Bacillus

Probl Tuberk, 1998, (5), 6 - 8
{Organization of care for tuberculosis children who had contact with maternal hospital worker suffering from bacillar tuberculosis}; Guseva EM et al.; While contacting a patient with bacillar tuberculosis, neonates and their mother are at risk for the disease, which makes it necessary to make up a programme that involves emergency antituberculosis measures . Among them primary specific drug therapy in all children in contacts, which may prevent tuberculosis in them, is highly effective.

Biochemistry (Mosc), 1998 Oct, 63(10), 1178 - 82
Isolation and properties of extracellular alkaline phosphatase from Bacillus intermedius; Sharipova MR et al.; Alkaline phosphatase (APase) was isolated from the culture liquid of the streptomycin-resistant strain of Bacillus intermedius S3-19 and purified as a homogeneous preparation by ion-exchange chromatography and FPLC . Electrophoresis and gel-filtration revealed that the active enzyme is a monomer with molecular weight of 46-47 kD . The enzyme possessed phosphomonoesterase and phosphodiesterase activities with maximal levels at pH 9.5 and 55 degreesC and was stable until 60 degreesC at pH 8.0-10.0 . The isolated APase exhibits a broad specificity towards a wide variety of substrates . The effect of divalent metal ions and other reagents on its catalytic activities was studied . It was concluded that alkaline phosphatase of B . intermedius is similar to the secreted alkaline phosphatases from other Bacillus species in its physicochemical and catalytic properties.

J Bacteriol, 1999 Jan, 181(1), 133 - 40
Molecular and biochemical characterization of the protein template controlling biosynthesis of the lipopeptide lichenysin; Konz D et al.; Lichenysins are surface-active lipopeptides with antibiotic properties produced nonribosomally by several strains of Bacillus licheniformis . Here, we report the cloning and sequencing of an entire 26.6-kb lichenysin biosynthesis operon from B . licheniformis ATCC 10716 . Three large open reading frames coding for peptide synthetases, designated licA, licB (three modules each), and licC (one module), could be detected, followed by a gene, licTE, coding for a thioesterase-like protein . The domain structure of the seven identified modules, which resembles that of the surfactin synthetases SrfA-A to -C, showed two epimerization domains attached to the third and sixth modules . The substrate specificity of the first, fifth, and seventh recombinant adenylation domains of LicA to -C (cloned and expressed in Escherichia coli) was determined to be Gln, Asp, and Ile (with minor Val and Leu substitutions), respectively . Therefore, we suppose that the identified biosynthesis operon is responsible for the production of a lichenysin variant with the primary amino acid sequence L-Gln-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Ile, with minor Leu and Val substitutions at the seventh position.

Infect Immun, 1999 Jan, 67(1), 395 - 402
Generation of multinucleated giant cells in vitro by culture of human monocytes with Mycobacterium bovis BCG in combination with cytokine-containing supernatants; Gasser A et al.; Multinucleated giant cells (MGC), a characteristic feature of tuberculous granulomas, form by fusion of monocytes or macrophages, but little is known about the mechanism of the fusion process itself . Several studies report an indirect effect of mycobacteria, i.e., induction of a soluble lymphocyte-derived fusion factor following stimulation by mycobacteria or mycobacterial products . The aim of our study was to determine whether contact with mycobacteria can induce MGC formation from human monocytes in vitro . Stimulation of monocytes with Mycobacterium bovis bacillus Calmette-Guerin (BCG) in combination with cytokine-containing supernatants of herpesvirus saimiri-transformed human T-cell clones (T-SN) led to MGC formation with fusion rates of about 27% . In contrast, stimulation with one component alone induced only low fusion rates of up to 10% . Heat-killed BCG in combination with T-SN induced monocyte fusion to the same extent as live mycobacteria . BCG culture supernatant, BCG lysate, or inert particles in combination with T-SN did not induce MGC formation . Experiments using transwell plates containing a semipermeable membrane revealed that induction of the fusion process is dependent on direct contact of monocytes and mycobacteria . MGC formation induced by BCG plus T-SN could be inhibited by addition of monoclonal antibodies to gamma interferon (but not tumor necrosis factor alpha) as well as to the beta chain (CD18) of beta2-integrins . These results demonstrate that contact with mycobacteria in combination with cytokine-containing supernatants is able to induce human monocytes to form MGC and that membrane-bound molecules of mycobacteria and monocytes are involved in the fusion process.

Infect Immun, 1999 Jan, 67(1), 131 - 9
Expression of listeriolysin O and ActA by intracellular and extracellular Listeria monocytogenes; Moors MA et al.; Listeria monocytogenes requires listeriolysin O (LLO) and ActA, the products of hly and actA, respectively, to establish a productive intracellular infection . LLO is essential for vacuolar lysis and entry into the cytosol, while ActA is required for bacterial spread to adjacent cells . We have used a transcriptional reporter gene system to compare the expression of actA and hly during intracellular growth to that during growth in broth cultures . The hly and actA genes were transcriptionally fused to Escherichia coli lacZ and Bacillus pumilus cat-86 (cat), and the fusions were integrated in single copies into the L . monocytogenes chromosome . A chloramphenicol resistance assay indicated that the hly fusion but not the actA fusion was significantly activated in Luria-Bertani (LB) broth, and this finding correlated with LLO and ActA levels detectable in broth cultures . Quantitation of promoter activity on the basis of beta-galactosidase activity revealed up to 10-fold-higher level of expression of the hly fusion relative to the actA fusion in LB broth . In contrast, both fusions were active in the cytosol of J774 cells, and the activity of the actA fusion was approximately 3-fold higher than that of the hly fusion under these conditions . However, quantitative immunoprecipitation of ActA and LLO from infected J774 cells demonstrated approximately 70-fold more cytosolic ActA than cytosolic LLO . Finally, in comparison to induction in broth cultures, actA was highly induced (226-fold) and hly was moderately induced (20-fold) in J774 cells . Collectively, these results indicate that actA and hly are differentially regulated in response to the growth environment and that both genes are preferentially expressed during intracellular growth . Further, while the lower level of production of ActA than of LLO in broth can be accounted for by transcriptional regulation, the relative abundance of intracellular ActA compared to that of intracellular LLO is a function of additional, possibly host-mediated, factors.

Wei Sheng Wu Xue Bao, 1997 Apr, 37(2), 83 - 6
{Morphogenesis of AcMNPV nucleocapsid}; Chen J et al.; In this paper we reported that the nucleocapsid morphogenetic process of AcMNPV in which polyhedrin gene was deleted in the nuclei of sf9 cells . First, the capsid proteins entered the nuclei and assembled many 34nm diameter long fascicularly arranged hollow-tube structures . Then viral DNA entered these tubes that turned into solid structure with high electronic density . The solid structure separated at a certain distance to form 34 x 26 nm fascicular bacilliform structure known as nucleocapsid . Finally, fascicular nucleocapsids were wrapped by envelope and became complete multicapsid morphotype viron.

FEBS Lett, 1998 Nov 27, 440(1-2), 226 - 30
Biotin synthase mechanism: on the origin of sulphur; Bui BT et al.; Biotin synthase catalyses the last step of the biosynthesis of biotin in microorganisms and plants . The active protein isolated from Bacillus sphaericus and Escherichia coli contains an iron-sulphur (FeS) cluster . The native enzymes were depleted of their iron and inorganic sulphide and the resulting apoenzymes were chemically reconstituted with FeCl3 and Na2{34S} to give labelled (Fe34S) enzymes . These enzymes were functional and when assayed in vitro produced labelled biotin containing about 65% of 34S . These data strongly support the hypothesis that the sulphur of biotin is derived from the (FeS) centre of the enzyme.

FEBS Lett, 1998 Nov 27, 440(1-2), 208 - 12
From beta-glucanase to beta-glucansynthase: glycosyl transfer to alpha-glycosyl fluorides catalyzed by a mutant endoglucanase lacking its catalytic nucleophile; Malet C et al.; Removal of the catalytic nucleophile Glu134 of the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis by mutation to alanine yields an enzyme with no glycosidase activity . The mutant is able to catalyze the regio- and stereospecific glycosylation of alpha-laminaribiosyl fluoride with different glucoside acceptors through a single-step inverting mechanism . The main advantage of the mutant as glycosylation catalyst with respect to the kinetically controlled transglycosylation using the wild-type enzyme is that the reaction products cannot be hydrolyzed by the mutant enzyme, and glycosylation yields rise to 90%.

Mycoses, 1998 Sep-Oct, 41(7-8), 303 - 8
Humoral immunosuppressant activity of aflatoxin ingestion in rabbits measured by response to Mycobacterium bovis antigens using enzyme-linked immunosorbent assay and serum protein electrophoresis; Gabal MA et al.; A total of 30 New Zealand white rabbits were divided into five equal groups . Animals in groups 1 and 3 were sensitized with bacillus Calmette-Guerin (BCG), and those in groups 2 and 4 with inactivated cells of Mycobacterium bovis (Sensitinogen) . Group 1 and 2 rabbits were fed 2 ppm day-1 aflatoxin for 3 months . Group 5 served as control . Serum samples from animals in all groups were subjected to enzyme-linked immunosorbent assays (ELISAs) to determine antibody titre and to protein electrophoresis to determine immunoglobulin levels . The antibody titres and the immunoglobulin levels were significantly decreased in the aflatoxin-treated groups.

Biochemistry, 1998 Dec 15, 37(50), 17562 - 70
Sphingomyelinase induces lipid microdomain formation in a fluid phosphatidylcholine/sphingomyelin membrane; Holopainen JM et al.; The behaviors of two chemically well-defined sphingolipids, N-palmitoyl-sphingomyelin (C16:0-SM) and the corresponding ceramide (C16:0-Cer), in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) matrix were compared . Minor attenuation of lateral diffusion upon increasing the mole fraction of C16:0-SM (XSM, up to 0.25) was indicated by the slight decrement in the excimer/monomer intensity ratio (Ie/Im) for a trace amount (mole fraction X = 0.01) of a pyrene-labeled ceramide analogue (N-{(pyren)-1-yl}decanoyl-sphingosine, PDCer) in keeping with the miscibility of C16:0-SM in POPC . Increasing membrane order was revealed by the augmented polarization P for diphenylhexatriene (DPH) . In contrast, when C16:0-Cer was substituted for C16:0-SM an approximately 1.6-fold increase in Ie/Im for PDCer was evident upon increasing Xcer, with parallel increment in DPH polarization . In agreement with our recent data on natural ceramides in dimyristoylphosphatidylcholine (DMPC) bilayers {Holopainen et al . (1997) Chem . Phys . Lipids 88, 1-13}, we conclude that C16:0-Cer becomes enriched into microdomains in the fluid POPC membrane . Interestingly, enhanced formation of microdomains by ceramide was observed when the total sphingolipid content in tertiary alloys with POPC was maintained constant (Xcer + XSM = 0.25) and the SM/Cer stoichiometry was varied . Finally, when ceramide was generated enzymatically in POPC/C16:0-SM (3:1, molar fraction) LUVs by sphingomyelinase (SMase, Bacillus cereus), maximally approximately 85% of hydrolysis of sphingomyelin was measured within <3 min at 30 degreesC . The formation of ceramide was accompanied by a closely parallel increase in DPH polarization . There was also an increase in Ie/Im for PDCer; however, these changes in Ie/Im were significantly slower, requiring approximately 105 min to reach a steady state . These data show that the rapid enzymatic formation of ceramide under these conditions is followed by much slower reorganization process, resulting in the formation of microdomains enriched in this lipid.

Biochemistry, 1998 Dec 15, 37(50), 17537 - 44
The unusually slow unfolding rate causes the high stability of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus: equilibrium and kinetic studies of guanidine hydrochloride-induced unfolding and refolding; Ogasahara K et al.; To elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (GuHCl)-induced unfolding and refolding were carried out with CD measurements at 220 nm in comparison with those from the mesophile homologue (BaPCP) from Bacillus amyloliquefaciens . The mutant protein of PfPCP substituted with Ser at both Cys142 and Cys188 (PfC142/188S) was used . The GuHCl unfolding for PfC142/188S and BaPCP was reversible . It was difficult to obtain the equilibrated unfolding curve of the hyperthermophile proteins at temperatures below 50 degreesC and pH 7, because of the remarkably slow rate of the unfolding . The unfolding for PfC142/188S attained equilibrium after 7 days at 60 degreesC, resulting in the coincidence between the unfolding and refolding curves . The Gibbs energy change of unfolding, DeltaGH2O (56.6 kJ/mol), for PfC142/188S at 60 degreesC and pH 7 was dramatically higher than that (7.6 kJ/mol) for BaPCP at 40 degreesC and pH 7 . The unfolding and refolding kinetics for PfC142/188S and BaPCP at both 25 and 60 degreesC at pH 7 were approximated as a single exponential . The rate constant in water (kuH2O) of the unfolding reaction for PfC142/188S (1.6 x 10(-)15 s-1) at 25 degreesC and pH 7 was drastically reduced by 7 orders of magnitude compared to that (1.5 x 10(-)8 s-1) for BaPCP, whereas the refolding rates (krH2O) in water for PfC142/188S (9.3 x 10(-)2 s-1) and BaPCP (3.6 x 10(-)1 s-1) at 25 degreesC and pH 7 were similar . These results indicate that the greater stability of the hyperthermophile PCP was characterized by the drastically slow unfolding rate.

Biochemistry, 1998 Dec 8, 37(49), 17192 - 8
Reassessment of acarbose as a transition state analogue inhibitor of cyclodextrin glycosyltransferase; Mosi R et al.; The binding of several different active site mutants of Bacillus circulans cyclodextrin glycosyltransferase to the inhibitor acarbose has been investigated through measurement of Ki values . The mutations represent several key amino acid positions, most of which are believed to play important roles in governing the product specificity of cyclodextrin glycosyltransferase . Michaelis-Menten parameters for the substrates alpha-maltotriosyl fluoride (alphaG3F) and alpha-glucosyl fluoride (alphaGF) with each mutant have been determined by following the enzyme-catalyzed release of fluoride with an ion-selective fluoride electrode . In both cases, reasonable correlations are observed in logarithmic plots relating the Ki value for acarbose with each mutant and both kcat/Km and Km for the hydrolysis of either substrate by the corresponding mutants . This indicates that acarbose, as an inhibitor, is mimicking aspects of both the ground state and the transition state . A better correlation is observed for alphaGF (r = 0.98) than alphaG3F (r = 0.90), which can be explained in terms of the modes of binding of these substrates and acarbose . Re-refinement of the previously determined crystal structure of wild-type CGTase complexed with acarbose {Strokopytov, B., Penninga, D., Rozeboom, H . J., Kalk, K . H., Dijhuizen, L., and Dijkstra, B . W . (1995) Biochemistry 34, 2234-2240} reveals a binding mode consistent with the transition state analogue character of this inhibitor.

Clin Sci (Lond), 1999 Jan, 96(1), 89 - 97
Macrophage-mediated lysis of a beta-cell line, tumour necrosis factor-alpha release from bacillus Calmette-Guérin (BCG)-activated murine macrophages and interleukin-8 release from human monocytes are dependent on extracellular glutamine concentration and glutamine metabolism; Murphy C et al.; Macrophages and monocytes are cells with a large capacity for cytokine production . Cytokines produced by these cells are not preformed and released upon stimulation, but must be transcribed and translated . Although much is known concerning the regulation of the latter processes at the molecular level, the role of exogenous amino acids in the secretory process has not been actively investigated . Glutamine is utilized by macrophages at a much faster rate than any other amino acid . The role for high rates of glutamine utilization in macrophages or monocytes is not fully understood . We demonstrate here that the rates of lipopolysaccharide-stimulated tumour necrosis factor-alpha secretion from bacillus Calmette-Guerin (BCG)-activated murine peritoneal macrophages and lipopolysaccharide-stimulated interleukin-8 production from human monocytes are dependent upon extracellular glutamine concentration . We also demonstrate that potent inhibition of cytokine production can be achieved by incubating macrophages or monocytes in the presence of the glutaminase inhibitor 6-diazo-5-oxo-norleucine . On co-culture of BCG-activated macrophages and the clonal pancreatic beta-cell line BRIN-BD11, macrophage-specific beta-cell death was significantly reduced on prior exposure of macrophages to 6-diazo-5-oxo-norleucine . Thus glutamine metabolism may be essential for generation of cytotoxic products from macrophages, including tumour necrosis factor-alpha.

Int J Urol, 1998 Nov, 5(6), 534 - 9
Results of transurethral resection plus adjuvant intravesical chemotherapy for superficial bladder cancer; Nomi M et al.; BACKGROUND: We analyzed the results of conservative therapy for superficial bladder cancer to determine the risk factors for recurrence and progression . METHODS: Between May 1984 and February 1997, 111 patients with primary superficial bladder cancer were treated by a transurethral resection with or without intravesical instillation of chemotherapy, or for patients with concomitant carcinoma in situ (CIS), bacillus Calmette-Guerin . We examined the relationship between tumor stage, grade, incidence of concomitant CIS and recurrence-free survival according to pathologic findings and the drugs instilled . RESULTS: The incidence of concomitant CIS in pT1, grade 3 tumors was significantly higher than that in pTa, grade 1 tumors (42% vs . 3%, P= 0.006) . The 5-year recurrence-free survival rate of all patients was 73% . There was no significant difference in recurrence-free survival and pathologic stage, tumor grade, presence of concomitant CIS, or drugs used for instillation . However, the recurrence-free survival in patients with > or = 5 tumors was significantly lower than in patients with less than 5 tumors . Of the 111 patients, only 3 patients demonstrated disease progression and underwent a radical cystectomy, while 1 patient with a pT1b, grade 3 tumor developed a tumor in the ureter . No patient died of bladder cancer . CONCLUSION: Our results indicate that the prognosis of superficial bladder cancer patients with a high-stage, high-grade (pT1, grade 3) tumor is favorable when treated by a transurethral resection and intravesical instillation . Bacillus Calmette-Guerin therapy is useful to prevent the recurrence of tumors with concomitant CIS.

Arkh Patol, 1998 Sep-Oct, 60(5), 66 - 8
{Bacillary generalized angiomatosis in HIV infection}; Parkhomenko IuG et al.; A unique case of generalized bacillary angiomatosis (BA) in a patient who died of HIV infection is described . Apart from widely spread skin lesions there were also manifestations in the brain, lungs, heart, esophagus and intestine . Gram-negative bacteria were found in the histological sections . Oval and roundish bacteria with a predominantly perivascular location were found electron microscopically in the archives material.

Proteins, 1998 Dec 1, 33(4), 567 - 76
Structural basis of increased resistance to thermal denaturation induced by single amino acid substitution in the sequence of beta-glucosidase A from Bacillus polymyxa; Sanz-Aparicio J et al.; The increasing development of the biotechnology industry demands the design of enzymes suitable to be used in conditions that often require broad resistance against adverse conditions . beta-glucosidase A from Bacillus polymyxa is an interesting model for studies of protein engineering . This is a well-characterized enzyme, belonging to glycosyl hydrolase family 1 . Its natural substrate is cellobiose, but is also active against various artificial substrates . In its native state has an octameric structure . Its subunit conserves the general (alpha/beta)8 barrel topology of its family, with the active site being in a cavity defined along the axis of the barrel . Using random-mutagenesis, we have identified several mutations enhancing its stability and it was found that one them, the E96K substitution, involved structural changes . The crystal structure of this mutant has been determined by X-ray diffraction and compared with the native structure . The only difference founded between both structures is a new ion pair linking Lys96 introduced at the N-terminus of helix alpha2, to Asp28, located in one of the loops surrounding the active-site cavity . The new ion pair binds two segments of the chain that are distant in sequence and, therefore, this favorable interaction must exert a determinant influence in stabilizing the tertiary structure . Furthermore, analysis of the crystallographic isotropic temperature factors reveals that, as a direct consequence of the introduced ion pair, an unexpected decreased mobility of secondary structure units of the barrel which are proximal to the site of mutation is observed . However, this effect is observed only in the surrounding of one of the partners forming the salt bridge and not around the other . These results show that far-reaching effects can be achieved by a single amino acid replacement within the protein structure . Consequently, the identification and combination of a few single substitutions affecting stability may be sufficient to obtain a highly resistant enzyme, suitable to be used under extreme conditions.

Immunotechnology, 1998 Oct, 4(2), 127 - 40
A novel tumor-specific human single-chain Fv selected from an active specific immunotherapy phage display library; Hall BL et al.; A colon tumor-associated antigen, CTAA 28A32-32K (CTA # 2E), related to the annexin family of proteins, was initially identified by its reactivity with a low affinity human IgM monoclonal antibody (mAb), 28A32 . Both in vitro lymphoproliferative assays with human peripheral blood lymphocytes and delayed type hypersensitivity responses in patients immunized with autologous colon tumor cells indicated that CTA # 2E elicits potent T cell mediated responses and may be an important antigen in the development of a generic colorectal vaccine (Pomato et al . Vaccine Res 1994;3:145-161) . A CTA # 2E-specific, murine hybridoma-derived mAb, 5-11A, which recognizes the amino-terminus of the tumor-associated antigen, exhibited qualitative human colon tumor-specific immunohistochemical reactivity . To rapidly develop a human mAb with similar antigen specificity and tumor reactivity as the murine 5-11A mAb, antibody phage display technology was employed . Two human antibody phage display libraries with 3.1 x 10(7) and 2.3 x 10(8) members were prepared from the variable region genes expressed by circulating B cells of patients undergoing active specific immunotherapy (ASI) with autologous tumor cells, predominantly from the colon, admixed with Bacille Calmette-Guerin (BCG) . A CTA # 2E-reactive human single-chain (sc)Fv was selected by panning the larger library on decreasing concentrations of biotinylated tumor-associated antigen in solution . It exhibited similar antigen specificity as the murine hybridoma-derived 5-11A scFv, requiring the presence of the CTA # 2E amino-terminus for reactivity . This human scFv exhibited qualitative human colon tumor-specific immunohistochemical reactivity when displayed as a gene III fusion protein on phage . When reconstructed and expressed as an intact human IgG1, K mAb, its qualitative colon tumor-specificity was unaltered . Two other CTA # 2E-reactive human scFvs were selected from the smaller library by panning initially on decreasing concentrations of CTA # 2E coated to polystyrene and then on biotinylated CTA # 2E in solution . These human scFvs, which exhibited modest reactivity with different epitopes on the CTA # 2E antigen, did not exhibit human colon tumor-specific immunohistochemical reactivity.

Ann Biol Clin (Paris), 1998 Nov-Dec, 56(6), 681 - 92
{Bartonellosis: I . Bartonella henselae}; Piemont Y et al.; The recent discovery of the bacterium Bartonella henselae was mainly due to the development of molecular biology techniques adapted to microbial diagnosis and to the description of new human diseases linked to Aids . About 10% of pet cats and 33% of stray cats harbour that bacterium in their blood . In immunocompetent patients, that bacterium is responsible for human cat scratch disease, characterized essentially by a localized lymph nodes enlargement in the vicinity of the entry site of the bacteria . This disease occurs more likely in pet cats less than 1-year-old and infested with fleas . The bacterium is transmitted to humans by scratches or bites; the role of fleas is possible, but is not yet documented . In 5 to 13% of cases, the cat scratch disease appears as more severe, including health impairment, hepatitis, Parinaud's oculo-glandular syndrome, neurological complications or stellate retinitis . In immunocompromised patients, B . henselae is responsible for various clinical presentations: bacillary angiomatosis, bacillary peliosis, recurrent or persistent bacteremia or endocarditis . Diagnosis of infections due to B . henselae can be performed by serological specific testing with sensitivity and specificity values ranging from 75 to 100% . Cultivation of the bacterium is fastidious, particularly in cases of cat scratch disease . The most efficient diagnostic test is the in vitro DNA amplification which has the drawback to require a lymph node sample . Antibiotics are usually inefficient for the treatment of cat scratch disease . By contrast, in immunocompromised patients, these infections are successfully treated for a more or less long time by macrolides or tetracyclines or rifampin.

J Bacteriol, 1998 Dec, 180(24), 6780 - 3
Identification of two binding domains, one for peptidoglycan and another for a secondary cell wall polymer, on the N-terminal part of the S-layer protein SbsB from Bacillus stearothermophilus PV72/p2; Sara M et al.; First studies on the structure-function relationship of the S-layer protein from B . stearothermophilus PV72/p2 revealed the coexistence of two binding domains on its N-terminal part, one for peptidoglycan and another for a secondary cell wall polymer (SCWP) . The peptidoglycan binding domain is located between amino acids 1 to 138 of the mature S-layer protein comprising a typical S-layer homologous domain . The SCWP binding domain lies between amino acids 240 to 331 and possesses a high serine plus glycine content.

