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 n