested as a result . Compliance varied according to health service and was better amongst travellers to Africa . In addition, non-compliance was independently associated with male sex, work being the main travel purpose, and an undecided duration of travel on departure . CONCLUSIONS: Compliance of Dutch travellers with tuberculin skin-test screening is limited, particularly if no extra calls are issued . Bacillus Calmette-Guerin vaccination appears to be preferable for travellers with undecided travel duration and persons travelling for work on a frequent basis. Ned Tijdschr Geneeskd, 2003 Mar 22, 147(12), 543 - 6 {The tuberculin skin test in the Netherlands: new policies for an old test; guideline from the Netherlands Tuberculosis Control Policy Committee}; Lambregts-van Weezenbeek CS et al.; The primary function of tuberculin skintesting is to demonstrate latent tuberculosis infection . The Netherlands Tuberculosis Control Policy Committee revised the guideline for the use and interpretation of the test because of new insights and changes of the epidemiological situation . Tuberculin testing should target persons who are likely to benefit from treatment of latent tuberculosis infection, such as contacts of tuberculosis-source cases, persons with increased occupational risk of tuberculosis exposure and persons with an increased risk of breaking down from infection to active disease as a result of depressed cellular immunity . The contribution of the tuberculin skintest to the diagnosis of active tuberculosis is limited . Different cut-off values for a positive test result are recommended in order to obtain optimum positive and negative predictive values in different target groups . In screening programmes, if the initial test result is 3-9 mm, follow-up tests are only indicated after exclusion of boosting by the initial two-step method . In contact investigations and persons with immune disorders, a history of Bacillus Calmette Guerin (BCG) vaccination should not longer be regarded a contraindication for tuberculin testing. Ned Tijdschr Geneeskd, 2003 Mar 22, 147(12), 539 - 43 {Indications for, and the significance of, the tuberculin test in the Netherlands}; Verbon A et al.; The almost 100-year-old tuberculin skin test still is the gold standard for diagnosing Mycobacterium tuberculosis infection . The sensitivity of this test with the usual cut-off values is high, but may be decreased with impaired cellular immunity and at older age . The specificity is primarily determined by cross-reactivity to atypical mycobacterial infections and vaccination with Bacillus Calmette-Guerin (BCG) . Positivity of the skin test after BCG vaccination decreases with time after vaccination and depends on the age when vaccinated . The tuberculin reaction can be boosted by repeated tuberculin skin tests over a short time period, whereby the anamnestic immune response is stimulated . This boosting phenomenon occurs mostly with atypical mycobacterial infections, after BCG vaccination and at older age . Interpretation of the tuberculin skin test depends on the indication for the test, the expected risk of latent tuberculosis infection, higher prevalence of 'old' tuberculosis in elderly Dutch people and immigrants, BCG vaccination status and, if a baseline value is available, the boosting phenomenon . Its role in the diagnosis of tuberculosis is limited. Nat Med, 2003 May, 9(5), 533 - 9 Epub 2003 Apr 14. Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis; Pym AS et al.; The live tuberculosis vaccines Mycobacterium bovis BCG (bacille Calmette-Guerin) and Mycobacterium microti both lack the potent, secreted T-cell antigens ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) . This is a result of independent deletions in the region of deletion-1 (RD1) locus, which is intact in virulent members of the Mycobacterium tuberculosis complex . To increase their immunogenicity and protective capacity, we complemented both vaccines with different constructs containing the esxA and esxB genes, which encode ESAT-6 and CFP-10 respectively, as well as a variable number of flanking genes . Only reintroduction of the complete locus, comprising at least 11 genes, led to full secretion of the antigens and resulted in specific ESAT-6-dependent immune responses; this suggests that the flanking genes encode a secretory apparatus . Mice and guinea pigs vaccinated with the recombinant strain BCG::RD1-2F9 were better protected against challenge with M . tuberculosis, showing less severe pathology and reduced dissemination of the pathogen, as compared with control animals immunized with BCG alone. Biochem J, 2003 Jul 15, 373(Pt 2), 337 - 43 Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus alpha-amylase: a calorimetric investigation; Nielsen AD et al.; The effect of temperature and calcium ions on the denaturation of a recombinant alpha-amylase from Bacillus halmapalus alpha-amylase (BHA) has been studied using calorimetry . It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability . Thus an apparent denaturation temperature ( T (d)) of 83 degrees C in the presence of excess calcium ions was observed, whereas T (d) decreased to 48 degrees C when calcium was removed . The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA . An activation energy E (A) of 101 kJ/mol was found for the denaturation of calcium-depleted BHA . The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures . The two denaturation reactions are coupled with the calcium-binding equilibrium between calcium-bound and -depleted amylase . A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration . Thus it is estimated that the apparent E (A) can be increased to approx . 123 kJ/mol by increasing the free-calcium concentration. Bioresour Technol, 2003 Feb, 86(3), 239 - 43 Feather degradation by Bacillus sp . FK 46 in submerged cultivation; Suntornsuk W et al.; Cultivation conditions affecting feather degradation by Bacillus sp . FK 46 were investigated . The results showed that feather was almost completely degraded under the following conditions: 1% whole chicken feather as a substrate at the initial medium pH of 9 with 5% bacterial inoculum, at a temperature of 37 degrees C and a shaking speed of 250 rev/min . Glucose, methanol, Tween 80 and Triton X-100, however, had no effect on feather degradation . After feather was degraded, its residue and fermented broth would become a protein feed for animals.
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