J Bacteriol, 1998 Dec, 180(24), 6468 - 75
The arcABDC gene cluster, encoding the arginine deiminase pathway of Bacillus licheniformis, and its activation by the arginine repressor argR; Maghnouj A et al.; The arginine deiminase pathway enables Bacillus licheniformis to grow anaerobically on arginine . Both the presence of arginine and anaerobiosis are needed to trigger induction of the pathway . In this study we have cloned and sequenced the arc genes encoding the pathway . They appear clustered in an operon-like structure in the order arcA (arginine deiminase), arcB (ornithine carbamoyltransferase), arcD (putative arginine-ornithine antiporter), arcC (carbamate kinase) . It was found that B . licheniformis has an arginine repressor, ArgR, homologous to the B . subtilis arginine repressor AhrC . Mutants affected in argR were isolated . These mutants have lost both repression by arginine of the anabolic ornithine carbamoyltransferase and induction of the arginine deiminase pathway . Electrophoretic band shift experiments and DNase I footprinting revealed that in the presence of arginine, ArgR binds to a site upstream from the arc promoter . The binding site is centered 108 nucleotides upstream from the transcription start point and contains a single Arg box.

FEMS Microbiol Lett, 1998 Dec 1, 169(1), 117 - 24
Analysis of the Mycobacterium bovis hsp60 promoter activity in recombinant Mycobacterium avium; Batoni G et al.; A clinical isolate of Mycobacterium avium was transformed with a new shuttle plasmid containing the Escherichia coli beta-galactosidase reporter gene under the control of the Mycobacterium bovis bacillus Calmette-Guerin (BCG) hsp60 promoter . beta-Galactosidase activity was assayed spectrophotometrically in bacterial homogenates of the recombinant strain (M . avium::lacZ) and used for quantification of the hsp60 promoter strength in different conditions of extra- and intracellular growth . Very low levels of beta-galactosidase were recorded during the exponential phase of in vitro growth, while they increased progressively during the late exponential and stationary phases . A significant increase in enzyme activity was also induced in exponentially growing cells by shifting the incubation temperature from 37 to 45 degrees C, but not from 37 to 42 degrees C nor from 30 to 42 degrees C . No induction of the promoter was observed by adding hydrogen peroxide to the cultures . Finally, beta-galactosidase levels were quantified during growth of M . avium::lacZ in murine macrophages . Soon after phagocytosis and, to a lesser extent at 1, 5 and 7 days after infection, increased levels of bacterial beta-galactosidase were observed indicating an increment in transcriptional activity of hsp60 promoter both at early phases of infection and during the course of intracellular growth.

Int J Food Microbiol, 1998 Oct 20, 44(1-2), 125 - 32
Influence of several environmental factors on the initiation of germination and inactivation of Bacillus cereus by high hydrostatic pressure; Raso J et al.; The influence of pH, aw, L-alanine, and fat concentration of milk on the initiation of germination and inactivation by high hydrostatic pressure (HHP) (250 mPa at 25 degrees C for 15 min and 690 mPa at 40 degrees C for 2 min) of Bacillus cereus sporulated at 20, 30 and 37 degrees C was investigated . B . cereus sporulated at the lowest temperature was found to be the most resistant to the initiation of germination and inactivation by HHP . At ambient pressure, the rate and extension of germination induced by L-alanine were also lower in B . cereus sporulated at 20 than 30 or 37 degrees C . The optimum pH for the germination and inactivation of B . cereus depended on the sporulation temperature . At 250 mPa the extent of germination for the three suspensions increased with higher pH . At 690 mPa, the pH barely affected the germination of B . cereus sporulated at 20 degrees C (3 log cycles), but the inactivation increased as the pH of the medium was lowered . After the same treatment, pressure optimally initiated the germination of B . cereus sporulated at 30 and 37 degrees C (6-7 log cycles) around neutral pH . Higher inactivation was obtained at pH 6 . High concentrations of sucrose protected the spores from the germinating and inactivating effect of HHP . At aw 0.92, no germination was detected when the spores were pressurized at 250 mPa, and only 1 log cycle of B . cereus sporulated at 20 and 30 degrees C and 2 log cycles of B . cereus sporulated at 37 degrees C were germinated at 690 mPa . In addition, no inactivation was observed at aW 0.92 even at 690 mPa . The presence of L-alanine in the medium of pressurization increased the germination initiated by HHP at 250 mPa, but not at 690 mPa . A combination of 250 mPa at 25 degrees C with L-alanine (100 mM) was found to give an additive response . The initiation of germination and inactivation by HHP were not affected by the fat concentration of the milk.

Int J Food Microbiol, 1998 Oct 20, 44(1-2), 31 - 41
Bacillus cereus in a whey process; Pirttijarvi TS et al.; A cheese dairy and its whey manufacturing line were examined for Bacillus cereus . Colonies typical of B . cereus were detected in 120 (17%) samples out of 720 analysed . Only 3% of the sampled raw milk contained B . cereus ( > or = 10 cfu ml(-1)) whereas in evaporated whey concentrate B . cereus was present in 76% of the samples . Nitrate reductase negative and weakly casein hydrolysis isolates were rare in raw milk and the early parts of the process but these defective biotypes became increasingly frequent towards the end of the whey process . The composition of whole cell fatty acids of B . cereus isolates originating from the whey part of the process was different from that of the type strain and of the isolates originating from the raw materials of cheese making . The B . cereus strains in concentrated whey were 100% similar to the type strain in 16S rDNA sequence (500 bp) although they were not or only poorly recognized as B . cereus by a commercial whole cell fatty acid library . All of B . cereus isolates in raw milk were sensitive to one or more of the B . cereus group phages (n = 17) whereas 43% of the isolates from the whey process were sensitive to none . None of the 23 strains originating from the whey processing lines grew at < or = 8 degrees C . although strains with minimum growth temperatures of 5.3 degrees C and 7.0 degrees C were present in the raw materials . Our results indicate that the B . cereus population of the warm ( > 30 degrees C) parts of the cheese dairy process was separate from that of cold (2 degrees C to 4 degrees C) part of the process.

Int J Food Microbiol, 1998 Oct 20, 44(1-2), 21 - 30
Predictive model to describe the combined effect of pH and NaCl on apparent heat resistance of Bacillus stearothermophilus; Periago PM et al.; The combined effect of pH and NaCl on the apparent thermal resistance of Bacillus stearothermophilus ATCC 12980 spores was studied . Spores were heated at different temperatures (115-125 degrees C) in mushroom substrate, acidified using glucono-delta-lactone to different pH levels (from 5.75 to 6.7), which contained concentrations of NaCl that ranged from 0.5 to 3% (w/v) . The recovery medium was acidified to the same pH level and contained the same NaCl concentration as the heating menstruum . A factorial experimental design allowed a predictive model to be developed, which described the combined effect of heating temperature, pH and NaCl on the thermal resistance of B . stearothermophilus spores . Predictions from the model provided a valid description of the data used to generate the model, and agreed with observations from the literature and from an independent experiment performed using asparagus and bean substrates.

FEMS Immunol Med Microbiol, 1998 Nov, 22(3), 217 - 24
Non-random fragmentation of ribosomal RNA in Helicobacter pylori during conversion to the coccoid form; Monstein HJ et al.; The integrity of DNA and ribosomal RNAs in exponentially growing (bacillary) and ageing stationary phase (coccoid) cultures of Helicobacter pylori type strain CCUG 17874 was investigated . Extensive non-random fragmentation of rRNAs was observed during the conversion to the coccoid form . Beside a small proportion of full-length 16S and 23S rRNA that was always present, the majority of both 16S and 23S rRNA molecules showed distinct highly specific fragmentation patterns . The 16S rRNA fragmentation was characterised in detail by means of Northern blot and primer extension analysis . One cleavage site was located within the highly conserved U5 region (position about 920) . The results could not be attributed to the presence of intervening sequences in the 16S and 23S rRNA genes.

Int J Tuberc Lung Dis, 1998 Nov, 2(11), 898 - 903
Tuberculosis among health care workers in British Columbia; Pleszewski B et al.; OBJECTIVES: To compare the clinical features and prevalence of active TB in British Columbia (BC) health-care workers (HCWs) with those of the general population, between 1991 and 1996 . METHODS: Comparison of 25 HCWs and 50 controls randomly selected from the Centres for Disease Control registry, with respect to demographics, prevention, diagnosis and management . RESULTS: HCWs had fewer related risk factors, but more had initiated prior chemoprophylaxis (16% vs . 0%, P < 0.01) and knew their bacille Calmette-Guerin (BCG) (68% vs . 24%, P < 0.001) and purified protein derivative (PPD) status (60% vs 32%, P < 0.05) . There were no differences in symptom duration (3.3+/-3.6 vs . 3.0+/-3.4 months), mycobacteriology and diagnostic features, treatment duration (264.9+/-69.9 vs . 239.0+/-78.7 days) and completion rates (84% for both) . All HCWs used self-administered treatment (100% vs . 70%, P < 0.01), and fewer were hospitalized (8% vs . 28%, P < 0.05) . Disease rates in nurses (3.6+/-4.4 per 100 000) were lower than the general population rates (9.0+/-0.8), but did not differ among physiotherapists (8.96+/-21.95), general practitioners (7.60+/-11.78) and medical residents (30.75+/-75.32); CONCLUSIONS: Clinical features were similar in HCWs, but management strategies differed . BC HCWs are not at increased risk of tuberculosis, but the small sample size limited the power of our study to detect such an increase.

J, Mar . Biotechnol. . 1998 Aug, 6(3), 189 - 92
A novel marine Bacillus with multiple amino acid analog resistance and selenomethionine-dependent antibiotic productivity; Imada C et al.; Amino acid analogs (AAA) were used as selective pressures for isolation of marine bacteria with novel physiological properties and as effecters for antibiotic production . Relatively small numbers of isolates were obtained from a minimal medium containing aminoethylcysteine (AC), 3,4-dehydroproline (DP), 5-methyltryptophan (MT), and selenomethionine (SM) . These bacteria exhibited a high probability (68%) of antibiotic production in the presence of AAA, which was 10-fold higher than that (7%) in the absence of AAA . Among them, strain 14, obtained as the only SM-resistant and SM-dependent antibiotic (selenohomocystine) producer, was characterized for microbiological properties . It showed taxonomic properties falling into those of the genus Bacillus, required seawater for growth, and exhibited a high level (0.5 mM) of resistance to all the AAAs tested . Neither known Bacillus spp . nor other marine isolates showed such properties . Therefore, the strain 14 appears to be the first marine Bacillus strain with unique AAA resistance and AAA-dependent antibiotic productivity . The AAA-resistance-based strategy was thus demonstrated to be effective for isolation of novel bacteria as well as for screening for antibiotic production.

J Indiana Dent Assoc, 1997 Spring, 76(1), 45 - 50; quiz 52
Sterilization of slide sheath anesthetic injection systems placed within sharps containers; Palenik CJ et al.; The purpose of this study was to evaluate the effect that two steam autoclaves and an unsaturated chemical vapor sterilizer had on killing bacterial endospores present on commercial spore strips or applied to sterile anesthetic injection systems placed within sharps containers . Three types of sterilizers were used: a gravity steam autoclave, a high vacuum steam autoclave and an unsaturated chemical vapor sterilizer . The microbial challenge for the sterilizers were Bacillus stearothermophilus spores present on commercial spore strips or drawn into and applied onto sliding sheath anesthetic injection systems with anesthetic carpules attached . Spore-soiled items were placed into the middle of sharps containers three-quarters-filled with representative clinical waste and sterilized . If, after culturing, sterilization of all test items in a group was not achieved, additional sterilization time was applied . Spore strips were killed within a single cycle of each sterilizer . Spore-soiled injection systems and carpules could not be routinely sterilized in the gravity steam autoclave or unsaturated chemical vapor sterilizers, even after three consecutive sterilization cycles . These items, however, were sterilized by exposure to a single-treatment cycle in a high-vacuum steam autoclave . Results indicate that routine sterilization of spore contaminated anesthetic carpules or injection systems could not be accomplished in a reasonable amount of time using sterilizers commonly found in dental offices.

PDA J Pharm Sci Technol, 1998 Sep-Oct, 52(5), 198 - 208
Bacillus stearothermophilus sporulation response to different composition media; Penna TC et al.; To evaluate the effectiveness of 11 commonly used ingredients to improve Bacillus stearothermophilus ATCC 7953 sporulation, with high spore yields in a short period of incubation, 32 composition media were set up by a fractional factorial 2IV11-6 design at two levels: D-glucose (0.018-0.25%), L-glutamic acid (0.040-0.10%), yeast extract (0.050-0.40%), peptone (0.30-0.50%), sodium chloride (0.001-1.0%), magnesium sulfate (0.001-0.20%), ammonium phosphate (0.010-0.035%), potassium phosphate monobasic (0.050-0.25%), calcium chloride (0.001-0.05%), ferrous sulfate (0.0003-0.002%), manganese sulfate (0.001-0.50%) . The largest variation on Log10 CFU response took place due to sodium chloride main effect, by changing it from low to high levels . Magnesium sulfate, calcium chloride, and ferrous sulfate were split and exerted no detectable main effect influence on sporulation . Setting up two 16 runs for sodium chloride effect, in each of which the remainder levels were kept constant, other components contribution was studied . At low sodium chloride, best average 7.25 Log10 CFU yielded by fastening yeast extract and peptone at high level, and remainders at low level . Considering high level of sodium chloride, peptone, yeast extract and ammonium phosphate kept at high level and remainders at low level confirmed the best sporulation yield . Adjusted models evidenced a strong influence of joint yeast/peptone effect, associated to ammonium phosphate contributing positively . The reduced incubation period from 15 days to 3-6 days at 62 degrees C was attained for all 32 experimental runs.

Ann Diagn Pathol, 1998 Oct, 2(5), 335 - 49
James Carroll: a biography; del Regato JA; James Carroll was born in England in 1854; at the age of 15, he emigrated to Canada where he worked at various odd jobs . At age 20, he crossed the border and volunteered for the US Army, in which he remained for the rest of his life . Appointed as Hospital Steward, he became interested in medicine . He was permitted to take basic courses at St Paul University and later at Bellevue Hospital in New York . He received his MD degree in 1891 from the University of Maryland while still a sergeant . He then took the course in bacteriology offered by Welch at Hopkins . At an 1893 international exposition in Chicago, Carroll was put in charge of the Army's exhibit on bacteriology . He was then called to become Assistant Professor of Microscopy at the new Army Medical School; his senior there was Walter Reed . Both men were offered professorships in pathology and bacteriology at George Washington University, and in 1900, both were appointed to the US Board sent to Havana . After several weeks, the Board determined that the alleged agent causing yellow fever was Bacillus cholerae suis (Sanarelli) . Visiting British researchers informed the Board of their favorable view of Carlos Finlay's theory that the disease was transmitted by the mosquito . The Board then visited Finlay, who gave them eggs of the particular species of mosquito that he had discovered to be the culprit . Board members Lazear and Carroll submitted themselves to the bite of an infected mosquito; both developed severe fever and Lazear died . The Board then carried out a well-planned experiment which proved that Finlay had been right for 20 years . Further experiments by Carroll showed that the agent could pass through a Berkefeld filter and was not bacterial.

FEBS Lett, 1998 Nov 20, 439(3), 241 - 5
Substituting selenocysteine for active site cysteine 149 of phosphorylating glyceraldehyde 3-phosphate dehydrogenase reveals a peroxidase activity; Boschi-Muller S et al.; Replacing the essential Cys-149 by a selenocysteine into the active site of phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus leads to a selenoGAPDH that mimics a selenoperoxidase activity . Saturation kinetics were observed with cumenyl and tert-butyl hydroperoxides, with a better catalytic efficiency for the aromatic compound . The enzymatic mechanism fits a sequential model where the formation of a ternary complex between the holoselenoenzyme, the 3-carboxy 4-nitrobenzenethiol used as the reductant and the hydroperoxide precedes product release . The fact that the selenoGAPDH is NAD-saturated supports a binding of hydroperoxide and reductant in the substrate binding site . The catalytic efficiency is similar to selenosubtilisins but remains low compared to selenoglutathione peroxidase . This is discussed in relation to what is known from the X-ray crystal structures of selenoglutathione peroxidase and GAPDHs.

Neuropeptides, 1998 Oct, 32(5), 393 - 403
Systemic treatment with Mycobacterium bovis bacillus Calmette-Guérin (BCG) potentiates kinin B1 receptor agonist-induced nociception and oedema formation in the formalin test in mice; de Campos RO et al.; This study investigates the effect and some of the mechanisms involved following systemic treatment of mice with Mycobacterium bovis bacillus Calmette-Guerin (BCG) (1 dose per animal containing 6.4 x 10(4) colony-forming units (CFu) 20-60 days beforehand) on modulation of the kinin B1 receptor agonist-induced nociception and oedema formation in the formalin test . Intraplantar (i.p.l.) co-injection of des-Arg9-bradykinin (4-32 nmol/paw) or des-Arg10-kallidin (1-15 nmol/paw), together with sub-maximal concentrations of formalin (0.01 or 0.5%), potentiated (P < 0.01) both pain phases and the paw oedema caused by formalin in animals pre-treated with saline . However, when animals were pre-treated with BCG, the dose-response curves for both B1 agonists were shifted 2 to 8-fold to the left . These B1-mediated effects peaked at 30-45 days after BCG treatment and were still elevated at 60 days after BCG injection . The pain response and oedema formation caused by i.p.l . co-injection of des-Arg9-bradykinin, together with formalin in BCG-pre-treated animals, were dose-dependently antagonised by i.p.l . co-injection of the B1 antagonist des-Arg9{Leu8}bradykinin (1-15 nmol/paw), but were not affected by the B2 antagonist Hoe 140 (10 nmol/paw) . The i.p.l . co-injection of tyrosine8-bradykinin (a B2 agonist, 3-15 nmol/paw) with formalin (0.01 or 0.5%) potentiated the pain response and paw oedema in BCG and saline-pre-treated animals to the same extent (P < 0.01) . The actions caused by tyrosine8-bradykinin were antagonised by Hoe 140, while des- Arg9{Leu8}bradykinin (10 nmol/paw) had no effect . Dexamethasone (0.5 mg/kg, s.c.), given every 24 h, from day 0 to 30-45, inhibited significantly the potentiation of nociceptive response and oedema formation caused by i.p.l . co-injection of formalin plus des-Arg9-bradykinin, while indomethacin (2 mg/kg, i.p.) or phenidone (30 mg/kg, i.p.), given 1 h prior, caused less inhibition . These data show that the long-term systemic treatment of mice with BCG produced dose-related potentiation of B1 receptor agonist-mediated nociception and oedema formation, without affecting similar responses caused by the B2 receptor agonist tyrosine8-bradykinin . Thus, systemic treatment of mice with BCG induces upregulation of B1 receptors, without affecting B2-mediated responses, by a mechanism that seems to be secondary to cytokine release.

Rev Clin Esp, 1998 Oct, 198(10), 651 - 4
{Clinical evaluation of a new non-radiometric automatic system for the rapid diagnosis of tuberculosis}; Casal M et al.; BACKGROUND: Tuberculosis (TB) is again a public health problem un many countries and is considered a re-emerging disease . The fastest possible diagnosis in our patients is essential for TB control programs . ESP is a non-radioactive, totally automated, continuously monitored system designed to detect mycobacteria . METHODS: Clinical evaluation of this new system for the rapid diagnosis of tuberculosis . During 1997 a total of 1,022 clinical sputum specimens were investigated . Specimens were processed in triplicate for ESP, BACTEC 460 TB and Lowenstein-Jensen systems . The validity, isolates of Mycobacterium tuberculosis and time required for detecting M . tuberculosis by the three systems were determined . RESULTS: The sensitivity, specificity, positive predictive and negative predictive values of the new systems were 98%, 99.8%, 98% and 99.8%, respectively . No significant differences were found between the recovery rates by the three systems . The mean time for detection was 10 days (range: 7-13 days) for specimens with positive bacilloscopy and 14 days (range: 10-28 days) for specimens with negative bacilloscopy . The difference was statistically significant between ESP and Lowenstein-Jensen, but not between ESP and BACTEC . CONCLUSIONS: The new system proved to have an excellent sensitivity and specificity, which along with its total automation renders it a system of great clinical interest for the rapid diagnosis of TB and an alternative method for radiometric systems.

Clin Exp Immunol, 1998 Dec, 114(3), 347 - 54
Inflammatory cell infiltrate in a responding metastatic nodule after vaccine-based immunotherapy; Logan TF et al.; A patient with von Hippel Lindau disease, bilateral symmetric renal cell carcinoma and pulmonary metastases treated with immunotherapy is the subject of this study . A left kidney and tumour mass were removed and the tumour cells used to make an autologous tumour/bacille Calmette-Guerin (BCG) vaccine as part of the treatment protocol . The patient's pulmonary nodules responded, but the remaining renal nodule subsequently grew . Samples of both tumours were obtained allowing for an internally controlled evaluation of the histological and immunohistologic differences between a responding and non-responding tumour nodule after therapy . The immunotherapy protocol is designed to promote a T cell response to autologous tumour . Cellular infiltrates were demonstrated in both responding and non-responding nodules compared with the pretreatment tumour specimen, but the responding nodule contained proportionately more T cells as well as markedly increased numbers of plasma cells and granulocytes . This suggested that several arms of the immune system may have been operative in the responding nodule.

Biochemistry, 1998 Nov 10, 37(45), 15981 - 9
Crystal structure of thiaminase-I from Bacillus thiaminolyticus at 2.0 A resolution; Campobasso N et al.; Thiaminase-I catalyzes the replacement of the thiazole moiety of thiamin with a wide variety of nucleophiles, such as pyridine, aniline, catechols, quinoline, and cysteine . The crystal structure of the enzyme from Bacillus thiaminolyticus was determined at 2.5 A resolution by multiple isomorphous replacement and refined to an R factor of 0.195 (Rfree = 0.272) . Two other structures, one native and one containing a covalently bound inhibitor, were determined at 2.0 A resolution by molecular replacement from a second crystal form and were refined to R factors of 0.205 and 0.217 (Rfree = 0.255 and 0.263), respectively . The overall structure contains two alpha/beta-type domains separated by a large cleft . At the base of the cleft lies Cys113, previously identified as a key active site nucleophile . The structure with a covalently bound thiamin analogue, which functions as a mechanism-based inactivating agent, confirms the location of the active site . Glu241 appears to function as an active site base to increase the nucleophilicity of Cys113 . The mutant Glu241Gln was made and shows no activity . Thiaminase-I shows no sequence identity to other proteins in the sequence databases, but the three-dimensional structure shows very high structural homology to the periplasmic binding proteins and the transferrins.

Biochemistry, 1998 Nov 10, 37(45), 15799 - 807
Imidazolyl carboxylic acids as mechanistic probes of flavocytochrome P-450 BM3; Noble MA et al.; omega-Imidazolyl carboxylic acids (C10-C12) have been used as probes of the active site and catalytic mechanism of the fatty acid hydroxylase P-450 BM3 from Bacillus megaterium . These compounds are the most potent inhibitors of P-450 BM3 yet reported . All are mixed inhibitors, increasing the Km and decreasing the kcat for laurate oxidation . All ligate the P-450 BM3 ferric heme iron, inducing a type II shift in the Soret absorbance band from 419 to 424 nm . Binding to the ferrous form is much weaker . 10-(Imidazolyl)decanoic acid was the best inhibitor (Kic = 0.9 microM, Kiu = 5.7 microM), while 12-(imidazolyl)dodecanoic acid (Kic = 1.35 microM, Kiu = 6.9 microM) was superior to 11-(imidazolyl)undecanoic acid (Kic = 7.5 microM, Kiu = 16 microM) . Dissociation constants for binding to oxidized P-450 BM3 heme iron were determined spectrophotometrically as 8 microM (C12 azole) and 27 microM (C11 azole) . The binding of 10-(imidazolyl)decanoic acid was too tight for an absolute Kd to be determined spectrophotometrically, but this value is <0.2 microM . The binding of different fatty acids to the enzyme was found to have distinct effects on the Kd for the azoles . Laurate induced tighter binding (Kd for the C12 azole lowered to 4.7 microM), while arachidonate weakened the affinity (Kd increased to 23 microM) . Arachidonate diminished the affinity for the C10 azole sufficiently that a Kd could be determined by spectrophotometric titration (11 microM) . Affinity for the C12 azole was decreased in active-site-mutants R47G (R47 tethers the fatty acid carboxylate group) and F87Y but increased in mutant F87G-indicating an important role for this residue in determining heme accessibility . The C10 azole binds much more weakly to the spin-state-insensitive F87Y (32 . 2 microM), suggesting that the inhibitors may bind preferentially to different conformers of P-450 BM3 . NADP+ binding in the reductase also tightened affinity of these inhibitors for P-450 BM3 (Kd for the C12 azole decreased to 2.7 microM), but this effect was not observed for FMN-deficient mutant W574D, suggesting that the interdomain effect of NADP+ on inhibitor binding was mediated via flavin mononucleotide . Resonance Raman spectroscopy indicates that the inhibitors form low-spin complexes with P-450 BM3 and that their binding induces movements of the heme vinyls relative to the ring.

Biosens Bioelectron, 1998 Nov 1, 13(10), 1077 - 82
Amperometric phenol biosensor based on a thermostable phenol hydroxylase; Metzger J et al.; Phenol hydroxylase (EC 1.14.13.7) was produced using Bacillus stearothermophilus in a 5-1 batch fermentation leading to approximately 17 units after 6 h . The partially purified phenol hydroxylase was entrapped in a sol-gel matrix . The enzyme-loaded silica gel was attached to the sensitive top of a Clark-type oxygen electrode and its application as a phenol biosensor was tested . There was linearity between the maximal rate of oxygen consumption and phenol concentration in the range between 2.5 and 400 microM at 40 degrees C and pH 7.6 . The signal could be read off after 10 s at a concentration of 400 microM phenol . The sensor lost 20% of its activity within 7 days . Para-substituted phenols were not detectable.

Ophthal Plast Reconstr Surg, 1998 Nov, 14(6), 398 - 402
Infection of a porous polyethylene orbital implant with Capnocytophaga; Wilson MW et al.; A 68-year-old woman experienced an infection of a porous polyethylene orbital implant caused by Capnocytophaga after a dental procedure . The infection was unresponsive to both topical and oral antibiotics and required removal of the porous polyethylene orbital implant . Capnocytophaga is a capnophilic, gram-negative bacillus . Capnocytophaga is a normal commensal of the mouth and is responsible for both gingivitis and periodontal disease . Capnocytophaga is a rare cause of ocular infections . This is the first reported patient with an infection of a porous polyethylene orbital implant caused by Capnocytophaga . The authors believe infected integrated orbital implants must be removed because neither topical or systemic therapy provide effective treatment.

Arch Bronconeumol, 1998 Oct, 34(9), 421 - 4
{The tuberculin skin test in BCG-vaccinated individualse}; Miret Cuadras P et al.; The aim of this study was to evaluate the tuberculin skin test in individuals vaccinated with bacillus Calmette-Guerin (BCG) using 2 IU of RT-23 . One hundred ninety-six individuals aged 22-40 years-old who had been vaccinated with BCG between 1965 and 1974 were enrolled along with 375 non-vaccinated individuals of the same age and with similar level of risk of infection . The positive predictive value of the test was assessed for three levels of response as indicated by areas of thickening in three diameters: 5, 10 and 15 mm . Vaccinated individuals with negative results were given a second skin test 7 days later to detect a booster effect . Positive diameters 5 mm were observed in 66% of the vaccinated individuals and 24% of the non-vaccinated subjects . Positive diameters 10 mm were observed in 51% of the vaccinated individuals and 19% of the non vaccinated ones . Positive diameters 15 mm were observed in 29% of the vaccinated subjects and in 13% of the non vaccinated ones . The differences were significant for all diameters . The positive predictive value of the test was 36.4% for a diameter 5 mm, 37.6% for diameter 10 mm and 44.8% for diameter 15 mm . The booster effect was detected in 25.8% of the vaccinated individuals who had tested negative at first . In vaccinated individuals, no guidelines can be established to guarantee that a positive reaction is due to infection by Mycobacterium tuberculosis infection, although the likelihood of infection (increased positive predictive value) increases with diameter . It is also impossible to fix a time limit . A second skin test is needed to detect a booster effect in all vaccinated individuals whose first test is negative.

Eur J Biochem, 1998 Nov 1, 257(3), 570 - 6
Bacterial pro-transglutaminase from Streptoverticillium mobaraense--purification, characterisation and sequence of the zymogen; Pasternack R et al.; The zymogen of bacterial transglutaminase was found during cultivation of Streptoverticillium mobaraense (DSMZ strain) using rabbit antibodies raised against the active enzyme . Ion-exchange chromatography at pH 5.0 yielded a highly purified pro-enzyme . Structure information was obtained by means of Edman degradation and analysis of PCR amplified nucleotide fragments . The data revealed an excess of negatively charged amino acids in the pro-region resulting in a decreased isoelectric point of the zymogen . Additionally, the new sequence gave rise to some modifications to the previously published hypothetical structure of prepro-transglutaminase derived from genomic DNA {Washizu, K., Ando, K., Koikeda, S., Hirose, S., Matsuura, A., Takagi, H., Motoki, M . & Takeuchi, K . (1994) Biosci . Biotechnol . Biochem . 58, 82-87} . Inactive transglutaminase, which carries an activation peptide of 45 amino acids, has a calculated molecular mass of 42445 Da . Its pro-region provides for both suppression of activity and increased thermostability . Furthermore, it could be shown that the micro-organism produces a protease which cleaves pro-transglutaminase at the C-side of Pro45 . Rapid transformation of the mature enzyme also occurs by addition of other proteases . During conversion, 43 and 41 amino acid peptides are released by bovine trypsin and dispase from Bacillus polymyxa, respectively . The detection of endogenous substrates in the murein layer makes discussion of the physiological role of bacterial transglutaminases necessary.

Vet Immunol Immunopathol, 1998 Oct 23, 65(2-4), 191 - 204
Homologous protection but lack of heterologous-protection by various species and types of Bartonella in specific pathogen-free cats; Yamamoto K et al.; Cat-scratch disease (CSD) is caused by Bartonella henselae, and possibly by B . clarridgeiae . In immuno-compromised persons, B . henselae is one of the agents causing bacillary angiomatosis . Domestic cats are the main reservoir of these bacteria, which are transmitted primarily from cat to cat by fleas . Possible strategies to prevent the spread of infection among cats are to eliminate flea infestation or to prophylactically immunize cats . In order to develop an appropriate vaccine, it is important to determine if cats become resistant to re-infection by the same strain or various types or species of Bartonella . In a series of experiments, 21 SPF cats were experimentally infected by the intradermal route with 10(5)-10(10) colony-forming units/ml of either B . henselae type II (17 cats), or a new strain 'Humboldt' isolated from a mountain lion (4 cats) . The cats were bled weekly to every other week for determination of bacteremia and specific antibody production . After they cleared their infection, they were challenged by a homologous or heterologous strain of Bartonella: 10 cats were challenged with B . henselae type II, three cats with B . henselae type I, four cats with B . clarridgeiae and four cats with the 'Humboldt' strain . Seven of these cats received a third inoculum dose resulting in three cats sequentially infected with sequence B . henselae type II/B . henselae type II/'Humboldt', two cats with sequence B . henselae type II/'Humboldt'/B . clarridgeiae, and two cats with the sequence 'Humboldt'/B . henselae type II/'Humboldt' . All cats challenged with a homologous strain remained abacteremic after challenge and had an increased IgG antibody titer . All cats challenged with either a different Bartonella species or type became bacteremic . The few cats receiving a third inoculum with a strain homologous to the initial strain remained abacteremicafter that challenge . All cats infected with B . clarridgeiae suffered relapsing bacteremia compared to only 36% of the B . henselae infected cats and 22% of the 'Humboldt'-infected cats (p=0.008) . The duration of bacteremia was significantly longer in B . henselae primary-infected cats (mean: 34 weeks) than B . henselae heterologously challenged cats (mean: 9 weeks) (p=0.014) . These data clearly indicate the lack of cross-protection between B . henselae and B . clarridgeiae and furthermore, indicate the lack of protection between B . henselae types I and II, and a wildlife isolate . A vaccine strategy for CSD prevention in domestic cats will require a multivalent vaccine approach.

Urology, 1998 Dec, 52(6), 1009 - 13; discussion 1013-4
Microstaging of pT1 transitional cell carcinoma of the bladder: identification of subgroups with distinct risks of progression; Smits G et al.; OBJECTIVES: To evaluate microstaging by means of quantifying the depth of invasion of the subepithelial connective tissue in pT1 transitional cell carcinoma (TCC) of the bladder for its additional prognostic value with respect to disease recurrence and progression . METHODS: We reviewed the pathologic findings of a consecutive series of 124 patients with pT1 tumors entered in a prospective randomized multicenter trial comparing mitomycin C and bacillus Calmette-Guerin treatment, with at least 3 years of follow-up and clinical outcome hidden from reviewers . The depth of invasion was established by identifying submucosal tumor invasion up to, in, or beyond the muscularis mucosae or vascular plexus and classified as pT1a, pT1b, or pT1c, respectively . In addition to tumor grade, the presence of carcinoma in situ (CIS) near the primary tumor or in biopsy specimens taken from abnormal looking mucosa was taken into account . The risks of recurrence and progression were calculated using Kaplan-Meier curves and modeled with proportional hazard models . RESULTS: pT1 subclassification was possible in more than 90% of the specimens . The 3-year risk of recurrence was not different in any of the subgroups . By contrast, the Kaplan-Meier 3-year risk for progression was 6%, 33%, and 55% for pT1a, pT1b (hazard ratio {HR} 5.51), and pT1c (HR 12.35) tumors, respectively (log-rank test P < 0.001) . The Kaplan-Meier 3-year risk of progression was 9% versus 39% (HR 5.62) for the absence or presence of CIS in the tumor (P=0.001) and 8% versus 49% (HR 6.72) for CIS in biopsy specimens (P < 0.001) . Tumor grade had no statistically significant prognostic value with respect to progression, nor had tumor volume or multifocality . The combination of the parameters (pT1c and CIS) increased the risk of progression by a factor of 27 (P < 0.0001) compared with the absence of pT1c and CIS . CONCLUSIONS: These data show that the extent of lamina propria invasion (pT1a, pT1b, pT1c) is a clinically relevant prognostic factor for progression of pT1 TCC of the bladder . With the combination of this pT1 subclassification and the presence of CIS subgroups, distinct risks of progression can be identified that may give additional information for follow-up and treatment policies.

Appl Environ Microbiol, 1998 Dec, 64(12), 4965 - 72
Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection; Bravo A et al.; Mexico is located in a transition zone between the Nearctic and Neotropical biogeographical regions and contains a rich and unique biodiversity . A total of 496 Bacillus thuringiensis strains were isolated from 503 soil samples collected from the five macroregions of the country . The characterization of the strain collection provided useful information on the ecological patterns of distribution of B . thuringiensis and opportunities for the selection of strains to develop novel bioinsecticidal products . The analysis of the strains was based on multiplex PCR with novel general and specific primers that could detect the cry1, cry3, cry5, cry7, cry8, cry9, cry11, cry12, cry13, cry14, cry21, and cyt genes . The proteins belonging to the Cry1 and Cry9 groups are toxic for lepidopteran insects . The Cry3, Cry7, and Cry8 proteins are active against coleopteran insects . The Cry5, Cry12, Cry13, and Cry14 proteins are nematocidal . The Cry11, Cry21, and Cyt proteins are toxic for dipteran insects . Six pairs of general primers are used in this method . Strains for which unique PCR product profiles were obtained with the general primers were further characterized by additional PCRs with specific primers . Strains containing cry1 genes were the most abundant in our collection (49.5%) . Thirty-three different cry1-type profiles were identified . B . thuringiensis strains harboring cry3 genes represented 21.5% of the strains, and 7.9% of the strains contained cry11 and cyt genes . cry7, cry8, and cry9 genes were found in 0.6, 2.4, and 2.6% of the strains, respectively . No strains carrying cry5, cry12, cry13, cry14, or cry21 genes were found . Finally, 14% of the strains did not give any PCR product and did not react with any polyclonal antisera . Our results indicate the presence of strains that may harbor potentially novel Cry proteins as well as strains with combinations of less frequently observed cry genes.

Appl Environ Microbiol, 1998 Dec, 64(12), 4782 - 8
A holistic approach for determining the entomopathogenic potential of Bacillus thuringiensis strains; Masson L et al.; The cry gene content of Bacillus thuringiensis subsp . aizawai HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR . A total of six cry genes were detected in genomic DNA purified from HD-133, four from the cry1 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families . To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC . Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio . Dot blot analysis of total mRNA purified from HD-133 showed that both the cry2B and cry1I genes, but not the cry1Aa gene, were transcribed . Cloning and sequencing of the cry1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame . Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of B . thuringiensis isolates for new insecticidal genes and specificities . Furthermore, based on the number of cryptic genes found in HD-133, the total cry gene content of B . thuringiensis strains may be higher than previously thought.

Rev Mal Respir, 1998 Oct, 15(5), 643 - 7
{Prevalence of nocardiosis in an area of endemic tuberculosis}; Koffi N et al.; This is a prospective study recruiting 120 patients successively who were admitted to the chest department in hospital for respiratory infections irrespective of their aetiology . The aim of the study was to assess the frequency of nocardiosis in respiratory pathology in the era of AIDS and in an area where tuberculosis is endemic . The HIV serology was carried out on all 120 patients . A systemic search was made for nocardiosis and Koch's bacillus in the sputum and also in the broncho-alveolar lavage liquid obtained by endoscopy . The HIV serology was positive in 74 patients (61.7%) . Pulmonary nocardiosis was diagnosed in five patients (4.2%), of whom four patients were HIV positive (80%) . Tuberculosis was diagnosed in 58 cases (48.3%) of whom 40 were HIV positive (70%) . The association of nocardiosis and tuberculosis was present in only one patient . The radioclinical aspect of nocardiosis in our service was suggestive of tuberculosis . The prevalence of nocardiosis in our series at 4.2% is in agreement with that obtained in autopsy studies in the Ivory Coast . The similarity of the radioclinical appearance between tuberculosis and nocardiosis demands that a search is made for the latter on all HIV positive patients and in negative cases a search for Koch's bacillus and empirical antibiotic therapy ought to have a spectrum of activity that would include nocardia.

J Biochem (Tokyo), 1998 Dec 1, 124(6), 1178 - 87
Mg2+ binding and catalytic function of sphingomyelinase from Bacillus cereus; Fujii S et al.; The modes of Mg2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity . This enzyme was shown to possess at least two binding sites for Mg2+ with low and high affinities . The effects of Mg2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied . The results indicated that the binding of Mg2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme . It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg2+ binding, whereas no significant protective effect was observed against the denaturation by urea . The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B . cereus, was studied in the presence of a large amount of Mg2+ to saturate both the low- and high-affinity sites . The pH dependence curves of the logarithm of 1/Km for these two kinds of substrates were similar in shape to each other, and showed a single transition . On the other hand, the shapes of the pH dependence curves of the logarithm of kcat for these two kinds of substrates were different from each other . The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively . On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition) . On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B . cereus SMase, we proposed a catalytic mechanism for B . cereus SMase based on general-base catalysis.

J Biochem (Tokyo), 1998 Dec 1, 124(6), 1163 - 9
Transamination as a side-reaction catalyzed by alanine racemase of Bacillus stearothermophilus; Kurokawa Y et al.; The pyridoxal form of alanine racemase of Bacillus stearothermophilus was converted to the pyridoxamine form by incubation with its natural substrate, D- or L-alanine, under acidic conditions: the enzyme loses its racemase activity concomitantly . The pyridoxamine form of the enzyme returned to the pyridoxal form by incubation with pyruvate at alkaline pH . Thus, alanine racemase catalyzes transamination as a side function . In fact, the apo-form of the enzyme abstracted tritium from {4'-3H}pyridoxamine in the presence of pyruvate . A mutant enzyme containing alanine substituted for Lys39, whose epsilon-amino group forms a Schiff base with the C4' aldehyde of pyridoxal 5'-phosphate in the wild-type enzyme, was inactive as a catalyst for racemization as well as transamination . However, when methylamine was added to the mutant enzyme, it became active in both reactions . These results suggest that the epsilon-amino group of Lys39 participates in both racemization and transamination when catalyzed by the wild-type enzyme.

Infect Control Hosp Epidemiol, 1998 Nov, 19(11), 829 - 35
Rates of tuberculosis infection in healthcare workers providing services to HIV-infected populations . Terry Beirn Community Programs for Clinical Research on AIDS; Zahnow K et al.; OBJECTIVE: To assess the prevalence of tuberculosis (TB) or a positive skin test in healthcare workers (HCWs) providing services to human immunodeficiency virus (HIV)-infected individuals and to determine prospectively the incidence of new infections in this population . DESIGN: This prospective cohort study enrolled 1,014 HCWs working with HIV-infected populations from 10 metropolitan areas . Purified protein derivative (PPD) tuberculin skin tests were placed at baseline and every 6 months afterwards on those without a history of TB or a positive PPD . Demographic, occupational, and TB exposure data also were collected . SETTING: Outpatient clinics, hospitals, private practice offices, and drug treatment programs providing HIV-related healthcare and research programs . PARTICIPANTS: A voluntary sample of staff and volunteers from 16 Community Programs for Clinical Research on AIDS units . RESULTS: Factors related to prior TB or a positive skin test at baseline included being foreign-born, increased length of time in health care, living in New York City, or previous bacille Calmette-Guerin vaccination . The rate of PPD conversion was 1.8 per 100 person years of follow-up . No independent relation was found between the amount or type of contact with HIV-infected populations and the risk of TB infection . CONCLUSION: These data provide some reassurance that caring for HIV-infected patients is not related to an increased rate of TB infection among HCWs in these settings.

Int J Antimicrob Agents, 1998 Aug, 10(3), 191 - 205
Ventilator-associated pneumonia; Visnegarwala F et al.; Mechanically ventilated patients are at a substantially higher risk for developing nosocomial pneumonia . Overall, there is a relatively constant 1&!TN!150;3% risk per day of developing pneumonia while receiving mechanical ventilation . The sensitivity and specificity of clinical criteria alone for diagnosis of ventilator-associated pneumonias (VAP) is low . Several techniques have been developed to sample and quantitate the lower respiratory tract to improve the diagnostic yield . Gram-negative bacillary pneumonias account for the majority of the VAP . Strategies for prevention of VAP such as use of sucralfate for stress ulcer prophylaxis and selective decontamination of the digestive tract have been the focus of many clinical studies . Cost-effective preventive measures are needed to combat the increasing antimicrobial resistance, growing population of immunocompromised patients and increasing number of mechanically ventilated patients.

Infect Control Hosp Epidemiol, 1998 Nov, 19(11), 856 - 8
An outbreak of Bacillus species in a cancer hospital; Thuler LC et al.; Bacillus species were recovered from the blood cultures of 39 oncology patients over 14 weeks . A matched case-control study showed a strong association of Bacillus species bacteremia with use of calcium gluconate solution (odds ratio=25.0) and of central venous lines (odds ratio=8.8) . Stopping use of the implicated calcium gluconate vials controlled the outbreak.

J Appl Microbiol, 1998 Nov, 85(5), 865 - 74
Biological indicators for steam sterilization: characterization of a rapid biological indicator utilizing Bacillus stearothermophilus spore-associated alpha-glucosidase enzyme; Albert H et al.; The alpha-glucosidase enzyme was isolated from vegetative cells and spores of Bacillus stearothermophilus, ATCC 7953 . Spore-associated enzyme had a molecular weight of approximately 92,700, a temperature optimum of 60 degrees C, and a pH optimum of 7.0-7.5 . The enzyme in crude aqueous spore extract was stable for 30 min up to a temperature of 65 degrees C, above which the enzyme was rapidly denatured . The optimal pH for stability of the enzyme was approximately 7.2 . The alpha-glucosidase in crude vegetative cell extract had similar characteristics to the spore-associated enzyme but its molecular weight was 86,700 . The vegetative cell and spore-associated enzymes were cross-reactive . The enzymes are postulated to derive from a single gene product, which undergoes modification to produce the spore-associated form . The location of alpha-glucosidase in the spore coats (outside the spore protoplast) is consistent with the location of most enzymes involved in activation, germination and outgrowth.

J Biotechnol, 1998 Oct 27, 65(2-3), 191 - 202
Effects of mutations in the starch-binding domain of Bacillus macerans cyclodextrin glycosyltransferase; Chang HY et al.; Cyclodextrin glycosyltransferase (CGTase) is an industrially important enzyme that produces cyclodextrins (CD) from starch by intramolecular transglycosylation . CGTase consists of five globular domains labeled A through E . To better understand the role of domain E in CGTase catalysis, we have constructed several mutants of Bacillus macerans CGTase . Removing the entire E domain resulted in an inactive enzyme . Adding six amino acids between domains D and E caused a decrease in activity and thermostability . Replacing domain E with the similar starch-binding domain from Aspergillus awamori glucoamylase I caused a drastic decrease in activity, indicating the necessity of correct alignment of bound substrate . Substituting tyrosine residue 634 (Tyr634) with phenylalanine had very little effect on activity or thermostability . Substituting Tyr634 with glycine resulted in a 25% increase of specific cyclization and starch-hydrolyzing activities compared with that of the wild-type enzyme . The latter mutant was less thermostable . The results of this study indicate that domain E is important for the stability and integrity of B . macerans CGTase.

Biochem Biophys Res Commun, 1998 Nov 18, 252(2), 402 - 6
Modulation of Cry IV A toxin protein expression by glucose in Bacillus thuringiensis israelensis; Banerjee-Bhatnagar N; Bacillus thuringiensis subsp . israelensis (Bti) produces Cry IV A protoxin protein as part of the insecticidal crystal toxin during sporulation . This study was conducted with the objective of identifying environmental signals which regulate toxin synthesis by Bti . Glucose was found to repress Cry IV A toxin induction at the mRNA level . The repressive effect of glucose was dependent on a phosphorylation step since protein kinase inhibitor calphostin c relieved the 130-kD protoxin synthesis at both the mRNA and protein level . Phosphorylation of HPr, the phosphocarrier protein of the phosphotransferase system, occurred during glucose repression of Cry IV A toxin synthesis in Bti cells was seen by Western blotting with anti-phosphoserine antibody and rabbit anti-HPr serum . Phosphorylation of HPr in vivo as well as in the in vitro assay was inhibited by calphostin c, a specific inhibitor of serine/threonine kinase . Calphostin c had no effect on sporulation efficiency of Bti cells .

Protein Sci, 1998 Nov, 7(11), 2405 - 12
Global analysis of the thermal and chemical denaturation of the N-terminal domain of the ribosomal protein L9 in H2O and D2O . Determination of the thermodynamic parameters, deltaH(o), deltaS(o), and deltaC(o)p and evaluation of solvent isotope effects; Kuhlman B et al.; The stability of the N-terminal domain of the ribosomal protein L9, NTL9, from Bacillus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O . The basic thermodynamic parameters for the unfolding reaction, deltaH(o), deltaS(o), and deltaC(o)p, were determined by global analysis of temperature and denaturant effects on stability . The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs-Helmholtz equation to model changes in stability with temperature . The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations . NTL9 has a maximum stability of 3.78 +/- 0.25 kcal mol(-1) at 14 degrees C . DeltaH(o)(25 degrees C) for protein unfolding equals 9.9 +/- 0.7 kcal mol(-1) and TdeltaS(o)++(25 degrees C) equals 6.2 +/- 0.6 kcal mol(-1) . DeltaC(o)p equals 0.53 +/- 0.06 kcal mol(-1) deg(-1) . There is a small increase in stability when D2O is substituted for H2O . Based on the results from global analysis, NTL9 is 1.06 +/- 0.60 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 5.8 +/- 3.6 degrees C in D2O . Based on the results from individual denaturation experiments, NTL9 is 0.68 +/- 0.68 kcal mol(-1) more stable in D2O at 25 degrees C and Tm is increased by 3.5 +/- 2.1 degrees C in D2O . Within experimental error there are no changes in deltaH(o) (25 degrees C) when D2O is substituted for H2O.

Extremophiles, 1998 Nov, 2(4), 447 - 53
Physical map of alkaliphilic Bacillus firmus OF4 and detection of a large endogenous plasmid; Gronstad A et al.; Extremely alkaliphilic Bacillus firmus OF4 is among the best characterized of this group of alkaliphiles . Together with alkaliphilic Bacillus C-125 and numerous non-alkaliphilic Bacillus species whose chromosomes and gene organizations are currently being studied in detail, work on B . firmus OF4 offers the opportunity to discern whether there are features of chromosome and gene organization that are associated with alkaliphily . A physical map of the B . firmus OF4 is consistent with a circular chromosome of approximately 4 Mb, with an extrachromosomal element of 110 kb also detected . The previously identified cadmium-resistance locus and transposition functions in B . firmus OF4 were localized to the extrachromosomal element, whose genes exhibit a slightly different pattern of codon usage from chromosomal genes . No clustering of genes thus far identified with roles in alkaliphily has been found . Direct repeat sequences (DRS) were previously reported upstream of a gene encoding a Na+/H+ antiporter that has a role in pH homeostasis . In the current analyses, these sequences were found to be present in multiple copies on the chromosome, most of which are present in one 920-kb fragment . Such sequences might play a role in DNA rearrangements that allow amplification of important genes in this region.

Arch Biochem Biophys, 1998 Dec 1, 360(1), 1 - 9
Molecular cloning of two genes for beta-D-glucosidase in Bacillus sp . GL1 and identification of one as a gellan-degrading enzyme; Hashimoto W et al.; In the bacterium Bacillus sp . GL1, gellan is depolymerized to give a tetrasaccharide by extracellular gellan lyase and then the tetrasaccharide is converted to constituent monosaccharides by intracellular glycosidases . Two genes encoding one of the glycosidases, beta-D-glucosidase (Bgl), were cloned in a genomic DNA library of the bacterium constructed in Escherichia coli and nucleotide sequences of the genes were determined . One of the genes, termed bglA, contained an open reading frame (ORF) consisting of 1344 base pairs coding a polypeptide (BglA) with a molecular mass of 51 kDa and the other, termed bglB, 2268 base pairs coding a protein (BglB) with a molecular mass of 82 kDa . By homology analyses of the ORFs against protein sequence databases, beta-D-glucosidase A (BglA) and beta-D-glucosidase B (BglB) were found to be classified into subfamilies BGA and BGB of cellulase family BG, respectively . BglA and BglB purified from E . coli were monomeric enzymes with molecular masses of 50 and 82 kDa and most active at pH 6.0 and 8.0, respectively . BglA showed broader substrate specificity than BglB . Only BglA acted on the tetrasaccharide produced from gellan by gellan lyase and released glucose from the molecule .

Infect Immun, 1998 Dec, 66(12), 5743 - 50
Mycobacterial dose defines the Th1/Th2 nature of the immune response independently of whether immunization is administered by the intravenous, subcutaneous, or intradermal route; Power CA et al.; It is believed that cell-mediated immunity alone can contain Mycobacterium tuberculosis, the pathogen responsible for tuberculosis . The induction of antibody, or of a mixed cell-mediated/humoral response, is associated with tuberculous disease . It is therefore important to determine the conditions of immunization with bacille Calmette Guerin (BCG), the attenuated strain of Mycobacterium bovis used to vaccinate humans against tuberculosis, that optimally induces an exclusive cell-mediated, Th1 response . Such a determination will then allow an assessment of whether the generation of such an exclusive Th1 response results in the generation of a Th1 imprint against mycobacteria . This Th1 imprint would ensure that the Th1 response is predominant following any challenge . We therefore tested the proposition that the dose of mycobacteria used for immunization generally determines the Th1/Th2 nature of the ensuing response . Our results demonstrate that relatively low doses lead to an almost exclusive cell-mediated, Th1 response, while higher doses induce a mixed Th1/Th2 response . Furthermore, the dependence on dose is independent of whether BCG is administered intravenously, subcutaneously, or intradermally . The implications of our findings to understanding how different classes of immunity are induced, to the epidemiology of tuberculosis, and to the design of effective vaccination strategies are discussed.

Infect Immun, 1998 Dec, 66(12), 5669 - 76
Systemic and mucosal immune responses after intranasal administration of recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing glutathione S-transferase from Schistosoma haematobium; Kremer L et al.; A major goal of current vaccine development is the induction of strong immune responses against protective antigens delivered by mucosal routes . One of the most promising approaches in that respect relies on the use of live recombinant vaccine carriers . In this study, Mycobacterium bovis BCG was engineered to produce an intracellular glutathione S-transferase from Schistosoma haematobium (Sh28GST) . The gene encoding Sh28GST was placed under the control of the mycobacterial hsp60 promoter on a replicative shuttle plasmid containing a mercury resistance operon as the only selectable marker . The recombinant Sh28GST produced in BCG bound glutathione and expressed enzymatic activity, indicating that its active site was properly folded . Both intraperitoneal and intranasal immunizations of BALB/c mice with the recombinant BCG resulted in strong anti-Sh28GST antibody responses, which were enhanced by a boost . Mice immunized intranasally produced a mixed response with the production of Sh28GST-specific immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgA in the serum . In addition, high levels of anti-Sh28GST IgA were also found in the bronchoalveolar lavage fluids, demonstrating that intranasal delivery of the recombinant BCG was able to induce long-lasting secretory and systemic immune responses to antigens expressed intracellularly . Surprisingly, intranasal immunization with the BCG producing the Sh28GST induced a much stronger specific humoral response than intranasal immunization with BCG producing the glutathione S-transferase from Schistosoma mansoni, although the two antigens have over 90% identity . This difference was not observed after intraperitoneal administration.

Eur J Biochem, 1998 Oct 15, 257(2), 500 - 5
Mutational analysis of the conserved cationic residues of Bacillus stearothermophilus 6-phosphoglucose isomerase; Meng M et al.; The importance in catalysis of the conserved arginine (R207) and lysine residues (K144, K294, K356, and K425) of 6-phosphoglucose isomerase from Bacillus stearothermophilus was assessed by site-directed mutagenesis and kinetic analysis . In general mutations had minor effects on the Km for fructose 6-phosphate . More dramatic effects were seen on kcat . The R207A mutant had a five orders of magnitude decrease in kcat relative to the wild-type enzyme . There was a significant recovery, by three orders of magnitude, in the kcat for the R207K mutant . The results suggest that the positive charge provided by R207 plays a critical role in the isomerization reaction . K425 was substituted with alanine, valine, phenylalanine, tryptophan and aspartate . All mutant enzymes at position 425 had kcat decreased in the range of several-hundred-fold . For the other mutants, K294A and K144A, the kcat values were 3.5% and 27% of the wild-type enzyme, respectively . No effects on catalysis were observed for the K356A mutant . The results suggest that R207, K144, K294, and K425 are located in the active site of the enzyme . The active-site location and the catalytic roles of K425 and K294 are supported further by the inhibitory effects of pyridoxal 5'-phosphate on enzymatic activities . The data also confirm the importance of K425 and K144 anticipated by the affinity labeling studies of the corresponding residues by pyridoxal 5'-phosphate in pig muscle phosphoglucose isomerase.

Eur J Biochem, 1998 Oct 15, 257(2), 495 - 9
Carbon dioxide fixation by reversible pyrrole-2-carboxylate decarboxylase from Bacillus megaterium PYR2910; Wieser M et al.; Pyrrole-2-carboxylate decarboxylase from Bacillus megaterium PYR2910 attains a balanced reaction equilibrium with an equilibrium constant of 0.3-0.4 M . Therefore, the enzyme catalyzes the reverse carboxylation of pyrrole after addition of bicarbonate . For the synthesis of pyrrole-2-carboxylate, the reverse reaction was optimized and the equilibrium was shifted towards the carboxylate . The product yield was 230 mM (25.5 g/l) pyrrole-2-carboxylate from 300 mM pyrrole in a batch reaction and 325 mM (36.1 g/l) from 400 mM pyrrole in a fed-batch reaction, using both whole cells and the purified enzyme in a pH 8.0 reaction mixture with bicarbonate saturation of 1.9 M . Kinetic studies indicated, that bicarbonate is the reactive species used by this carbon dioxide-fixation enzyme.

Eur J Biochem, 1998 Oct 15, 257(2), 309 - 18
Crystal structure of subtilisin DY, a random mutant of subtilisin Carlsberg; Eschenburg S et al.; The crystal structure of subtilisin DY inhibited by N-benzyloxycarbonyl-Ala-Pro-Phe-chloromethyl ketone has been solved by molecular replacement with subtilisin Carlsberg as the starting model . The model has been refined to a crystallographic R factor (= sigma absolute value {(absolute value Fo) - (absolute value Fc)} / sigma (absolute value of Fo) of 15.1% using X-ray diffraction data to 0.175 nm resolution . Subtilisin DY is an alkaline proteinase from the X-irradiated Japanese strain DY of Bacillus licheniformis, which normally produces subtilisin Carlsberg . It has very similar properties to subtilisin Carlsberg, with a slightly enhanced resistance to heat and guanidine hydrochloride-induced denaturation, in spite of the fact that the sequences of the two enzymes differ in 31 positions out of 274 residues . The close similarity in overall three-dimensional structure of subtilisins DY and Carlsberg and also their physicochemical properties, such as activity and stability, shows that nature aided by X-irradiation for rapid 'evolution' is able to accommodate considerable changes in sequence without substantial changes in property.

Tijdschr Diergeneeskd, 1998 Nov 1, 123(21), 628 - 32
{Isolation, identification and characterization of Bacillus cereus in the dairy industry}; te Giffel MC et al.; In order to determine the major contamination sources of milk with (psychrotrophic) Bacillus cereus, the incidence of vegetative cells and spores of B . cereus on dairy farms, at two dairy processing plants and in pasteurized milk in household refrigerators was investigated . On dairy farms the major contamination sources were soil and faeces . In winter, when the cows were housed, used bedding probably also participates in this contamination route . The udder will be contaminated, finally resulting in the presence of B . cereus in raw milk . The organism could be detected in 35% of the raw milk samples analyzed . During processing, an increase in the percentage of positive samples was observed . These results suggest that B . cereus can be introduced via sources other than raw milk; equipment may play an important role in this . Biochemical and molecular typing showed that selection of strains takes place in the milk production chain . It was demonstrated that some types were found in the raw milk, during processing and in the end products, indicating that raw milk is an important source of contamination . Other types could only be detected after the pasteurization step in the production process supporting the assumption that additional contamination occurs during processing . If stored under proper conditions, maximum storage temperature 7 degrees C, and consumed within the expiration date, the levels of B . cereus in pasteurized milk will, in general, not exceed 10(5) per ml and cause no problems for healthy adults.

J Infect . 1998 Sep;37(2):193.
Muscular bacillary angiomatosis in AIDS; Blanche P et al.; We describe an unusual case of bacillary angiomatosis first misdiagnosed as Kaposi's sarcoma in muscle in a patient with HIV infection.

Immunology, 1998 Oct, 95(2), 278 - 82
Expression of NO-synthase in cells of foreign-body and BCG-induced granulomata in mice: influence of L-NAME on the evolution of the lesion; Kreuger MR et al.; The microbicidal activity of macrophages in an inflammatory milieu has been related to the production of a large number of cytokins and intermediary metabolites of oxygen and nitrogen among them, nitric oxide (NO) . Considering that granulomatous inflammation is predominantly composed of macrophages and epithelioid cells, we decided to investigate the participation of NO in this peculiar type of inflammation . Two models were used: glass cover slip implantation into the subcutaneous tissue of mice and, the inoculation of live bacillus Calmette-Guerin (BCG) into the footpad of the animals . Using a histochemical method for the detection of NO synthase and of the concentration of citrulin metabolized by cells obtained from cover slips implanted on different time intervals or BCG-activated peritoneal cells, it was possible to demonstrate that epithelioid cells do not produce NO . Cells from granuloma induced by BCG inoculation express NO synthase, with different degrees of reactivity with a higher intensity in the cytoplasm of cells located in the edge of the lesions . The expression of NO synthase in the cytoplasm of these cells decreases with the age of the lesions . It could also be demonstrated that in mice treated with l-name, an inhibitor of NO metabolism, the lesions induced by BCG lost the granulomatous architecture, were necrotic, and had a significant increase in the bacillary load of the lesion . These data allow us to conclude that NO production by macrophages is a determining factor in the organization of the granulomatous lesion and that it also controls the bacterial load in BCG-induced lesions in mice.

Arch Ital Urol Androl, 1998 Sep, 70(4), 177 - 82
{Granulomatous prostatitis as collateral effect of intravesical immunotherapy with BCG}; Mattei FM et al.; Twenty-five male patients with superficial bladder cancer underwent intravesical Bacillus Calmette Guerin immunotherapy . A high incidence of side effects has occurred using three different substrains of BCG . Our interest has been focused on BCG related granulomatous prostatitis: we have found four asymptomatic patients with histologically diagnosed disease . We suppose therefore that its incidence is underestimated.

Scand J Immunol, 1998 Nov, 48(5), 535 - 43
Comparison of antigen-specific T-cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis; Mustafa AS et al.; We have screened peripheral blood mononuclear cells (PBMC) from tuberculosis (TB) patients for proliferative reactivity and interferon-gamma (IFN-gamma) secretion against a panel of purified recombinant (r) and natural (n) culture filtrate (rESAT-6, nMPT59, nMPT64 and nMPB70) and somatic-derived (rGroES, rPstS, rGroEL and rDnaK) antigens of Mycobacterium tuberculosis . The responses of PBMC to these defined antigens were compared with the corresponding results obtained with complex antigens, such as whole-cell M . tuberculosis, M . tuberculosis culture filtrate (MT-CF) and cell wall antigens, as well as the vaccine strain, Mycobacterium bovis bacillus Calmette-Guerin (BCG) . In addition, M . tuberculosis and MT-CF-induced T-cell lines were tested in the same assays against the panel of purified and complex antigens . The compiled data from PBMC and T-cell lines tested for antigen-induced proliferation and IFN-gamma secretion showed that the most frequently recognized antigen was ESAT-6, followed by MPT59, GroES, MPB70, MPT64, DnaK, GroEL and PstS . The frequency of ESAT-6 responders, as measured both by proliferation (18/19) and secretion of IFN-gamma (16/19) was comparable to the results obtained with whole-cell M . tuberculosis, MT-CF and M . bovis BCG . We also observed that most of the high responders to complex antigens recognized all of the antigens tested (covariation), demonstrating that the repertoire of human T-cell specificities induced by natural infection is directed towards several unrelated culture filtrate as well as somatic-derived protein antigens . In conclusion, the results obtained suggest that the cellular immune response in humans is directed against several important target antigens of M . tuberculosis and that some antigens, such as ESAT-6, are recognized by a high number of individuals . Such antigens represent candidates to be used for development of specific diagnostic reagents or in subunit vaccines.

Carbohydr Res, 1998 Sep, 311(1-2), 95 - 9
Expeditious synthesis of a new hexasaccharide using transglycosylation reaction catalyzed by Bacillus (1-->3),(1-->4)-Beta-D-glucan 4-glucanohydrolase; Viladot JL et al.; Enzymatic hydrolysis of barley (1-->3),(1-->4)-beta-D-glucan using a recombinant (1-->3),(1-->4)-beta-glucanase from Bacillus licheniformis gives Glc beta 4Glc beta 3Glc isolated after acetylation in 49% yield . Conventional treatment produced the corresponding beta-fluoride which was carefully de-O-acetylated . A transglycosylation reaction with this substrate, catalyzed by the title enzyme, gave Glc beta 4Glc beta 3Glc beta 4Glc beta 4Glc beta 3Glc in 20% yield.

Biochemistry, 1998 Nov 17, 37(46), 16430 - 9
Nonessential activation and competitive inhibition of bacterial phosphatidylinositol-specific phospholipase C by short-chain phospholipids and analogues; Zhou C et al.; Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is an allosteric enzyme with both a phospholipid activator site and an active site . The activation of PI-PLC enzyme is optimal with phosphatidylcholine (PC) binding to the activator site and anchoring the enzyme to the interface {Zhou, C., et al . (1997) Biochemistry 36, 347-355; Zhou, C., et al . (1997) Biochemistry 36, 10089-10091} . In contrast to PC, anionic short-chain phospholipids with smaller headgroups {phosphatidylmethanol (PMe) and phosphatidic acid (PA)} as well as phosphatidylglycerol (PG) can bind to both sites playing dual roles: nonessential activation and competitive inhibition of cyclic-(1, 2)-inositol phosphate hydrolysis . PG is also a substrate, albeit a poor one, for PI-PLC, and is cleaved slowly to form alpha-glycerol phosphate . Analysis of enzyme kinetics using cIP as the substrate coupled with effects of different short-chain phospholipids on enzyme intrinsic fluorescence indicates that anionic phospholipids with small headgroups bind to the two sites with different affinities . If no interface is present, all dihexanoylphospholipids bind to the activator site more strongly than to the active site . When the activator site is occupied, it is likely that the enzyme undergoes a conformational change that allows phospholipids to bind easily to the active site . Such behavior is consistent with the observation that enzyme activation is detected at low short-chain anionic phospholipid concentrations with inhibition observed at higher concentrations, and that only inhibition is seen with these phospholipids added as monomers in the presence of a PC interface that optimally activates the PI-PLC . A kinetic model is used to extract the affinity of short-chain lipids for the active site from experimental data.

Structure, 1998 Nov 15, 6(11), 1467 - 79
The crystal structure of pyrimidine nucleoside phosphorylase in a closed conformation; Pugmire MJ et al.; BACKGROUND: Pyrimidine nucleoside phosphorylase (PYNP) catalyzes the reversible phosphorolysis of pyrimidines in the nucleotide synthesis salvage pathway . In lower organisms (e.g . Bacillus stearothermophilus) PYNP accepts both thymidine and uridine, whereas in mammalian and other higher organisms it is specific for thymidine (designated thymidine phosphorylase, TP) . PYNP shares 40% sequence similarity (and presumably significant structural similarity) with human TP, which has been implicated as a growth factor in tumor angiogenesis . It is thought that TP undergoes a major conformational change upon substrate binding that consequently produces an active conformation . RESULTS: The crystal structure of PYNP from B . stearothermophilus with the substrate analog pseudouridine in its active site has been solved to 2.1 A resolution . This structure confirms the similarity of PYNP to TP and supports the idea of a closed active conformation, which is the result of rigid body movement of the alpha and alpha/beta domains . The active-site cleft, where the pyrimidine and phosphate substrates bind, is between the two domains . The structure reveals an asymmetric dimer in which one subunit is fully closed and the other is only partially closed . CONCLUSIONS: The closed conformation of PYNP serves as a good model to better understand the domain movement and overall function of TP . Active-site residues are confirmed and a possible mechanism for substrate binding and subsequent domain movement is suggested . Potent inhibitors of TP might have significant therapeutic value in various chemotherapeutic strategies, and the structure of PYNP should provide valuable insight into the rational design of such inhibitors.

Clin Cancer Res, 1996 Jan, 2(1), 21 - 8
Newcastle disease virus-infected intact autologous tumor cell vaccine for adjuvant active specific immunotherapy of resected colorectal carcinoma; Ockert D et al.; An active specific immunization (ASI) procedure with two types of autologous tumor cell vaccines (ATVs) is tested for adjuvant immunotherapy of resected colorectal carcinoma to provide preliminary information on local immunological skin responses, side effects, and 2-year survival rates . For vaccine preparation, the tumor-derived freshly isolated and cryopreserved cells were thawed, purified by Percoll density centrifugation, and depleted of tumor-infiltrating lymphocytes by immunomagnetic beads . After inactivation by 200 Gy, the cells of this ATV were either infected by Newcastle disease virus (NDV) or they were admixed with Bacillus Calmette Guerin (BCG) organisms . Vaccination was performed in the arm beginning 6-8 weeks after operation, three times at 2-week intervals . Of 57 patients that received ASI, 48 were treated by virus-infected ATV (ATV-NDV) and 9 were treated with the BCG-admixed vaccine (ATV/BCG) . The mean value of delayed hypersensitivity skin reactions from ATV-NDV-treated patients was 18 mm for the first vaccination and 26 and 29 mm for the succeeding ones . Although the application of ATV-NDV was associated with only mild side effects, the ATV/BCG vaccine led to long-lasting ulcers and to more serious side effects . The 2-year survival rate obtained with ATV-NDV was 97.9%, whereas the survival rate with ATV/BCG was 66.7% . The mean survival of 661 patients from a historical control was 73.8% . These data suggest that the type and quality of the tumor vaccine for ASI treatment is important . The findings with ATV-NDV necessitate corroboration in a prospective, randomized controlled study.

Clin Cancer Res, 1995 Jul, 1(7), 705 - 13
Human high molecular weight-melanoma associated antigen mimicry by mouse anti-idiotypic monoclonal antibody MK2-23: modulation of the immunogenicity in patients with malignant melanoma; Mittelman A et al.; The mouse anti-idiotypic (anti-id) mAb MK2-23 bears the mirror image of the antigenic determinant defined by antihuman high molecular weight-melanoma associated antigen (HMW-MAA) mAb 763.74 . The purpose of this study was to evaluate the effect of conjugation to a carrier and administration with an adjuvant and cyclophosphamide (CTX) on the immunogenicity of anti-id mAb MK2-23 in patients with malignant melanoma and to analyze the relationship between development of humoral immunity and survival time of patients . Fifty-eight patients were sequentially entered into four immunization protocols which included administration of mAb MK2-23, mAb MK2-23 conjugated to keyhole limpet hemocyanin (KLH) and mixed with Bacillus Calmette-Guerin (BCG), mAb MK2-23 and CTX, and mAb MK2-23 conjugated to KLH and mixed with BCG and CTX . Six patients could not be evaluated since they withdrew from the clinical trial after the first immunization . Sera were tested for the development of anti-anti-id antibodies, including those reacting with HMW-MAA . Testing of sera for development of antimouse Ig antibodies was used to monitor the immune competence of patients . Conjugation to KLH and administration with BCG markedly enhanced the ability of mAb MK2-23 to induce anti-anti-id antibodies, including those reacting with HMW-MAA . In contrast, pretreatment with CTX had no detectable effect on the ability of mAb MK2-23 to elicit a humoral anti-anti-id response . Kaplan-Meier survival analysis showed that the performance status of patients, anti-anti-id antibody level, and development of anti-HMW-MAA antibodies had an effect on survival time . This effect was found when the survival time was calculated both from the day of the first immunization and from 4 weeks after the first immunization to the end of the study . A multivariate analysis by Cox regression showed that the development of anti-HMW-MAA antibodies was the most important variable for predicting survival, and that performance status was the only variable that significantly added to the prediction of survival . These data have to be interpreted with caution because of the retrospective nature of the analysis . Nevertheless, the present study suggests that mAb MK2-23 represents a useful immunogen to implement active, specific immunotherapy in patients with malignant melanoma.

J Travel Med, 1997 Jun 1, 4(2), 76 - 82
Tuberculosis Risk and Prevention in Travelers-What about BCG?
Houston S.
Tuberculosis (TB) is one of the most important health problems in many tropical and developing countries, particularly since the advent of the human immuno- deficiency virus (HIV) epidemic . The level of TB transmission is much greater in these countries than in most of western Europe or North America . For example, the annual risk of infection with Mycobacterium tuberculosis is estimated to be 300 times higher in some subSaharan African countries1 than in the Netherlands.2 Travel guidelines and advice vary widely in the emphasis placed on TB and on specific recommendations for prevention . American sources generally advise that use of the bacille Calmette-Guerin (BCG) vaccine in travelers be limited to exceptional circumstances3 while some European authorities advocate broader use.4-6 This article reviews the risk of TB in travelers and possible approaches to its prevention, including the use of BCG vaccination.

J Am Mosq Control Assoc, 1998 Sep, 14(3), 351 - 2
Bacillus sphaericus inhibits hatching of phlebotomine sand fly eggs; Robert LL et al.; The effect of Bacillus sphaericus, at various concentrations, on hatching of phlebotomine sand fly eggs was examined using laboratory bioassays . Aqueous suspensions of B . sphaericus, strain 2362, inhibited hatching of eggs of Phlebotomus duboscqi and Sergentomyia schwetzi by 95% at concentrations as low as 0.05 and 0.11 mg/cm2, respectively . In contrast, B . sphaericus did not affect the ability of pupae to emerge as adults.

J Am Mosq Control Assoc, 1998 Sep, 14(3), 298 - 304
Potency of products based on Bacillus thuringiensis var . israelensis: interlaboratory variations; Skovmand O et al.; Six quality-control laboratories in 4 countries independently bioassayed aliquots of a flowable formulation of Bacillus thuringiensis var . israelensis (B.t.i.) against the international standard powder IPS-82 . All laboratories substantially followed World Health Organization or U.S . Department of Agriculture standard protocols . Significant differences were found in resulting potency values between laboratories . Factors that may have influenced results, such as age, stage, and strain of larvae used, amount and type of food provided to larvae, and processing of samples were examined . Use of different rearing temperatures, different strains of Aedes aegypti L., or late 3rd instars vs . the recommended early 4th instars did not explain the inconsistencies . The slope of the dose-response curve of the IPS-82 powder was influenced by particle size, which varied with the nature and duration of sample homogenization . Laboratories using low-intensity processing obtained a greater slope in the dose-response curve for the flowable product than for the powder standard . The type and quantity of food provided to larvae affected susceptibility . Larvae fed an excess of protein-rich food became 4th instars in 3 days and were less susceptible to B.t.i . than those fed smaller quantities of carbohydrate-rich food that became 4th instars in 5-7 days . Overall, deviations from standard protocols with regard to larval stage, holding temperature, and lighting regime may not be as important as differences in sample processing and pretest rearing conditions . The need to improve standardization in these areas, which are not clearly specified in current protocols, is discussed.

New Microbiol, 1998 Oct, 21(4), 379 - 89
Distribution of Synechococcus sp . and Synechococcus bacillaris in the waters of the Straits of Magellan (April 1995-early austral autumn); Caruso G et al.; During the last oceanographic cruise carried out in the Straits of Magellan (April 1995), a serological approach was used in order to determine the distribution and composition of the picophytoplankton community with respect to two cyanobacteria species, Synechococcus sp . and bacillaris, characterized respectively by phycoerythrin and phycocyanin as the main accessory photosynthetic pigment . In the period examined, the Straits were characterized by generally low concentrations of total picophytoplankton (10(5)-10(6) cells/l) . The qualitative composition of the community showed the prevalence of the species Synechococcus sp . in the Pacific basin, whereas S . bacillaris appears to be predominant in the central area . The immunofluorescence method proved to be effective in the study of the diversity of these microorganisms in aquatic environments.

Zentralbl Bakteriol, 1995 Oct, 282(4), 409 - 15
Efficacy of local Bacillus Calmette-Guérin treatment in superficial bladder cancer relapsing under Keyhole-Limpet Hemocyanin immunotherapy; Jurincic-Winkler C et al.; The efficacy of local immunotherapy with Keyhole-Limpet Hemocyanin (KLH) and Bacillus Calmette-Guerin (BCG) in preventing recurrence of superficial bladder cancer (stages pTa to pT1; grades 1 to 3) was checked in 96 patients . All tumours were resected and all patients were presumed to be free of malignant disease at initiation of prophylactic KLH instillations . Before starting KLH instillations (20 mg/administration week for 6 weeks, followed by regular (bi)monthly instillations for 3 years altogether) all patients were intracutaneously immunized with 1 mg KLH . Tumour relapse under this therapeutic schedule was the indication for BCG instillations (120 mg BCG-Connaught; administration in analogy to KLH treatment) . This study has proved that (1) prophylactic KLH treatment reduced superficial bladder cancer relapse rate after surgical intervention without considerable local/systemic side effects and (2) local BCG-administration was therapeutically effective in relapsing/progressive disease under KLH treatment . There were, however, pronounced side effects.

Planta Med, 1998 Oct, 64(7), 598 - 602
(+)-rhododendrol and epi-rhododendrin suppress the NO production by activated macrophages in vivo; Fushiya S et al.; In this study, we investigated the effect of (+)-rhododendrol (1) and epi-rhododendrin (2) isolated from Acer nikoense Maxim . (Aceraceae) on nitric oxide (NO) production in mouse peritoneal macrophages elicited by bacillus Calmette-Guerin and in vitro stimulated by lipopolysaccharide . The NO production was not affected by an oral administration of methanol extract at a dose of 100 mg/kg/day . However, the AcOEt soluble fraction significantly reduced the NO production . (+)-Rhododendrol (1) isolated as an active substance from the AcOEt fraction suppressed the NO production . epi-Rhododendrin (2), the glucoside of (+)-rhododendrol (1) isolated from the n-BuOH fraction, also suppressed the NO production . As NO is one of the critical mediators in inflammation, these results suggest that (+)-rhododendrol (1) and epi-rhododendrin (2) contribute in part to the anti-inflammatory effect of A . nikoense.

Arch Pediatr, 1998 Oct, 5(10), 1103 - 6
{Meningitis due to Bacillus cereus in an infant with Reye syndrome}; Ferroni A et al.; OBSERVATION: We report the case of a 2.5-month-old infant with Bacillus cereus meningitis who was initially admitted for Reye syndrome . Gram positive bacteria was isolated in CSF and shown to be located inside the polymorphonuclears . This pathogen was further identified by sequencing of the 16S RNA . Early administration of imipenem in association with amikacin resulted in a rapid recovery . No obvious immune defect or invasive procedure could be assessed . CONCLUSION: Although Bacillus cereus is mainly associated with contamination, repeated isolations of this bacteria may be due to true infection.

Curr Microbiol, 1998 Dec, 37(6), 408 - 11
Assignment of delta-endotoxin genes of the four lepidoptera-specific Bacillus thuringiensis strains that produce spherical parasporal inclusions; Wasano N et al.; Unique strains of Bacillus thuringiensis, that belong to the four H serogroups (serovar sumiyoshiensis, serovar fukuokaensis, serovar darmstadiensis, and serovar japonensis) and produce spherical parasporal inclusions specifically toxic to lepidopteran larvae, were examined for comparative analysis of the genes encoding delta-endotoxin proteins . Gene analysis revealed that there is no difference between the four strains in nucleotide sequences of the 1, 937-bp DNA segment covering the four conserved regions and a partial sequence of the block 5 region . Surprisingly, the nucleotide sequence of the four strains showed a 100% homology with that of the corresponding region of the cry9D gene encoding a delta-endotoxin protein, which had been reported to be active on the scarabaeid coleopterans . Alignment analysis revealed that the N-terminal half (16-660) amino acid sequence of the four proteins shared relatively high homologies (27.7-35.8%) with those of the Cry9Ba, Cry9Ca, and Cry1Ba proteins, while lower homologies with those of the Cry3Aa, Cry8Ca, and Cry1Aa proteins . The results show that the cry9D gene is retained in multiple heterogeneous H serovars of Lepidoptera-specific B . thuringiensis populations naturally occurring in soil environments of Japan.

Plasmid, 1998 Nov, 40(3), 175 - 89
Characterization of a theta plasmid replicon with homology to all four large plasmids of Bacillus megaterium QM B1551; Stevenson DM et al.; A replicon from one of an array of seven indigenous compatible plasmids of Bacillus megaterium QM B1551 has been cloned and sequenced . The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551 . The cloned 2374-bp HindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3' end . Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF . The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication . The ORF product was shown to act in trans . A small region with similarity to the B . subtilis chromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins . Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream . The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%) . There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF . The data suggest that this is a theta replicon with an expressed rep gene required for replication . The replicon contains its iterons within the gene and has no homology to reported replicons . It is the first characterization of a B . megaterium replicon .

Hinyokika Kiyo, 1998 Sep, 44(9), 627 - 31
{Intravesical BCG therapy for superficial bladder cancer}; Segawa N et al.; Intravesical instillation of Tokyo 172 strain bacillus Calmette-Guerin (BCG) after transurethral resection of bladder tumor (TUR-Bt) was applied to 35 patients with superficial bladder cancer . The patients received 80 mg of BCG in 40 ml saline infused into the bladder once a week for 6 weeks . Actuarial non-recurrence rates for all 35 patients were 81.7% and 58.4% at one and two years after the BCG therapy, respectively . The recurrence of the bladder cancer after the BCG therapy was observed in 12 patients 3-27 months later . Seven patients received an additional course of BCG instillation, and 6 (86%) showed no further recurrence . Thus, the overall success rate of 2 cycles of BCG instillation was 83% (29 of 35 patients) . Moreover, in some cases intravesical BCG instillation was effective for recurrent superficial bladder cancer after intravesical instillation of anti-cancer agents and prolonged the period until recurrence . The progression rate was only 5% (2 of 35 patients) . These results suggest that intravesical BCG therapy for superficial bladder cancer helps prevent disease progression.

J Econ Entomol, 1998 Oct, 91(5), 1089 - 95
Suppression of diamondback moth (Lepidoptera: Plutellidae) with an entomopathogenic nematode (Rhabditida: Steinernematidae) and Bacillus thuringiensis Berliner; Baur ME et al.; We tested the efficacy of the All strain of Steinernema carpocapsae (Weiser) against larvae of the diamondback moth, Plutella xylostella (L.) . In laboratory bioassays we found that (1) commercially formulated nematodes produced in vitro were as effective as nematodes produced in vivo, (2) resistance of P . xylostella to Bacillus thuringiensis Berliner subsp . kurstaki did not confer cross-resistance to nematodes, (3) mortality caused by nematodes was higher for early than late 3rd-instar P . xylostella larvae, and (4) no interaction occurred when B . thuringiensis and nematodes were combined against a susceptible strain of P . xylostella, but an antagonistic interaction occurred between the 2 pathogens against a strain of P . xylostella resistant to B . thuringiensis . In field trials conducted on 2 watercress {Rorippa Nasturtium-aquaticum (L.) Hayek} farms in Hawaii, nematodes provided 41% control, B . thuringiensis subsp . aizawai gave 44% control, and the combined treatment (B . thuringiensis plus nematodes both at half rate) resulted in 58% control . Using nemodes to control diamondback moth can theoretically reduce resistance development in diamondback moth populations to B . thuringiensis products, but repeated applications of nematodes will probably be ineffective in attaining control (suggested in simulation model) . The results of this study demonstrate that nematodes may be a useful component of integrated pest management programs if efficacy can be increased, especially for populations of P . xylostella that are resistant to B . thuringiensis.

Biosci Biotechnol Biochem, 1998 Sep, 62(9), 1791 - 4
Structures of novel acidic galactooligosaccharides synthesized by Bacillus circulans beta-galactosidase; Yanahira S et al.; The structures of acidic oligosaccharides synthesized by a transglycosylation reaction by Bacillus circulans beta-galactosidase, using lactose as the galactosyl donor, and N-acetylneuraminic acid (NeuAc) and glucuronic acid (GlcUA) as the acceptors were investigated . Acidic oligosaccharides thus synthesized were purified by anion exchange chromatography and charcoal chromatography . The MS and NMR studies indicated that the acidic oligosaccharides from NeuAc were Gal beta-(1-->8)-NeuAc, Gal beta-(1-->9)-NeuAc, and Gal beta-(1-->3)-Gal beta-(1-->8)-NeuAc, and those from GlcUA were Gal beta-(1-->3)-GlcUA and Gal beta-(1-->4)-Gal beta-(1-->3)-GlcUA . These are novel acidic galactooligosaccharides.

Appl Microbiol Biotechnol, 1998 Sep, 50(3), 314 - 7
Production of cyclomaltononaose (delta-cyclodextrin) by cyclodextrin glycosyltransferases from Bacillus spp . and bacterial isolates; Larsen KL et al.; The conversion of soluble starch to cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD) and cyclomaltononaose (delta-CD) by cyclodextrin glycosyltransferases (E.C . 2.4.1.19) from Bacillus spp . and bacterial isolates was studied . The results show that delta-CD was formed by all the enzymes investigated in the range of 5%-11.5% of the total amount of alpha-, beta-, gamma-, and delta-CD produced.

Clin Diagn Lab Immunol, 1998 Nov, 5(6), 766 - 72
Use of the cell division protein FtsZ as a means of differentiating among Bartonella species; Kelly TM et al.; Genes coding for homologs of the highly conserved cell division protein FtsZ were isolated from Bartonella henselae and Bartonella quintana, the causative agents of cat scratch disease and trench fever, respectively . DNA fragments coding for the ftsZ open reading frames (ORFs) were cloned into Escherichia coli following PCR amplification with primers based on the ftsZ sequence of the closely related species Bartonella bacilliformis . The amino acid sequences predicted from the cloned B . henselae and B . quintana ftsZ ORFs are 81 to 83% identical to the corresponding protein in B . bacilliformis . Like the FtsZ protein of B . bacilliformis, the B . henselae and B . quintana homologs are about twice as large as the FtsZ proteins reported in most other organisms . Localized sequence differences within the C-terminal coding regions of the Bartonella ftsZ genes were used as the basis for species-specific identification of these organisms at both the DNA and protein levels . Oligonucleotide primers which permit the amplification of an ftsZ fragment from each of the Bartonella species without amplifying DNA from the other two species were designed . Anti-FtsZ antisera raised in rabbits against synthetic peptides corresponding to the relatively divergent C-terminal regions were shown via Western blot analysis to react only with the FtsZ protein from the cognate Bartonella species . These observations raise the possibility that the differences in ftsZ sequences can be used as the basis for diagnostic tests to differentiate among these closely related pathogens.

Int J Food Microbiol, 1998 Sep 8, 43(3), 159 - 71
Identification of contamination sources of Bacillus cereus in pasteurized milk; Lin S et al.; In order to determine the sources of Bacillus cereus in pasteurized milk, a total of 232 milk samples from various sampling points along milk processing lines and 122 environmental swabs were collected in two dairy plants between March and September, 1996 . The incidence of B . cereus vegetative cells in raw milk from the plants was low (< or = 10%) . However, the incidence and the average counts of B . cereus spores in the raw milk were very high and similar to those of B . cereus vegetative cells in pasteurized milk or final products after enrichment (> 80% and 1.1 x 10(5) cfu ml(-1), respectively) . The incidence and average count of both vegetative cells and spores of B . cereus in environmental swabs was low . Using the microbial identification system (MIDI), a library of B . cereus fatty acid profiles comprising 229 B . cereus isolates from milk samples and environmental swabs was constructed using a critical Euclidian distance of 6.0 units as the cut-off value . Using this library, the relationship between 546 B . cereus isolates from the different sampling points along the milk processing lines and the environmental swabs was determined . Most B . cereus isolates obtained from the pasteurized milk and final products belonged to the same sub-groups as the B . cereus strains germinated from spores in raw milk . Furthermore, specific sub-groups were found in pasteurized milk, different dairy plants and at different sampling times . The results suggested that B . cereus spores in raw milk were the major source of B . cereus in pasteurized milk and that post-pasteurization contamination along the milk processing lines was possibly a minor source of B . cereus in pasteurized milk.

Urology, 1998 Nov, 52(5), 785 - 9
Intravesical bacillus Calmette-Guérin treatment for Stage T1 grade 3 transitional cell carcinoma of the bladder; Baniel J et al.; OBJECTIVES: To retrospectively analyze intravesical bacillus Calmette-Guerin (BCG) treatment for Stage T1 grade 3 (T1G3) transitional cell carcinoma (TCC) of the bladder . METHODS: Between 1984 and 1995, 78 patients with Stage T1 grade 3 tumor were treated by transurethral resection of all visible tumors and adjuvant BCG intravesical instillations . Median follow-up was 56 months (range 12 to 141) . RESULTS: After an initial induction course, 52 patients (67%) were tumor-free . Twenty-two patients (28%) had recurrent tumor after a median of 7 months (range 5 to 62) . Progression occurred in 6 patients (7.7%) after a median of 18 months (range 5 to 56) . CONCLUSIONS: Intravesical BCG appears to be an effective treatment for patients with Stage T1 grade 3 TCC . Patients whose tumors recur after an initial induction course may benefit from a second course of BCG . Intravesical BCG treatment may lower the tumor progression rate . Late recurrence, beyond 2 years, warrants long-term follow-up.

Br Poult Sci, 1998 Sep, 39(4), 526 - 9
Performance of broiler chickens supplemented with Bacillus coagulans as probiotic; Cavazzoni V et al.; 1 . A newly isolated Bacillus coagulans strain as probiotic was assayed as the only dietary additive for chickens . 2 . Chickens receiving no additive at all or only virginiamycin were used for comparison . 3 . Two trials each carried out on 75 chickens showed that, in terms of efficacy in growth and food conversion ratio, the B . coagulans biomass as a probiotic had a growth-promoting, prophylactic effect comparable to that of virginiamycin.

Biotechnol Appl Biochem, 1998 Dec, 28 ( Pt 3), 235 - 42
Characterization of Bacillus sp . endo-beta-N-acetylglucosaminidase and its application to deglycosylation of hen ovomucoid; Yamamoto K et al.; A bacterial strain isolated from soil and identified as a Bacillus species produced two endo-beta-N-acetylglucosaminidases in the culture broth when it was cultivated on medium containing only hen ovomucoid . Almost no production of the enzymes occurred when the bacterium was grown on glucose medium . The two endo-beta-N-acetylglucosaminidases, named Endo-BI and Endo-BII, were separated and purified to homogeneity by preparative gel electrophoresis after partial purification by column chromatography on DEAE-resins . Endo-BI hydrolysed oligosaccharides of both hen ovalbumin and ovomucoid . In contrast, Endo-BII could act only on oligosaccharides of hen ovalbumin and showed almost no activity towards those of hen ovomucoid . Deglycosylation of hen ovomucoid was performed with the partly purified endo-beta-N-acetylglucosaminidase preparation, with the aid of contaminating beta-N-acetylhexosaminidase . The deglycosylated ovomucoid exhibited no changes in trypsin inhibitory activity but was very unstable to heat treatment in comparison with native ovomucoid . These results suggest that oligosaccharides of ovomucoid have an important role in the stabilization of the protein against heat.

Biotechnol Appl Biochem, 1998 Dec, 28 ( Pt 3), 219 - 28
Conversion of cyclodextrin into high-amylose starch of low molecular mass by means of cyclodextrin glucanotransferase; Rendleman JA Jr et al.; alpha-Cyclodextrin (CD) was converted by Bacillus macerans cyclodextrin glucanotransferase into highly insoluble high-amylose starch with a low average degree of polymerization (56-73), in yields as high as 78% over a wide range of temperatures (25-70 degreesC) . Ability to undergo this conversion was highly concentration-dependent . gamma-CD was also convertible in good yield; however, beta-CD was relatively resistant to conversion . Degrees of polymerization and the percentages of amylose in the high-amylose products were estimated from spectrophotometric measurements on their iodine complexes . A plausible mechanism for conversion of CD into starch is proposed.

Appl Environ Microbiol, 1998 Nov, 64(11), 4368 - 71
Cyt1Aa protein of bacillus thuringiensis is toxic to the cottonwood leaf beetle, chrysomela scripta, and suppresses high levels of resistance to Cry3Aa
Federici BA, Bauer LS.
The insecticidal activity of Bacillus thuringiensis is due primarily to Cry and Cyt proteins . Cry proteins are typically toxic to lepidopterous, coleopterous, or dipterous insects, whereas the known toxicity of Cyt proteins is limited to dipterans . We report here that a Cyt protein, Cyt1Aa, is also highly toxic to the cottonwood leaf beetle, Chrysomela scripta, with a median lethal concentration of 2.5 ng/mm2 of leaf surface for second-instar larvae . Additionally, we show that Cyt1Aa suppresses resistance to Cry3Aa greater than 5, 000-fold in C . scripta, a level only partially overcome by Cry1Ba due to cross-resistance . Studies of the histopathology of C . scripta larvae treated with Cyt1Aa revealed disruption and sloughing of midgut epithelial cells, indicating that its mechanism of action against C . scripta is similar to that observed in mosquito and blackfly larvae . These novel properties suggest that Cyt proteins may have an even broader spectrum of activity against insects and, owing to their different mechanism of action in comparison to Cry proteins, might be useful in managing resistance to Cry3 and possibly other Cry toxins used in microbial insecticides and transgenic plants.

Appl Environ Microbiol, 1998 Nov, 64(11), 4588 - 90
In situ detection of an uncultured predominant bacillus in Dutch grassland soils; Felske A et al.; Uncultured predominant Bacillus ribotype DA001 in Dutch Drentse A grassland soils, as revealed by its 16S rRNA sequence, was detected in soil by fluorescent whole-cell in situ hybridization . A prominent rod-shaped cell type was identified in bacterial suspensions prepared from soil by a multiple 16S rRNA probing approach.

Appl Environ Microbiol, 1998 Nov, 64(11), 4174 - 9
Variable cross-resistance to Cry11B from Bacillus thuringiensis subsp . jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to single or multiple toxins of Bacillus thuringiensis subsp . israelensis; Wirth MC et al.; A novel mosquitocidal bacterium, Bacillus thuringiensis subsp . jegathesan, and one of its toxins, Cry11B, in a recombinant B . thuringiensis strain were evaluated for cross-resistance with strains of the mosquito Culex quinquefasciatus that are resistant to single and multiple toxins of Bacillus thuringiensis subsp . israelensis . The levels of cross-resistance (resistance ratios {RR}) at concentrations which caused 95% mortality (LC95) between B . thuringiensis subsp . jegathesan and the different B . thuringiensis subsp . israelensis-resistant mosquito strains were low, ranging from 2.3 to 5.1 . However, the levels of cross-resistance to Cry11B were much higher and were directly related to the complexity of the B . thuringiensis subsp . israelensis Cry toxin mixtures used to select the resistant mosquito strains . The LC95 RR obtained with the mosquito strains were as follows: 53.1 against Cq4D, which was resistant to Cry11A; 80.7 against Cq4AB, which was resistant to Cry4A plus Cry4B; and 347 against Cq4ABD, which was resistant to Cry4A plus Cry4B plus Cry11A . Combining Cyt1A with Cry11B at a 1:3 ratio had little effect on suppressing Cry11A resistance in Cq4D but resulted in synergism factors of 4.8 and 11.2 against strains Cq4AB and Cq4ABD, respectively; this procedure eliminated cross-resistance in the former mosquito strain and reduced it markedly in the latter strain . The high levels of activity of B . thuringiensis subsp . jegathesan and B . thuringiensis subsp . israelensis, both of which contain a complex mixture of Cry and Cyt proteins, against Cry4- and Cry11-resistant mosquitoes suggest that novel bacterial strains with multiple Cry and Cyt proteins may be useful in managing resistance to bacterial insecticides in mosquito populations.

Antimicrob Agents Chemother, 1998 Nov, 42(11), 2824 - 9
Suppression of murine endotoxin response by E5531, a novel synthetic lipid A antagonist; Kobayashi S et al.; As a consequence of blood-borne bacterial sepsis, endotoxin or lipopolysaccharide (LPS) from the cell walls of gram-negative bacteria can trigger an acute inflammatory response, leading to a series of pathological events and often resulting in death . To block this inflammatory response to endotoxin, a novel lipid A analogue, E5531, was designed and synthesized as an LPS antagonist, and its biological properties were examined in vitro and in vivo . In murine peritoneal macrophages, E5531 inhibited the release of tumor necrosis factor alpha (TNF-alpha) by Escherichia coli LPS with a 50% inhibitory concentration (IC50) of 2.2 nM, while E5531 elicited no significant increases in TNF-alpha on its own . In support of a mechanism consistent with antagonism of binding to a cell surface receptor for LPS, E5531 inhibited equilibrium binding of radioiodinated LPS ({125I}2-(r-azidosalicylamido)-1, 3'-dithiopropionate-LPS) to mouse macrophages with an IC50 of 0.50 microM . E5531 inhibited LPS-induced increases in TNF-alpha in vivo when it was coinjected with LPS into C57BL/6 mice primed with Mycobacterium bovis bacillus Calmette-Guerin (BCG) . In this model, the efficacy of E5531 was inversely correlated to the LPS challenge dose, consistent with a competitive antagonist-like mechanism of action . Blockade of the inflammatory response by E5531 could further be demonstrated in other in vivo models: E5531 protected BCG-primed mice from LPS-induced lethality in a dose-dependent manner and suppressed LPS-induced hepatic injury in Propionibacterium acnes-primed or galactosamine-sensitized mice . These results argue that the novel synthetic lipid A analogue E5531 can antagonize the action of LPS in in vitro and suppress the pathological effects of LPS in vivo in mice.

Protein Eng, 1998 Sep, 11(9), 789 - 95
Nonadditivity of mutational effects on the properties of catalase I and its application to efficient directed evolution; Matsuura T et al.; Catalase I of Bacillus stearothermophilus has high catalatic and low peroxidatic activities . The mutant from the first random mutant population, D130N, which has higher peroxidatic and lower catalatic activities than those exhibited by the wild-type enzyme, was subjected to second random mutagenesis in observance of the change in reaction specificity . From the second mutant population, the mutant I108T/D130N/I222T was selected and examined . The reaction specificity of the purified enzymes revealed that catalase I being originally 98% catalase and 2% peroxidase was brought to 58% specificity to peroxidase after two-step adaptive walks . From the statistical analysis of the two random mutant populations, the average degree of nonadditivity of the mutational effects was estimated to be 0.13 irrespective of the properties of the enzyme . It was demonstrated that the distribution pattern of a property of the second mutant population can be predicted well from the data of the first mutant population by taking into consideration the degree of nonadditivity . The strategy for an efficient adaptive walk in directed evolution of enzymes through the prediction of appropriate mutation rate and effective sample size for further mutation and selection was presented and discussed.

Vaccine, 1998 Dec, 16(20), 1982 - 9
Enhancing activity of mycobacterial cell-derived adjuvants on immunogenicity of recombinant human hepatitis B virus vaccine; Koike Y et al.; In a previous study, we demonstrated that a lipophilic derivative of muramyl dipeptide {MDP-Lys(L18)} augmented antibody response to recombinant human hepatitis B surface antigen (rhHBsAg) when it was co-immunized with rhHBsAg solubilized in PBS . Here, we examined adjuvant activity of two bacterial cell-derived adjuvants such as Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) and trehalose-6,6'-dimycolate (TDM), to enhance immunogenicity of rhHBsAg, comparing their activity with that of MDP-Lys(L18) . In an animal model where mice were immunized subcutaneously (s.c.) with rhHBsAg (25 micrograms/mouse) admixed with 100 micrograms/mouse of BCG-CWS (Vac/BCG-CWS) or 50 micrograms/mouse of TDM (Vac/TDM) in o/w emulsion formulation, both mice immunized with Vac/BCG-CWS and Vac/TDM showed higher antibody titres to HB antigen than those of mice immunized with the recombinant vaccine alone . The activity of BCG-CWS and TDM to enhance antibody induction seemed to be almost the same with that of MDP-Lys(L18) . Furthermore, the enhanced antibody response raised by these adjuvants was shown to be due to high titres of HB antigen-specific IgG1 . In addition, the activity of these three adjuvants to enhance antibody response was shown to be higher than that of the present clinical vaccine, aluminium hydroxide-attached rhHBsAg (rhHBsAg-alum) . In an analysis of delayed-type hypersensitivity (DTH) reaction where mice were immunized with rhHBsAg admixed with or without each adjuvant in o/w emulsion and followed by intrafootpad (i.f.) injection of rhHBsAg 4 weeks after immunization, mice immunized with Vac/BCG-CWS and Vac/TDM as well as Vac/MDP-Lys(L18) showed a significant increment of swelling reaction . These results suggest that BCG-CWS, TDM and MDP-Lys(L18) are potential adjuvants to enhance the immunogenicity of rhHBsAg to induce humoral and cellular responses.

Vaccine, 1998 Dec, 16(20), 1923 - 8
The effect of heterologous immunity upon the apparent efficacy of (e.g . BCG) vaccines; Fine PE et al.; Exposure of populations to microbes which share antigens with pathogens can influence the apparent efficacy of vaccines . This may explain the great variation (from below 0 to 80%) observed in protection by Bacillus Calmette Guerin (BCG) against tuberculosis . This paper explores three models for the effect of such heterologous immunity, and demonstrates that: (a) if the immune responses to the microbial antigens in nature and in the vaccines differ qualitatively, there will be no effect on observed efficacy; (b) if the immune responses differ only quantitatively, the observed vaccine efficacy will be reduced, and it will be minimal when vaccine-induced and heterologous protection are of similar magnitude; and (c) if the heterologous exposure can block the vaccine action, then observed efficacy will be reduced and may even appear negative . These results provide important guidance for the interpretation of BCG's utility and for the development and evaluation of new vaccines, in particular against tuberculosis.

Biochim Biophys Acta, 1998 Oct 23, 1425(2), 300 - 10
Characterization of a novel exo-N-acetyl-beta-D-glucosaminidase from the thermotolerant Bacillus sp . NCIM 5120; Amutha B et al.; An exo-N-acetyl-beta-d-glucosaminidase from the thermotolerant Bacillus sp . NCIM 5120 was purified to homogeneity by chromatography on CM-cellulose, Sephacryl S-300 and phenyl-Sepharose . The enzyme has a Mr of 230000 as determined by size exclusion chromatography on Sephacryl S-300/Sephadex G-200 and exhibited a relative subunit Mr of 60000 on denaturing gel electrophoresis . It is a neutral protein with a pI of 6.79 . The optimum pH and temperature for the enzyme activity are 6.0 and 70 degreesC, respectively . Determination of the reaction stereochemistry indicates that the enzyme is a retaining glycosidase with the beta anomer of GlcNAc formed as the initial product . Determination of the energy of activation with different leaving groups (p-nitrophenol and 4-methyl-umbelliferone) reveals that the enzyme exhibits a biphasic Arrhenius plot with two characteristic energy of activation with an inflection temperature of 50 degreesC . The activation energy at temperatures below the inflection point was found to be higher than that above the inflection point . The energy of activation for 4-Me-Umb-beta-d-GlcNAc was higher at temperatures below the inflection point than for pNP-beta-d-GlcNAc (60.3 and 43.2 kJ mol-1, respectively) . It hydrolyzes specifically, terminally linked beta(1-4) GlcNAc residues from the non-reducing end of oligosaccharides . Comparative studies on the hydrolysis of chito-oligosaccharides by the exo-N-acetyl-beta-d-glucosaminidase indicates that chitobiose is the best substrate with a Km and kcat of 0.34 mM and 24 microoff min-1mg-1, respectively . It also exhibits strict substrate specificity with respect to the glycone substitution as well as anomeric linkage.

J Am Soc Mass Spectrom, 1998 Nov, 9(11), 1222 - 5
High-resolution electrospray ionization Fourier transform mass spectrometry with infrared multiphoton dissociation of glucokinase from Bacillus Stearothermophilus; Dufresne CP et al.; Glucokinase (GK, EC 2.7.1.2), a member of the enzyme family of hexokinases, has been shown to be linked to maturity-onset diabetes of the young type II (MODY-2) . Although nucleotide and amino acid sequence information are available for the human varieties, they are not known for the variety from Bacillus stearothermophilus, which is often used in protein binding studies . Here, a combination of electrospray Fourier transform mass spectrometry (FTMS) and infrared multiphoton dissociation (IRMPD) is used to obtain accurate molecular weight and preliminary amino acid sequence information for the protein . Electrospray FTMS provides evidence of a solution phase dimer . In addition, dithiothreitol reduction shows no shift in high-resolution isotopic distributions, indicating a probable absence of disulfide bonds in the protein . The partial sequence information obtained from IRMPD could be the basis for creating a DNA probe for the protein.

Carbohydr Res, 1998 Aug, 310(1-2), 53 - 64
Synthesis of aryl 3-O-beta-cellobiosyl-beta-D-glucopyranosides for reactivity studies of 1,3-1,4-beta-glucanases; Planas A et al.; A series of substituted aryl beta-glycosides derived from 3-O-beta-cellobiosyl-D-glucopyranose with different phenol-leaving group abilities as measured by the pKa of the free phenol group upon enzymatic hydrolysis has been synthesised . Aryl beta-glycosides with a pKa of the free phenol leaving group > 5 were prepared by phase-transfer glycosidation of the per-O-acetylated alpha-glycosyl bromide with the corresponding phenol, whereas the 2,4-dinitrophenyl beta-glycoside was obtained by condensation of 1-fluoro-2,4-dinitrobenzene with the partially acetylated trisaccharide followed by acid de-O-acetylation . The aryl beta-glycosides have been used for reactivity studies of the wild-type Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase . The Hammett plot log kcat versus pKa is biphasic with an upward curvature at low pKa values suggesting a change in transition-state structure depending on the aglycon.

Rev Argent Microbiol, 1998 Jul-Sep, 30(3), 122 - 9
Influence of pH on the toxicity and survival of total cells and spores of Bacillus thuringiensis; Dias SC et al.; B . thuringiensis (Bt) is one of the most important microorganism among those studied because of their entomopathogenic potential against insect plagues and vectors . In this work, the pathogenicity and survival of total cells and spores of three autochthonous strains of B . thuringiensis isolated from Argentine soils (A61, A27 and A68) and the commercial strain HD-1 were studied at different ion hidrogen concentrations (pH = 4.4; 5.4; 7.4; 8.4; 9.4 and 10.4) under laboratory conditions . The greatest antimicrobial effect on the number of spores and total cells was observed at pH 4.4 with a great decrease after 72 hours' growth . Comparing the survival percentage of total cells and spores; pH 5.4 was the one which allowed the higher relative tolerance (survival) among the studied strains . No decrease in pathogenicity was observed in the investigated serotypes at different pHs in bioassays against Cydia pomonella . Two soil strains of Bt (A61/A27) and the Bt (HD-1) of commercial origin caused the highest mortality of the target insect . The sotto serotype (A68), however, did not produce this effect.

J Biochem (Tokyo), 1998 Nov, 124(5), 905 - 10
Construction and properties of a fragmentary D-amino acid aminotransferase; Fuchikami Y et al.; D-Amino acid aminotransferase {EC 2.6.1.21} catalyzes the inter-conversion between various D-amino acids and alpha-keto acids . The subunit of the homodimeric enzyme from Bacillus sp . YM-1 consists of two domains connected by a single loop, which has no direct contact with the active site residues or the cofactor, pyridoxal 5'-phosphate {Sugio, S., Petsko, G.A., Manning, J.M., Soda, K., and Ringe, D . (1995) Biochemistry 34, 9661-9669} . We constructed two plasmids, one encoding a polypeptide fragment corresponding to the N-terminal domain, and the other a fragment corresponding to the C-terminal domain . When both polypeptide fragments were expressed together in the same host cell, an active fragmentary enzyme consisting of two sets of the two polypeptide fragments was produced . When the two polypeptide fragments were expressed separately, each of them provided a soluble protein but with no activity . However, D-amino acid aminotransferase activity appeared upon incubation of a mixture of the two fragments . The active fragmentary enzyme was purified to homogeneity and characterized; it was found to be similar to the wild-type enzyme in various enzymological properties except substrate specificity, inhibition by alpha-ketoglutarate, and thermostability . The fragmentary enzyme showed higher catalytic activity toward several substrates, such as D-lysine and D-arginine, than the wild-type enzyme.

AIDS, 1998 Oct 1, 12(14), 1793 - 803
Bacillary angiomatosis in immunocompromised patients; Gasquet S et al.; OBJECTIVES: To report seven cases of bacillary angiomatosis; to evaluate the most useful diagnostic tools; to analyse the clinical and epidemiological features associated with Bartonella quintana or Bartonella henselae infections . DESIGN: Clinical, diagnostic and epidemiological evaluation of 37 speciated bacillary angiomatosis cases in the literature, including the seven patients in our study . METHODS: Pathological examination of tissue samples, including Warthin-Starry staining and immunohistology; titre of antibodies to Bartonella sp.; detection of Bartonella sp . in blood and biopsy materials by culture or PCR; and statistical analysis of clinical and epidemiological features associated with B . quintana or B . henselae bacillary angiomatosis cases . RESULTS: Seven immunocompromised patients (six with AIDS and one patient with acute leukaemia) had bacillary angiomatosis confirmed by histology . B . quintana was cultured in three patients, whereas B . henselae DNA was amplified by PCR in the remaining four patients . Serum from only one patient reacted with Bartonella antigens . Amongst the 14 B . quintana and 23 B . henselae bacillary angiomatosis cases now reported in the literature, lymphadenopathies were significantly more frequent in B . henselae-infected patients, and neurological disorders of the central nervous system in B . quintana-infected patients . Risk factors were contact with cats, and homelessness or poor socioeconomic status in B . henselae and B . quintana bacillary angiomatosis cases, respectively . CONCLUSIONS: Although diagnosis of bacillary angiomatosis often remains solely based upon histology, culture or PCR-based methods are useful for the detection of Bartonella sp., and allow identification of the species involved, which is necessary to further characterize clinical and epidemiological features associated with B . quintana or B . henselae infections.

Virology, 1998 Sep 30, 249(2), 231 - 7
Mushroom bacilliform virus RNA: the initiation of translation at the 5' end of the genome and identification of the VPg; Revill PA et al.; Mushroom bacilliform virus (MBV) is often found in cultivated mushrooms (Agaricus bisporus) with La France disease . MBV has a 4-kb ssRNA genome of positive-sense encoding four major open reading frames (ORFs) . The arrangement of ORFs at the 5' end of the genome and the deduced amino-acid sequences of two of the putative gene products (protease and RNA-dependent RNA polymerase) show remarkable similarity to some plant viruses, particularly subgroup II luteoviruses . We show that this similarity extends to the translation strategy at the 5' end of the genome, the presence of a genome-linked protein (VPg), and the location of the VPg downstream of the protease motifs in the polypeptide encoded by ORF2 .

J Mol Biol, 1998 Nov 6, 283(4), 863 - 82
Conformational analysis of GpA and GpAp in aqueous solution by molecular dynamics and statistical methods; Giraldo J et al.; Barnase, an extracellular endoribonuclease from Bacillus amyloliquefaciens, hydrolyses single-stranded RNA . Its very low catalytic activity toward GpN dinucleotides, where N stands for any nucleoside, is markedly increased when a phosphate is added to the 3'-end, as in GpNp . Here we investigate the conformational properties of GpA and GpAp in solution, in order to determine whether differences in these properties may be related to the changes in enzymatic activity . Two independent 1.3 ns molecular dynamics trajectories are generated for each dinucleotide in the presence of explicit water molecules and counter ions . These trajectories are analysed by monitoring molecular properties, such as the solvent accessible surface area, the distance and orientation between the bases, the behaviour of torsion angles and formation of intramolecular H-bonds . To identify relevant correlations between these parameters, statistical techniques, comprising multiple regression, clustering and discriminant analysis are used . Results show that GpA has a significant propensity to form folded conformations (approximately 50%), fostered by a small number of intramolecular H-bonds, whereas GpAp remains essentially extended . The latter behaviour seems to be due to an H-bond between the terminal phosphate and adenosine ribose group, which restricts rotation about the adenine Agamma angle . We also find that GpA folding is induced by a concerted motion of specific torsion angles, which is closely coupled to the formation of a network of flexible hydrogen bonds . Finally, on the basis of an expression for barnase KM, which incorporates the folded/extended conformational equilibria of the dinucleotide substrates, it is argued that our findings on the differences between these equilibria, can qualitatively rationalize the experimentally measured differences in enzymatic properties .

Scand J Immunol, 1998 Oct, 48(4), 403 - 9
Human T- and B-cell reactivity to the 16kDa alpha-crystallin protein of Mycobacterium tuberculosis; Wilkinson RJ et al.; The attributes of immunodominance, predominant expression during mycobacterial dormancy and restriction to the Mycobacterium tuberculosis complex make the 16 kDa protein an important candidate for the study of the immune response in humans . We therefore investigated the relationship between T- and B-cell reactivity to the recombinant antigen and disease in a total of 127 subjects . The percentage of T-cell responders towards both the intact antigen and its permissively recognised peptide 16p91-110 was highest in healthy bacillus Calmette-Guerin (BCG)-sensitized controls (96% and 68%, respectively) and lowest in those with extensive untreated tuberculosis (26% and 18%) (P < 0.001) . By contrast, antibody levels (ABT50 > 100) were highest in patients with extensive disease (46-50%) (P < 0.005) . There was significantly higher production of IFN-gamma in the BCG-sensitized controls by comparison with untreated patients (P < 0.05), but complete antituberculous chemotherapy abolished this deficit in patients . The significance of these findings to immunodiagnosis and protective immunity is discussed.

Proc Natl Acad Sci U S A, 1998 Oct 27, 95(22), 12950 - 5
The conducting form of gramicidin A is a right-handed double-stranded double helix; Burkhart BM et al.; The linear pentadecapeptide antibiotic, gramicidin D, is a naturally occurring product of Bacillus brevis known to form ion channels in synthetic and natural membranes . The x-ray crystal structures of the right-handed double-stranded double-helical dimers (DSDH) reported here agree with 15N-NMR and CD data on the functional gramicidin D channel in lipid bilayers . These structures demonstrate single-file ion transfer through the channels . The results also indicate that previous crystal structure reports of a left-handed double-stranded double-helical dimer in complex with Cs+ and K+ salts may be in error and that our evidence points to the DSDH as the major conformer responsible for ion transport in membranes.

Comp Biochem Physiol B Biochem Mol Biol, 1998 May, 120(1), 197 - 204
Purification and partial amino acid sequences of the binding protein from Bombyx mori for CryIAa delta-endotoxin of Bacillus thuringiensis; Ihara H et al.; The binding protein for Bacillus thuringiensis delta-endotoxin, CryIAa, from the brush border membrane of the midgut of Bombyx mori was purified by the dot blot method and delta-endotoxin affinity chromatography . The binding protein was purified to 235-fold enrichment from cholic acid extracts of brush border membranes from B . mori midgut by activated CryIAa-affinity chromatography and DEAE ion-exchange chromatography . The purified binding protein showed a single band of 180 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and this band specifically reacted to 125I-labeled CryIAa on Immobilon membrane . The affinity of the binding protein for CryIAa was equivalent to that of the brush border membrane vesicles and solubilized membrane proteins . Partial amino acid sequences of the binding protein showed sequence similarity to the cadherin-like binding protein for CryIAb from Manduca sexta, but not for CryIAc binding protein from M . sexta and Heliothis virescens.

Mol Biol Evol, 1998 Oct, 15(10), 1288 - 97
Satellite sequence turnover in parthenogenetic systems: the apomictic triploid hybrid Bacillus lynceorum (Insecta, Phasmatodea); Mantovani B; In the genus Bacillus (Insecta, Phasmatodea) the Bag320 satellite DNA family is present in the bisexual B . grandii and in the related automictic nonhybrid B . atticus; it is lacking in the other bisexual taxon of the genus, B . rossius . This family of highly repeated sequences was analyzed for 11 populations of the apomictic triploid hybrid B . lynceorum . In the neighbor-joining dendrogram, B . lynceorum nucleotide sequences distribute, regardless of geographical origin, among two clusters, one also including all clones of the three B . atticus races, and the other including sequences of the B . grandii grandii subspecies . Thus, B . lynceorum is a trihybrid taxon: as the molecular approach definitively demonstrates, it embodies one haploid complement each of both B . grandii grandii and B . atticus, which must be added to that of B . rossius . The contribution of the latter species has already been assessed on karyological and allozymic grounds . A statistical analysis performed on p-distances shows that for the parental taxa, nucleotide substitution values are of comparable magnitudes at the population level but differ at the subspecific level, being higher for the bisexual taxon . In the apomictic hybrid, atticus- and grandii grandii- like sequences coexist with significantly different p-distance values . For three clones, the nucleotide compositions at the diagnostic loci suggest that gene conversion can occur between atticus- and grandii grandii-like monomers . On the whole, this supports bisexuality as a driving force in variant fixation and suggests that in Bacillus, different gametogenetic processes and different origins of the unisexuals are mirrored in genomic turnover rates of satellite DNA.

J Biol Chem, 1998 Oct 30, 273(44), 29127 - 34
Sphingomyelinase induces aggregation and fusion, but phospholipase A2 only aggregation, of low density lipoprotein (LDL) particles . Two distinct mechanisms leading to increased binding strength of LDL to human aortic proteoglycans; Oorni K et al.; During atherogenesis, low density lipoprotein (LDL) particles bind to extracellular matrix proteoglycans in the arterial wall, become modified, and appear as aggregated and fused particles . Sphingomyelinase (SMase) and phospholipase A2 (PLA2) have been found in the arterial wall, and, moreover, lesional LDL shows signs of hydrolysis of both sphingomyelin and phosphatidylcholine . We have now studied the effects of these two lipolytic modifications on the aggregation and fusion of LDL particles by hydrolyzing the particles with Bacillus cereus SMase or bee venom PLA2 . In addition, the binding strengths of the modified LDL to human aortic proteoglycans (PG) were analyzed on an affinity column . We found that SMase induced aggregation and fusion of LDL, but PLA2 induced only aggregation of the particles . In addition, the SMase-induced aggregation and fusion of LDL was promoted by pretreatment of LDL with PLA2 . Determination of the binding strengths of the hydrolyzed LDL revealed that mere lipolysis of LDL without aggregation or fusion, either by SMase or PLA2, did not affect the binding of the particles to PG . Aggregation and fusion of lipolyzed LDL particles, however, increased their strength of binding to PG . Active lysine residues in apolipoprotein B-100 (apoB-100) appear to be involved in the binding of LDL to PG, and, in fact, quantitative 13C NMR analysis revealed that, in the fused LDL particles, the number of active lysine residues per apoB-100 moiety was increased . Moreover, aggregation and fusion of LDL increased the number of apoB-100 copies and, consequently, the number of active lysine residues per aggregate or fused particle . Our present findings therefore (i) show that treatment of LDL with SMase and PLA2 generates modified LDL particles, which then bind to human aortic PG with increased strength, and (ii) suggest that SMase- and PLA2-induced aggregation and fusion of LDL are potential mechanisms leading to focal retention of extracellular lipid in the arterial wall.

J Dairy Sci, 1998 Sep, 81(9), 2341 - 5
Testing of raw milk for tetracycline residues; Nouws JF et al.; A newly improved Bacillus calidolactis tube diffusion test and two postscreening test systems--a receptor assay (Charm HVS; Charm Sciences, Inc., Malden, MA) and a newly developed Bacillus cereus ATCC 11778 mycoides test system--were evaluated for the detection and identification of tetracycline residues using 973 samples of bulk milk taken at random in The Netherlands . All milk samples were assayed with the B . calidolactis tube and the receptor test . The milk samples testing as suspect or positive with one or both of the test systems were analyzed with HPLC (limit of detection, 10 ng/ml) and the recently developed B . cereus test system . The B . calidolactis tube diffusion test detected tetracycline residues > 45 ng/ml in milk . With the B . cereus test plate, residues of oxytetracycline and tetracycline of > 30 ng/ml milk were detected; for chlortetracycline and doxycycline, the detection limit was 10 ng/ml . Raw milk exhibiting inhibition diameters of < 20 mm on the B . cereus test plate fulfilled the European Union criterion for maximum residue level for tetracyclines of < 100 ng/ml (including their 4-epimer derivatives) . The detection limits of the receptor assay depended on the type of milk used . The scintillation counts that were obtained for control samples of bulk milk were considerably lower than for the milks obtained from Charm Sciences, Inc . or processed using UHT pasteurization . One of 973 milk samples was suspect for tetracycline residues by means of the B . calidolactis tube test as well as by the receptor assay; 8 other samples were also considered to be positive using the receptor assay alone . The presence of tetracycline residues could not be proved for these 9 samples (residue concentration, < 10 ng/ml) with HPLC . We concluded that the receptor assay was not reliable to detect tetracycline residues in raw milk at < 150 ng/ml . The B . cereus test plate was determined to be an inexpensive, reliable alternative for the receptor assay for detection of tetracycline residues.

Infect Immun, 1998 Nov, 66(11), 5537 - 42
Protection by CD4 or CD8 T cells against pulmonary Mycobacterium bovis bacillus Calmette-Guérin infection; Xing Z et al.; Mice deficient in CD8 T cells demonstrated levels of Th1 cytokines and granulomatous responses in the lungs very similar to those demonstrated by normal control mice and were fully capable of controlling pulmonary mycobacterial infection by Mycobacterium bovis BCG as assessed at day 37 postinfection . In comparison, mice deficient in CD4 T cells had similar levels of interleukin-12 (IL-12) and tumor necrosis factor alpha but lower levels of gamma interferon in the lungs and were still able to mount tissue granulomatous responses and control pulmonary mycobacterial infection . In contrast, IL-12(-/-) mice with impaired CD4 and CD8 T-cell responses had a markedly weakened control of infection, whereas SCID mice deficient in all T cells succumbed to such pulmonary mycobacterial infections.

Infect Immun, 1998 Nov, 66(11), 5534 - 6
Murine model of Bartonella henselae infection in the immunocompetent host; Regnath T et al.; Bartonella henselae is an emerging pathogen causing cat scratch disease, bacillary angiomatosis, and peliosis hepatis . Progress in understanding the pathogenesis of and the immune response to these infections has been limited by the lack of an animal model . Following intraperitoneal infection of C57BL/6 mice with B . henselae, organs were cleared of cultivatable bacteria within 6 days . In contrast, B . henselae DNA could be detected in liver tissue for at least 3 months . Liver tissue showed granulomatous inflammation reaching its highest degree of intensity during the fourth week of infection and resolving within 12 weeks postinfection . This mouse model is applicable to the study of the pathogenesis of B . henselae and the immune response to this pathogen in the immunocompetent host.

Infect Immun, 1998 Nov, 66(11), 5268 - 74
Acquired resistance but not innate resistance to Mycobacterium bovis bacillus Calmette-Guérin is compromised by interleukin-12 ablation; Thompson-Snipes L et al.; Interleukin-12 (IL-12) is one of the first cytokines produced by macrophages, key mediators of innate resistance, during the host's immune response to infections . Therefore, in this study we propose that IL-12 has an important role in the early phase of the immune response to Mycobacterium bovis BCG . IL-12 has been shown to enhance the maturation of protective Th1 cells and gamma interferon (IFN-gamma) production during mycobacterial infection . Therefore, it may play a crucial role during the immune phase of infection as well . To examine the role of IL-12 in both the innate and the immune phase of infection, we compared BCG-resistant mice, B10.A (Bcgr), to the susceptible congenic strain B10.A (Bcgs) following administration of a blocking monoclonal antibody to IL-12 (10F6) . Anti-IL-12-treated susceptible animals exhibited a two- to threefold increase in spleen CFU by day 21 . In contrast, anti-IL-12 treatment had little or no effect on the response of the genetically resistant animals to infection . The B10.A (Bcgr) but not the B10.A (Bcgs) mice had an increase in IFN-gamma mRNA relative to baseline levels as early as day 1 of infection irrespective of anti-IL-12 treatment . By day 14, B10.A (Bcgr) mice showed a decrease in IFN-gamma mRNA while the B10.A (Bcgs) mice showed a significant increase in IFN-gamma mRNA levels . Thus, during BCG infection, the B10.A (Bcgr) mice mount an early IFN-gamma response against BCG whereas the B10.A (Bcgs) mice have a delayed IFN-gamma response correlating with their genetic permissiveness expressed as an increased mycobacterial load by day 21 . Overall, our data demonstrate that the inherent resistance of B10.A (Bcgr) mice to mycobacteria does not depend on optimal levels of IL-12 to maintain effective control of the bacteria, whereas IL-12 is important for the susceptible animals' response to BCG during the peak of infection.

J Invertebr Pathol, 1998 Nov, 72(3), 262 - 8
Effects of bacillus thuringiensis and destruxins (Metarhizium anisopliae mycotoxins) combinations on spruce budworm (Lepidoptera: tortricidae)
Brousseau C, Charpentier G, Belloncik S.
Laboratory bioassays were performed in order to assess the efficacy of a combination of a commercial preparation of Bacillus thuringiensis var . kurstaki (B.t.k.) (Thuricide) with destruxins (Metarhizium anisopliae mycotoxins) on the fifth instar of Choristoneura fumiferana Clemens . Lethal doses were determined for each microbial agent and used as a basis for the bioassays involving a combination of the two agents . Interaction or the lack of interaction was determined by comparing the observed and the theoretically expected mortality rates . Seven out of 10 different combinations demonstrated synergism between the two agents . The modelization applied on results allowed us to establish the following general equation: LDmixture = 1.259(LDDx) + 1.129(LDBt) - 0.016(LDDx)(LDBt) + 12.196 . Such an equation explains the relationship which exists between the two lethal agents (R2 = 0.99) . Our results suggest that the two agents contribute to the synergism in the system and that a combination of both could be an efficient means of controlling C . fumiferana populations and of reducing the dose of B.t.k . which is usually required for such a control .

J Invertebr Pathol, 1998 Nov, 72(3), 206 - 13
Expression of chromosomally inserted bacillus thuringiensis israelensis toxin genes in bacillus sphaericus
Bar E, Sandler N, Makayoto M, Keynan A.
Bacillus thuringiensis israelensis delta-endotoxin genes were inserted into transposon Tn917 in plasmid pTV51Ts and cloned into the chromosome of Bacillus sphaericus 2362 . Many of the transformants reacted with antibody to the 135-, 128-, 65-, and 28-kDa B.t.israelensis toxin proteins and were approximately 10 times more toxic to A . aegypti larvae than the untransformed host . Some of the transformants differed physiologically and morphologically from the wild-type B . sphaericus . The toxicity of the transformed phenotype was maintained through many transfers in the absence of selective pressure .

J Nat Prod, 1998 Oct, 61(10), 1313 - 4
Biotransformation of resveratrol to piceid by Bacillus cereus; Cichewicz RH et al.; Microbial transformation of resveratrol (1), trans-3,4', 5-trihydroxystilbene, was studied . Preparative scale biotransformation of 1 with whole-cell suspensions of Bacillus cereus UI 1477 resulted in the production of metabolite 2 which was identical in all respects to an authentic sample of piceid, resveratrol 3-O-beta-D-glucoside.

Science, 1998 Oct 23, 282(5389), 759 - 62
Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene; Berthet FX et al.; The virulence of the mycobacteria that cause tuberculosis depends on their ability to multiply in mammalian hosts . Disruption of the bacterial erp gene, which encodes the exported repetitive protein, impaired multiplication of M . tuberculosis and M . bovis Bacille Calmette-Guerin in cultured macrophages and mice . Reintroduction of erp into the mutants restored their ability to multiply . These results indicate that erp contributes to the virulence of M . tuberculosis.

J Urol, 1998 Nov, 160(5), 1673 - 7
A phase I/II trial of transurethral surgery combined with concurrent cisplatin, 5-fluorouracil and twice daily radiation followed by selective bladder preservation in operable patients with muscle invading bladder cancer; Zietman AL et al.; PURPOSE: We describe a protocol designed to evaluate the use of twice daily radiation used together with cisplatin and 5 fluorouracil (5-FU) in the treatment of operable transitional cell carcinoma of the bladder with potential bladder preservation . MATERIALS AND METHODS: A total of 18 consecutive patients with T2-T4a bladder tumors underwent as complete a transurethral resection as possible, which was visibly complete in 14 cases . They then received twice daily radiation and infusion cisplatin and 5-FU during an induction phase . No therapy was given for 3 weeks, following which patients were reevaluated cystoscopically . Cases of clinical complete response by biopsy and cytology were consolidated with further chemotherapy/radiation using the same chemotherapeutic agents and radiation schedule . Patients who had incomplete responses were advised to undergo an immediate radical cystectomy . Of the 18 patients 15 subsequently received 3 cycles of adjuvant chemotherapy, consisting of methotrexate, cisplatin and vinblastine . Median followup for the entire group is 32 months . RESULTS: Of the 18 patients 14 had no detectable tumor after induction therapy . Of the 4 patients with persistent tumor 2 underwent radical cystectomy and 2 refused cystectomy, 1 of whom was treated with partial cystectomy and the other with consolidation chemotherapy/radiation . The actuarial overall survival at 3 years was 83% . The chance of a patient being alive at 3 years with a native bladder was 78% . No patient required cystectomy for hematuria or bladder shrinkage . Three patients in whom superficial tumors developed were treated successfully with bacillus Calmette-Guerin . Small bowel obstruction in 1 case was corrected surgically . CONCLUSIONS: This pilot study demonstrates a high rate of response to this combined chemotherapy/radiation regimen in conjunction with a visibly complete transurethral resection . Reevaluation after a short induction phase allows for the early selection of patients with persistent disease for radical cystectomy.

J Urol, 1998 Nov, 160(5), 1668 - 71; discussion 1671-2
Results of a randomized phase III trial of sequential intravesical therapy with mitomycin C and bacillus Calmette-Guerin versus mitomycin C alone in patients with superficial bladder cancer; Witjes JA et al.; PURPOSE: We study toxicity and efficacy of sequential intravesical therapy with mitomycin C and bacillus Calmette-Guerin (BCG) in patients with intermediate or high risk superficial bladder cancer compared to the use of intravesical mitomycin C alone . MATERIALS AND METHODS: Patients with intermediate and high risk papillary superficial bladder cancer and carcinoma in situ were randomized after transurethral resection between 4 weekly instillations with 40 mg . mitomycin C followed by 6 weekly instillations with BCG (group 1, 90 patients) or 10 weekly instillations with mitomycin C (group 2, 92 patients) . RESULTS: The frequency of bacterial and chemical cystitis, and other local side effects was similar in both groups . Allergic reactions, including skin rash, were more frequent in the mitomycin C only group (12 of 92 patients versus 5 of 90, p = 0.08), and other systemic side effects were more frequent in the sequential group (16 of 90 versus 8 of 92, p = 0.07) . After a median followup of 32 months the number of recurrences (sequential 35 of 90 patients versus mitomycin C only 42 of 92, p = 0.36) and progression (5 of 90 versus 4 of 92 respectively, p = 0.70) were similar in both groups . CONCLUSIONS: We did not find any major differences in toxicity or treatment efficacy with intravesical mitomycin C and the sequential use of BCG or mitomycin C for intermediate and high risk superficial papillary bladder cancer.

Int J Tuberc Lung Dis, 1998 Oct, 2(10), 836 - 43
The cytokine response to bacille Calmette Guérin vaccination in South India; Das SD et al.; SETTING: Evaluation of immune response after BCG vaccination in a population from South India where BCG vaccination was a failure . OBJECTIVE: To study the cell-mediated immune responses (CMI) in vitro by assessing skin test conversion, lymphocyte proliferation and cytokine patterns before and after BCG vaccination in 20 Mantoux negative subjects, and to compare this with that of 20 naturally Mantoux positive subjects . DESIGN: In vitro lymphocyte proliferation and cytokine responses to various mycobacterial antigens were studied in 12 subjects from each group . The cytokines interferon gamma (IFN-gamma), interleukin (IL)-4 and IL-10 were measured by both reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) . RESULTS: All of those who were initially Mantoux negative but one subject converted to positivity following vaccination, confirming that BCG vaccination does cause skin test conversion . However, in vitro proliferative responses to phytohaemagglutinin (PHA), purified protein derivative (PPD) and Mycobacterium tuberculosis remained largely unaltered by vaccination . The production of IFN-gamma was significantly higher in PPD-positive individuals compared to the PPD-negative group, and BCG vaccination of the latter did not change the levels of IFN-gamma, IL-4 or IL-10 . CONCLUSIONS: The finding that PPD-negative individuals did not produce IFN-gamma even following vaccination and skin test conversion suggests that BCG had little effect in driving the immune response towards a protective Th1 type.

Extremophiles, 1998 Aug, 2(3), 185 - 90
Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures; Ito S et al.; The cleaning power of detergents seems to have peaked; all detergents contain similar ingredients and are based on similar detergency mechanisms . To improve detergency, modern types of heavy-duty powder detergents and automatic dishwasher detergents usually contain one or more enzymes, such as protease, amylase, cellulase, and lipase . Alkaliphilic Bacillus strains are often good sources of alkaline extracellular enzymes, the properties of which fulfil the essential requirements for enzymes to be used in detergents . We have isolated numbers of alkaliphilic Bacillus that produce such alkaline detergent enzymes, including cellulase (CMCase), protease, alpha-amylase, and debranching enzymes, and have succeeded in large-scale industrial production of some of these enzymes . Here, we describe the enzymatic properties, genetics, and structures of the detergent enzymes that we have developed.

Microbiology, 1998 Sep, 144 ( Pt 9), 2573 - 8
Construction of a single-copy integration vector and its use to study gene expression in Bacillus licheniformis; Tran LS et al.; A versatile system consisting of an integrational vector and a bacitracin (Bt)-producing beta-galactosidase (beta-Gal)-negative (Lac-) Bacillus licheniformis TLH strain was constructed to quantify promoter activity and to study gene regulation in a single-copy set-up . The vector pTLH utilizes the promoterless Escherichia coli lacZ gene derived from pQF52 and contains the pBR322 origin of replication and a kanamycin-resistance gene for selection in both B . licheniformis and E . coli . The vector also contains an inner part of the first gene of the Bt synthetase (bts) operon which enables its integration into the bts of B . licheniformis by Campbell-type recombination . This recombination event can be easily tested on a Micrococcus flavus lawn where loss of Bt production, i.e . no clearing zone on the lawn, is indicative of the proper integration . The Lac- B . licheniformis TLH strain was developed by elimination of the natural beta-Gal activity of B . licheniformis strain ATCC 10716 UM12 using NTG mutagenesis.

Kekkaku, 1998 Aug, 73(8), 525 - 9
{The coexistence of pulmonary tuberculosis and thymoma a case report}; Murakami O et al.; A 33-year-old male was admitted with complaints of cough, dysphagia, and swelling of face and upper extremities . Chest X-ray and CT scan revealed a large mediastinal mass and infiltrates in the right upper lobe . Percutaneous biopsy proved the mediastinal tumor as thymoma with cellular atypia . After irradiation, the tumor was surgically removed . Caseous epitheloid granulomas were found in the dissected mediastinal lymph nodes . AFB (Acid fast bacillus) stain of the patient's gastric fluid was positive for Mycobacterium . The coexistence of these two diseases was incidental, however, this case suggested that clinicians should perform careful evaluation of lung parenchyma as well as mediastinum on chest X-ray to identify occult diseases including pulmonary tuberculosis in patients with mediastinal mass lesion.

J Infect Dis, 1998 Nov, 178(5), 1450 - 6
Mycobacterium-mediated chemokine expression in pleural mesothelial cells: role of C-C chemokines in tuberculous pleurisy; Mohammed KA et al.; Pulmonary tuberculosis is characterized by granulomatous inflammation with an extensive infiltration of mononuclear phagocytes, but the mechanisms of phagocyte recruitment to the pleural space is unknown . In this study, pleural fluid from patients with tuberculosis contained significantly (P<.001) more biologically active MIP-1alpha and MCP-1 (C-C cytokines) than did effusions from patients with congestive heart failure . Antigenic MIP-1alpha and MCP-1 was detected by immunocytochemistry in pleural biopsy sections of patients with tuberculous pleurisy . In vitro, pleural mesothelial cells stimulated with bacille Calmette-Guerin (BCG) or interferon (IFN)-gamma produced MIP-1alpha and MCP-1 . Reverse transcription-polymerase chain reaction studies confirmed that both BCG and IFN-gamma induced MIP-1alpha and MCP-1 expression in mesothelial cells, demonstrating that mesothelial cell-derived C-C chemokines play a biologically important role in the recruitment of mononuclear cells to the pleural space.

Comp Immunol Microbiol Infect Dis, 1998 Oct, 21(4), 305 - 18
Effect of vaccination and the immunomodulators "bacillus of Calmette-Guérin, avridine and Propionibacterium acnes" on rabies in mice; Megid J et al.; Responses of vaccination and treatment to immunomodulators against rabies in mice were evaluated through macrophage inhibition factor (MIF), intra-pad inoculation (IPI) and serum neutralization (SN) tests and by the detection of gamma-interferon (IFN-gamma) . Onco-BCG, Avridine and Propionibacterium acnes were administered to groups of mice . Higher survival rates were found in animals treated with P . acnes . Lower levels of IFN-gamma were observed in the groups of infected and vaccinated mice . The IPI was not effective on detecting the response of delayed-type hypersensitivity . Vaccine induced in the infected animals a more intense response to MIF reaction.

Biochim Biophys Acta, 1998 Oct 14, 1388(1), 267 - 72
Sequence of the cry11Bb11 gene from Bacillus thuringiensis subsp . medellin and toxicity analysis of its encoded protein; Orduz S et al.; The nucleotide sequence of the cry11Bb1 gene from Bacillus thuringiensis subsp . medellin was determined . The corresponding protein has a deduced molecular mass of 88.2 kDa, and is 60.9% and 83% identical to the proteins Cry11Aa1 and Cry11Ba1, respectively . The Cry11Bb1 protein contains five repetitive blocks of 16 amino acids at the C terminal part . It is highly toxic to first instar laboratory reared Aedes aegypti, Anopheles albimanus and Culex quinquefasciatus larvae.

Diabetes Care, 1998 Oct, 21(10), 1691 - 3
Effect of bacille Calmette-Guérin vaccination on C-peptide secretion in children newly diagnosed with IDDM; Elliott JF et al.; OBJECTIVE: To determine whether administration of bacille Calmette-Guerin (BCG) vaccination to newly diagnosed IDDM patients can help preserve C-peptide secretion over the subsequent 18 months . RESEARCH DESIGN AND METHODS: Twenty-six IDDM patients, all of whom had been diagnosed within the previous year, had basal C-peptide levels >0.06 nmol/l, and had negative reactions to Mantoux's test, were randomized pairwise as they presented and were given either 0.1 ml (100 microg) BCG vaccine or 0.1 ml saline intradermally Both the patients and the investigators were blinded to the treatment . Fasting and glucagon-induced C-peptide levels and HbA1c were measured in all patients at enrollment and at 1, 3, 6, 9, 12, and 18 months after vaccination, and insulin dose was recorded at each visit . RESULTS: At enrollment, there was no significant difference in age, duration of diabetes, insulin dose, HbA1c, or fasting C-peptide levels between the BCG-vaccinated and control groups . The mean basal and stimulated C-peptide levels in the BCG-treated group did not differ significantly from those in the control group at any time during the 18 months of follow-up, and there was no difference in insulin dose or HbA1c at any time between the groups . CONCLUSIONS: BCG vaccination in children who have been recently diagnosed with IDDM does not affect the progressive decline in C-peptide levels or alter the clinical course of the disease.

Probl Tuberk, 1998, (4), 7 - 10
{Social and hygienic problems of tuberculosis in the penal executive system}; Sannikov AL; Analyzing the main indices of tuberculosis epidemiology among the convicts showed that there is 1 steady-state upward trend of its morbidity, mortality, bacillary forms . The latters are characterized by the extent of tuberculosis and its severity, which is suggestive of a large infection reservoir among the convicts . This is due to deteriorating macro- and microsocial factors, so this situation should be considered to be extremely explosive both for the convicts and for the general population of the country.

Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12625 - 30
Molecular characterization of the mycobacterial heparin-binding hemagglutinin, a mycobacterial adhesin; Menozzi FD et al.; Although it generally is accepted that the interaction of Mycobacterium tuberculosis with alveolar macrophages is a key step in the pathogenesis of tuberculosis, interactions with other cell types, especially epithelial cells, also may be important . In this study we describe the molecular characterization of a mycobacterial heparin-binding hemagglutinin (HBHA), a protein that functions as an adhesin for epithelial cells . The structural gene was cloned from M . tuberculosis and bacillus Calmette-Guerin, and the sequence was found to be identical between the two species . The calculated Mr was smaller than the observed Mr when analyzed by SDS/PAGE . This difference can be attributed to the Lys/Pro-rich repeats that occur at the C-terminal end of the protein and to a putative carbohydrate moiety . Glycosylation of HBHA appears to protect the protein from proteolytic degradation, which results in the removal of the C-terminal Lys/Pro-rich region responsible for binding of HBHA to sulfated carbohydrates . Evidence suggests that glycosylation is also important for HBHA-mediated hemagglutination and for certain immunologic properties of the protein . Finally, the absence of a signal peptide in the coding region of HBHA raises the possibility that this protein is not secreted via the general secretion pathway.

Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12289 - 94
The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis delta-endotoxin are consistent with an "umbrella-like" structure of the pore; Gazit E et al.; The aim of this study was to elucidate the mechanism of membrane insertion and the structural organization of pores formed by Bacillus thuringiensis delta-endotoxin . We determined the relative affinities for membranes of peptides corresponding to the seven helices that compose the toxin pore-forming domain, their modes of membrane interaction, their structures within membranes, and their orientations relative to the membrane normal . In addition, we used resonance energy transfer measurements of all possible combinatorial pairs of membrane-bound helices to map the network of interactions between helices in their membrane-bound state . The interaction of the helices with the bilayer membrane was also probed by a Monte Carlo simulation protocol to determine lowest-energy orientations . Our results are consistent with a situation in which helices alpha4 and alpha5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like the ribs of an umbrella (the "umbrella model") . Our results also support the suggestion that alpha7 may serve as a binding sensor to initiate the structural rearrangement of the pore-forming domain.

Clin Infect Dis, 1998 Sep, 27(3), 639 - 40
Mycobacterium bovis osteomyelitis involving a hip arthroplasty after intravesicular bacille Calmette-Guérin for bladder cancer; Guerra CE et al.; Bacille Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis, is one of the most effective agents in the treatment of superficial bladder cancer . BCG osteomyelitis is an infrequent complication of intravesicular BCG therapy; only five cases of BCG vertebral osteomyelitis have been reported in the literature . Similarly, the infection of an indwelling extravascular device by BCG is rare; there is only one previous report documenting infection associated with an automated implantable cardiac defibrillator . We report a case of BCG osteomyelitis involving a hip arthroplasty that occurred after intravesicular administration of BCG for bladder cancer, and we review the risk factors predisposing to such infections and their treatment.

Pathol Biol (Paris), 1998 Jun, 46(6), 375 - 9
{Multifocal tuberculosis . Apropos of 49 cases in the midwest region . GERICCO (Group for Epidemiology and Research in Clinical Infections of the Central West of France), 1991-1993.}; Denis-Delpierre N et al.; Diffuse or multifocal tuberculosis (TB) accounts for 9% to 10% of cases of extrapulmonary TB and carries a poor prognosis with a mortality rate of 16% to 25% . Forty-nine cases of multifocal TB defined as involvement of two extrapulmonary sites with or without pulmonary TB were reviewed . Mean patient age (+/- SD) was 50 +/- 18 years . Twenty-three per cent of patients were immigrants . A history of TB and contact with a TB patient were found in 23% and 18% of cases, respectively . Of the 52% of immunocompromised patients, 38% were HIV-positive . The skin tuberculin test was positive in 67% of cases . Mean time from symptom onset to admission was 80 +/- 77 days (median, 58 days) . The 49 patients had a total of 128 TB foci . Six patients had positive blood cultures . The tubercle bacillus was recovered from the extrapulmonary sites in 88% of cases . Mean treatment duration was nine months . Recovery from the TB was achieved in 64% of cases . The overall mortality rate was 47%, and 33% of patients died as the direct result of TB . Most deaths occurred in immunocompromised patients . A high index of suspicion for multifocal TB should be maintained in immunocompromised patients, even those who test negative for the HIV.

Acta Microbiol Immunol Hung, 1998, 45(2), 229 - 37
Effect of different carbon sources on the production of amylase by Bacillus sp . MD 124; Jana M et al.; An extracellular, thermostable salt tolerant amylase has been obtained from Bacillus sp . MD 124 which was previously isolated from municipal garbage . Among the carbon sources used for amylase production, rhamnose, starch, glucose, lactose, galactose, maltose and sucrose favoured enzyme production whereas sorbose suppressed the enzyme production . Maximum amount of enzyme (15 unit/ml culture broth) was produced after 24 h of incubation at 45 degrees C in the medium containing 0.5% starch, at pH 6.5 . The effect of temperature, pH as well as effect of metal ions on enzyme activity was also studied.

Res Microbiol, 1998 Jun, 149(6), 389 - 97
Biochemical characterization of tellurite-reducing activities of Bacillus stearothermophilus V; Moscoso H et al.; Bacillus stearothermophilus V is a naturally occurring Gram-positive rod which exhibits resistance to potassium tellurite . Crude extracts of this bacterium catalyse the NADH-dependent, protease-sensitive reduction of K2TeO3 in vitro . Two fractions which showed the ability to reduce potassium tellurite (H1 and H2) were obtained . Fraction H1 behaved as a macroaggregate exhibiting a very high molecular mass that could not be estimated accurately . Upon electrophoresis in polyacrylamide gels in the presence of SDS, however, it was resolved into three distinct bands of 60, 41 and 37.5 kDa . On the other hand, an M(r) of 121 was determined for fraction H2 by means of gel filtration and high-pressure liquid chromatography . In SDS-PAGE a unique protein band of 60 kDa was observed, suggesting that it is actually a dimer . Both fractions showed pH and temperature optima of 7.5 and 57 degrees C, respectively . Concentrations of 2.5 M NaCl or 0.35 mM SDS inhibited fraction H2 almost completely, while fraction H1 retained 20% of its activity under the same conditions . Concentrations of 5 mM EDTA caused the activity of both fractions to increase 2-fold . In addition to reducing tellurite, they were also able to reduce Na2SeO3 and Na2SO3 in vitro.

Carbohydr Res, 1998 Jul, 309(4), 311 - 8
Mechanism of action and the substrate-dependent pH maximum shift of the alpha-amylase of Bacillus coagulans; Keating L et al.; The alpha-amylase of Bacillus coagulans is a saccharifying alpha-amylase which hydrolyses the disaccharide maltose {L . Keating, C . Kelly, and W . Fogarty, Biochem . Soc . Trans., 24 (1996) 44S} . The pH maximum for maltose hydrolysis is pH 5.0, differing from the pH maximum for starch hydrolysis which is pH 6.0 . Studies using reducing end 14C-labeled maltooligosaccharides revealed a substrate-dependent pH maximum shift; hydrolysis of radiolabeled maltotriose (G3*) was maximal at pH 5.0 while the pH maximum for hydrolysis of radiolabeled maltopentaose (G5*) and maltohexaose (G6*) was pH 6.0 . With maltotetraose (G4*) however, the pH maximum was pH 5.0-6.0 . In addition, the bond cleavage pattern of G4* was dependent on pH . At pH 5.0, the pH maximum for maltose hydrolysis, the frequency of hydrolysis of the reducing end terminal bond of G4* was maximal . Determination of the pH maximum of the productive binding modes of the cleavage patterns of G3* to G6* illustrated the possible role of the occupation of subsite r + 2 in the pH control mechanism of B . coagulans alpha-amylase.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 448 - 50
Crystallization and preliminary X-ray analysis of thiaminase I from Bacillus thiaminolyticus: space group change upon freezing of crystals; Campobasso N et al.; Thiaminase I (Mr = 42 100) from B . thiaminolyticus, expressed in E . coli, has been crystallized by the vapor-diffusion method . Three crystal forms, two of which grew from 0.1 M sodium acetate (pH = 4.6), 0.2 M ammonium sulfate and 30%(w/v) PEG 2000, have been examined by X-ray analysis . One crystal form diffracted to 2.5 A at room temperature, was orthorhombic, and had unit-cell edges of a = 87.7, b = 120.5 and c = 76.7 A with space group P212121 . A self-Patterson map showed a strong peak indicating noncrystallographic translational pseudosymmetry with (u, v, w) = (0.03, 0.0, 0.5) . When these crystals were frozen at liquid-nitrogen temperatures, a second crystal form was observed which had unit-cell dimensions a = 85.5, b = 117.5 and c = 36.6 A with space group P21212 . A third crystal form grew from 0.1 M Tris (pH = 8.5), 0.2 M sodium acetate trihydrate and 28%(w/v) PEG 6000 to produce orthorhombic crystals of space group P212121 with cell edges of a = 114.4, b = 123.1 and c = 92.5 A.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 446 - 7
Purification, crystallization and preliminary X-ray diffraction studies on the thermostable catechol 2,3-dioxygenase of Bacillus stearothermophilus expressed in Escherichia coli; Chen MQ et al.; The thermostable catechol 2,3-dioxygenase of Bacillus stearothermophilus has been crystallized . The crystal is probably in the space group I222 with unit-cell dimensions of a = 70.87, b = 74.60 and c = 133.69 A . A native data set has been collected with a completeness of 96% at 2.22 A resolution and an Rmerge value of 0.091.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 416 - 8
Preliminary X-ray crystallographic analysis of a novel maltogenic amylase from Bacillus stearothermophilus ET1; Cho MJ et al.; A novel maltogenic amylase from Bacillus stearothermophilus ET1, which has a dual activity of alpha-1,4- and alpha-1,6-glycosidic bond cleavages and alpha-1,6-glycosidic bond formation, was crystallized by using the hanging-drop vapor-diffusion method . The best crystals were obtained by employing a high concentration of protein (56 mg ml-1) and a precipitant containing 22% glycerol, 1.6 M ammonium sulfate in 0.1 M Tris-HCl (pH 8.5) . Native diffraction data to 2.66 A resolution have been obtained from crystals flash-frozen at 110 K . The crystals belong to the space group P212121 with unit-cell dimensions of a = 77.62, b = 121.23, c = 244 . 29 A, and contain three or four protomers per asymmetric unit . Structure determination by multiple isomorphous replacement is in progress.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 409 - 12
Crystallization and preliminary high-resolution X-ray diffraction analysis of native and beta-mercaptoethanol-inhibited urease from Bacillus pasteurii; Benini S et al.; Hexagonal crystals of urease from Bacillus pasteurii have been obtained by vapour diffusion at 293 K in 20 mM Tris-HCl, neutral pH, containing 50 mM Na2SO3 . Isomorphous crystals of urease inhibited with beta-mercaptoethanol were also obtained by including 4 mM of the inhibitor in the enzyme solution . Crystals of the native and inhibited enzyme diffract respectively to 2.00 A (96.7% completeness) and to 1.65 A (98.7% completeness) using synchrotron X-ray cryogenic (100 K) conditions . The space group is P6322 for both forms, and the unit-cell parameters are a = b = 131.36, c = 189 . 76 A for native urease and a = b = 131.34, c = 190.01 A for inhibited urease . Under the same conditions, single crystals of B . pasteurii urease inhibited with acetohydroxamic acid, cisteamine, and phenylphosphorodiamidate were also obtained.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 403 - 4
Crystallization and preliminary X-ray investigation of the complex of RNase Sa with wild-type barstar; Urbanikova L et al.; RNase Sa, an extracellular ribonuclease produced by Streptomyces aureofaciens, is inhibited by barstar, the natural protein inhibitor of barnase, the ribonuclease of Bacillus amyloliquefaciens . The complex of RNase Sa with wild-type barstar was crystallized by hanging-drop vapour diffusion . It was shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis that RNase Sa and barstar are present in equimolar proportions in the crystals . The crystals are in the hexagonal space group P65 with unit cell dimensions a = b = 56.95, c = 135.8 A . They diffract to 1.7 A resolution at the DESY synchronton source . The asymmetric unit contains one molecule of the complex.

Acta Crystallogr D Biol Crystallogr, 1998 May 1, 54 ( Pt 3), 313 - 23
1.85 A resolution structure of the zinc (II) beta-lactamase from Bacillus cereus; Carfi A et al.; Class B beta-lactamases are wide spectrum enzymes which require bivalent metal ions for activity . The structure of the class B zinc-ion-dependent beta-lactamase from Bacillus cereus (BCII) has been refined at 1.85 A resolution using data collected on cryocooled crystals (100 K) . The enzyme from B . cereus has a molecular mass of 24 946 Da and is folded into a beta-sandwich structure with helices on the external faces . The active site is located in a groove running between the two beta-sheets {Carfi et al . (1995) . EMBO J . 14, 4914-4921} . The 100 K high-resolution BCII structure shows one fully and one partially occupied zinc sites . The zinc ion in the fully occupied site (the catalytic zinc) is coordinated by three histidines and one water molecule . The second zinc ion is at 3.7 A from the first one and is coordinated by one histidine, one cysteine, one aspartate and one unknown molecule (most likely a carbonate ion) . In the B . cereus zinc beta-lactamase the affinity for the second metal-ion is low at the pH of crystallization (Kd = 25 mM, 293 K; {Baldwin et al . (1978) . Biochem . J . 175, 441-447} and the dissociation constant of the second zinc ion was thus apparently decreased at the cryogenic temperature . In addition, the structure of the apo enzyme was determined at 2.5 A resolution . The removal of the zinc ion by chelating agents causes small changes in the active-site environment.

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 45 - 57
X-ray structure of the ZnII beta-lactamase from Bacteroides fragilis in an orthorhombic crystal form; Carfi A et al.; beta-Lactamases are extracellular or periplasmic bacterial enzymes which confer resistance to beta-lactam antibiotics . On the basis of their catalytic mechanisms, they can be divided into two major groups: active-site serine enzymes (classes A, C and D) and the ZnII enzymes (class B) . The first crystal structure of a class B enzyme, the metallo-beta-lactamase from Bacillus cereus, has been solved at 2.5 A resolution {Carfi, Pares, Duee, Galleni, Duez, Frere & Dideberg (1995) . EMBO J . 14, 4914-4921} . Recently, the crystal structure of the metallo-beta-lactamase from Bacteroides fragilis has been determined in a tetragonal space group {Concha, Rasmussen, Bush & Herzberg (1996) . Structure, 4, 823-836} . The structure of the metallo-beta-lactamase from B . fragilis in an orthorhombic crystal form at 2.0 A resolution is reported here . The final crystallographic R is 0.196 for all the 32501 observed reflections in the range 10-2.0 A . The refined model includes 458 residues, 437 water molecules, four zinc and two sodium ions . These structures are discussed with reference to Zn binding and activity . A catalytic mechanism is proposed which is coherent with metallo-beta-lactamases being active with either one Zn ion (as in Aeromonas hydrophila) or two Zn ions (as in B . fragilis) bound to the protein.

Biochem Mol Biol Int, 1998 Sep, 45(6), 1265 - 71
A new PCR-based approach to a fast search of a wide spectrum of cry genes from Bacillus thuringiensis strains; Shevelev AB et al.; A pair of highly degenerated primers was adapted to carry out a single-step PCR-detection of any known and probably unknown cry genes of classes cry1, cry4 and cry9 encoding for 130 kDa protein delta-endotoxins in the natural Bacillus thuringiensis (BT) strains . The Southern hybridization of the product has demonstrated that essentially remote cry-genes like cry1Aa and cry9A (cryIG) could be represented in the single amplificate if they are simultaneously present in the genome of the analyzed strain . Four genes were detected by the proposed scheme in the BT ssp . galleriae 11-67 . One of them, gene cry1Ga1 was originally found and cloned using the PCR-amplification product obtained from the genomic DNA of this strain as a probe . The new gene was completely identical to one cloned by B . Lambert (unpublished, EMBL accession number Z22510) and essentially related to cryIM (EMBL accession number Y09326), renamed according to the new nomenclature as cry1Ga2.

Biochem Mol Biol Int, 1998 Sep, 45(6), 1243 - 54
Glutathione metabolism and glutathione S-conjugate export ATPase (MRP1/GS-X pump) activity in cancer . II . Cell-to-cell variability, relation with cellular activation state and functional absence of GS-X pump in lymphocytes; de Bittencourt Junior PI et al.; A severe complication in late-stage cancer patients is host immunosuppression . It is suggested that overproduction of the highly cytostatic and cytotoxic antiproliferative cyclopentenone prostaglandins (CP-PGs) within the plasma of cancer-bearing subjects may contribute to immunosuppression . Lymphoid tissues of Walker 256 tumor-bearing rats accumulate large amounts of CP-PGs while the tumor tissue itself does not . Moreover, tumor cells may present differential sensitivity to CP-PGs due to the expression of the multidrug resistance-associated protein (MRP1) gene product which shows a Mg(2+)-dependent vanadate-sensitive glutathione S-conjugate export ATPase (GS-X pump) activity that extrudes CP-PGs from cells as glutathione S-conjugates . In this study, the possibility that deficient GS-X pump activity in immune cells that may be involved in the accumulation of CP-PGs is investigated . Rat lymph node lymphocytes do not exhibit any notable activity even when mitogen-stimulated . Conversely, although rat peritoneal resident (quiescent) or thioglycollate-stimulated (inflammatory) macrophages exhibit low GS-X pump activity, Bacillus Calmette-Guerin (BCG)-activated macrophages show a notable rise in the activity of the ATPase, suggesting that the cellular activation state may modulate GS-X pump activity/expression and that, under appropriate stimuli (e.g., during immune response) macrophages may provide a self-defense against electrophilic CP-PGs by forming GS-conjugates that can be extruded from cells through the GS-X pump . ras oncogene expression may be linked with MRP1/GS-X pump expression/activity, since C2C12 promyoblasts transformed by v-H-ras transfection doubled GS-X pump activity . These results support the proposition that the accumulation of CP-PGs and the immunosuppression of tumor-bearing subjects may be attributed to a lack of GS-X pump activity/expression in lymphocytes.

Anal Biochem, 1978 Nov, 91(1), 158 - 65
The enzymic synthesis of beta-{32P}UDP-N-acetylglucosamine; Heptinstall J et al.; Cells of Micrococcus sp . 2102 incorporate inorganic {32P}phosphate from the medium into the sugar-phosphate polymer of the wall . Controlled acid hydrolysis of sodium dodecyl sulphate-extracted cells gives N-acetylglucosamine 6-{32P}-phosphate which can be purified by ion-exchange chromatography and incubated with UTP in the presence of crude preparations of phosphoacetylglucosamine mutase from Neurospora crassa and UTP:N-acetylglucosamine 1-phosphate phosphotransferase from Bacillus licheniformis which act in concert to synthesise beta-{32P}UDP-N-acetylglucosamine.

Int J Food Microbiol, 1998 Aug 18, 43(1-2), 115 - 22
Microbial populations associated with commercially produced South African sorghum beer as determined by conventional and Petrifilm plating; Pattison TL et al.; Microbial populations of 46 commercially produced sorghum beer samples from retail outlets in Johannesburg, South Africa, were enumerated and characterized . Aerobic plate counts, lactic acid bacteria counts and yeast counts were performed by conventional and Petrifilm plating . Conventional methods yielded yeast counts of 7.84 log CFU/ml, lactic acid bacteria counts of 6.44 log CFU/ml and aerobic plate counts of 5.96 log CFU/ml . In comparison, Petrifilm counts were 7.85 log CFU/ml for yeasts, 5.31 log CFU/ml for lactic acid bacteria and 5.34 log CFU/ml for aerobic bacteria . Characterization of 419 predominant bacterial isolates from Standard One Nutrient Agar, MRS Agar and corresponding Petrifilm plates yielded 88.0% lactic acid bacteria, 8.4% Bacillus species, 2.9% Micrococcus species and 0.7% Gram negative bacteria . Composition of predominant lactic acid bacteria populations from Standard One Nutrient Agar and both types of Petrifilm plates showed marginal differences . Increased proportions of heterofermentative lactic acid bacteria were, however, isolated from conventional MRS Agar compared to the modified Petrifilm product which represented the equivalent to MRS Agar.

Chin J Biotechnol, 1998, 14(1), 45 - 51
Batch fermentation and optimization of media for Bacillus thuringiensis; Guan X et al.; The composition of No . II medium obtained with shaking cultivation contained three factors: nitrogen source, carbon source, and inorganic salts . The relationship between component factors (x(i)) of the media and spore numbers (y) of Bacillus thuringiensis (Bt) was demonstrated by the orthogonal-rotation-combination test . A response surface equation was formed as follows: y = 384 - 7.245x1 + 11.705x2 + 15.475x3 + 14.039x1(2) + 41.831x2(2) - 79.49x3(2) - 35.375x1x2 - 3.375x1x3 - 106.625x2x3 . The results showed that this method is simple, practical, and rapid enough for selecting fermentation media for Bt . In addition, the whole course of batch fermentation was also investigated.

Chin J Biotechnol, 1998, 14(1), 25 - 9
Molecular cloning of a chitinase gene from Bacillus circulans C-2; Zeng H et al.; The 2-10 kb DNA fragments of the PstI partially digested total DNA of Bacillus circulans C-2 were cloned into the PstI site of vector pUC19 and the resulting hybrid DNA molecules were then transformed into Escherichia coli . One chitinase gene-containing clone (named pCHT1) was selected from about 8000 recombinants on chitin overlay plates . Analysis of pCHT1 cut with 12 restriction enzymes showed that the inserted fragment in this clone was about 3.0 kb in size and contained one site for each of the three restriction enzymes: KpnI, SacI, and SspI . Cells harboring the recombinants plasmid (pCHT1-R) in which the insert was in an inverted orientation also displayed chitinase activity, indicating that the cloned fragment from B . circulans C-2 contained an intact chitinase gene and its own promoter could be recognized by the Escherichia coli transcriptional system . Southern hybridization analysis proved that the inserted fragment of pCHT1 was really from the genome of B . circulans C-2, and there was only one copy existing in the genome . This fragment could not hybridize with the total DNAs from the other seven chitinase-producing bacteria.

Appl Environ Microbiol, 1998 Oct, 64(10), 4060 - 1
Activities of Bacillus thuringiensis insecticidal crystal proteins Cyt1Aa and Cyt2Aa against three species of sheep blowfly; Chilcott CN et al.; The toxicity of Bacillus thuringiensis Cyt1Aa protein to sheep blowfly larvae depends on its solubilization and proteolytic activation . Cyt1Aa crystals were not toxic . Full-length and trypsin-digested Cyt1Aa proteins were toxic to larvae of three species of sheep blowfly . Neither full-length nor trypsin-digested Cyt2A soluble crystal proteins were toxic.

Appl Environ Microbiol, 1998 Oct, 64(10), 3932 - 8
Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence; Park HW et al.; The insecticidal activity of Bacillus thuringiensis strains toxic to coleopterous insects is due to Cry3 proteins assembled into small rectangular crystals . Toxin synthesis in these strains is dependent primarily upon a promoter that is active in the stationary phase and a STAB-SD sequence that stabilizes the cry3 transcript-ribosome complex . Here we show that significantly higher yields of Cry3A can be obtained by using dual sporulation-dependent cyt1Aa promoters to drive the expression of cry3Aa when the STAB-SD sequence is included in the construct . The Cry3A yield per unit of culture medium obtained with this expression system was 12.7-fold greater than that produced by DSM 2803, the wild-type strain of B . thuringiensis from which Cry3Aa was originally described, and 1.4-fold greater than that produced by NB176, a mutant of the same strain containing two or three copies of cry3Aa, which is the active ingredient of the commercial product Novodor, used for control of beetle pests . The toxicities of Cry3A produced with this construct or the wild-type strain were similar when assayed against larvae of the cottonwood leaf beetle, Chrysomela scripta . The volume of Cry3A crystals produced with cyt1Aa promoters and the STAB-SD sequence was 1.3-fold that of typical bipyramidal Cry1 crystals toxic to lepidopterous insects . The dual-promoter/STAB-SD system offers an additional method for potentially improving the efficacy of insecticides based on B . thuringiensis.

Appl Environ Microbiol, 1998 Oct, 64(10), 3910 - 6
The introduction into bacillus sphaericus of the Bacillus thuringiensis subsp . medellin Cyt1Ab1 gene results in higher susceptibility of resistant mosquito larva populations to B . sphaericus; Thiery I et al.; The fragment containing the gene encoding the cytolytic Cyt1Ab1 protein from Bacillus thuringiensis subsp . medellin and its flanking sequences (I . Thiery, A . Delecluse, M . C . Tamayo, and S . Orduz, Appl . Environ . Microbiol . 63:468-473, 1997) was introduced into Bacillus sphaericus toxic strains 2362, 2297, and Iab872 by electroporation with the shuttle vector pMK3 . Only small amounts of the protein were produced in recombinant strains 2362 and Iab872 . The protein was detected in these strains only by Western blotting and immunodetection with antibody raised against Cyt1Ab1 protein . Large amounts of Cyt1Ab1 protein were produced in B . sphaericus recombinant strain 2297, and there was an additional crystal, other than that of the binary toxin, within the exosporium . The production of the Cyt1Ab1 protein in addition to the binary toxin did not increase the larvicidal activity of the B . sphaericus recombinant strain against susceptible mosquito populations of Culex pipiens or Aedes aegypti . However, it partially restored (10 to 20 times) susceptibility of the resistant mosquito populations of C . pipiens (SPHAE) and Culex quinquefasciatus (GeoR) to the binary toxin . The Cyt1Ab1 protein produced in recombinant B . thuringiensis SPL407(pcyt1Ab1) was synthesized in two types of crystal-one round and with various dense areas, surrounded by an envelope, and the other a regular cuboid crystal, very similar to that found in the B . sphaericus recombinant strain.

Appl Environ Microbiol, 1998 Oct, 64(10), 3765 - 8
Xanthan lyase of Bacillus sp . strain GL1 liberates pyruvylated mannose from xanthan side chains; Hashimoto W et al.; When the bacterium Bacillus sp . strain GL1 was grown in a medium containing xanthan as the carbon source, the viscosity of the medium decreased in association with growth, showing that the bacterium had xanthan-depolymerizing enzymes . One of the xanthan-depolymerizing enzymes (xanthan lyase) was present in the medium and was found to be induced by xanthan . The xanthan lyase purified from the culture fluid was a monomer with a molecular mass of 75 kDa, and was most active at pH 5.5 and 50 degrees C . The enzyme was highly specific for xanthan and produced pyruvylated mannose . The result indicates that the enzyme cleaved the linkage between the terminal pyruvylated mannosyl and glucuronyl residues in the side chain of xanthan.

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 954 - 63
Recognition of RNase Sa by the inhibitor barstar: structure of the complex at 1.7 A resolution; Sevcik J et al.; We report the 1.7 A resolution structure of RNase Sa complexed with the polypeptide inhibitor barstar . The crystals are in the hexagonal space group P65 with unit-cell dimensions a = b = 56.9, c = 135.8 A and the asymmetric unit contains one molecule of the complex . RNase Sa is an extracellular microbial ribonuclease produced by Streptomyces aureofaciens . Barstar is the natural inhibitor of barnase, the ribonuclease of Bacillus amyloliquefaciens . It inhibits RNase Sa and barnase in a similar manner by steric blocking of the active site . The structure of RNase Sa is very similar to that observed in crystals of the native enzyme and its complexes with nucleotides . Barstar retains the structure found in its complex with barnase . The accessible surface area of protein buried in the complex is about 300 A2 smaller and there are fewer hydrogen bonds in the enzyme-inhibitor interface in RNase Sa-barstar than in barnase-barstar, providing an explanation of the reduced binding affinity in the former . Previous studies of barstar complexes have used mutants of the inhibitor and this is the first structure which includes wild-type barstar.

Int J Epidemiol, 1998 Aug, 27(4), 713 - 21
Delayed-type hypersensitivity to Mycobacterium leprae soluble antigens as a test for infection with the leprosy bacillus; Sterne JA et al.; BACKGROUND: Mycobacterium leprae (M . leprae) soluble antigen (MLSA) reagents have been developed with the aim of finding a reagent, comparable to tuberculin, which could identify individuals infected with the leprosy bacillus . They have yet to be evaluated fully in human populations . METHODS: More than 15000 individuals living in a leprosy endemic area of northern Malawi were skin tested with one of five batches of MLSA prepared using two different protocols . The main difference in preparation was the introduction of a high G centrifugation step in the preparation of the last three ('second-generation') batches . RESULTS: The prevalence of skin-test positivity (delayed-type hypersensitivity (DTH)) and association with the presence of a BCG scar were greater for first (batches A6, A22) than second (batches AB53, CD5, CD19) generation reagents . The association of positivity with M . leprae infection was investigated by comparing results among known (household) contacts of leprosy cases, and among newly diagnosed leprosy patients with those in the general population . While positivity to 'first-generation' antigens appeared to be associated with M . leprae infection, positivity to later antigens was unrelated either to exposure to leprosy cases or presence of leprosy disease . There were geographical differences in the prevalence of DTH to the various batches, probably reflecting exposure to various mycobacteria in the environment . CONCLUSIONS: Our results suggest that the 'second-generation' batches have lost antigens that can detect M . leprae infections, but that they retain one or more antigens which are shared between M . leprae and environmental mycobacteria . Natural exposure to these both sensitizes individuals and provides natural protection against M . leprae infection or disease . Identification of antigens present in these groups of skin test reagents may assist in production of improved skin test reagents.

J Photochem Photobiol B, 1998 Jul 10, 44(2), 151 - 8
Enhancement of tumour response to photodynamic therapy by adjuvant mycobacterium cell-wall treatment; Korbelik M et al.; Mycobacterium cell-wall extract (MCWE) is a potent non-specific immunostimulant that elicits a local inflammatory response associated with antitumour activity . Tumour-localized administration of MCWE has been examined as an adjuvant to photodynamic therapy (PDT) mediated by the photosensitizers Photofrin, benzoporphyrin derivative monoacid (BPD), metatetrahydroxyphenylchlorin (mTHPC), or zinc (II)-phthalocyanine (ZnPc) . A single MCWE treatment, given immediately after light treatment of murine EMT6 tumours, potentiates the curative effect of PDT . A similar enhancement of tumour response to Photofrin-based PDT is obtained with the live Bacillus Calmette-Guerin (BCG) vaccine . Despite differences in the kinetics/intensity of damage induction to tumour microvasculature and other characteristics underlying the mechanism of antitumour activity of Photofrin, BPD, mTHPC and ZnPc, there appear to be no marked differences in the therapeutic benefit of adjuvant MCWE therapy combined with the PDT mediated by these various photosensitizers . This may be related to the fact that MCWE elicits a wide range of immunomodulatory effects that could amplify and sustain the inflammatory/immune responses triggered by PDT . The enhancement of inflammatory effector cell activity is indicated by the increased infiltration of neutrophils and other myeloid cells at the expense of malignant cells found in the MCWE plus mTHPC-based PDT treatment group compared to the PDT-only group.

Science, 1998 Oct 2, 282(5386), 121 - 5
An antimicrobial activity of cytolytic T cells mediated by granulysin; Stenger S et al.; Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism . Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro . Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M . tuberculosis . The ability of CTLs to kill intracellular M . tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

Biotechnol Appl Biochem, 1998 Oct, 28 ( Pt 2), 145 - 54
Immobilization of alpha-amylase on poly(vinyl alcohol)-coated perfluoropolymer supports for use in enzyme reactors; Yang Y et al.; The suitability and potential for the use of poly(vinyl alcohol) (PVA)-coated solid perfluoropolymers in immobilized-enzyme engineering have been evaluated by using alpha-amylase from Bacillus licheniformis for the hydrolysis of starch . alpha-Amylase was covalently immobilized on PVA-coated poly(tetrafluoroethylene-hexafluoropropylene) (PVA-FEP) by covalent coupling with the use of p-beta-sulphate-(ethyl sulphonide)-aniline, 2,4,6-trichloro-1,3,5-triazine, 1.1'-carbonyldi-imidazole and 2,2, 2-trifluoroethanesulphonyl chloride activation procedures, and also for comparison with cyanogen bromide-activated Sepharose 4B . In all cases, immobilization greatly improved the thermostability of the alpha-amylase and its resistance to inactivation by 6 M urea . Also the enhancements of enzymic activities with increased temperature were higher for the immobilized enzymes than for the soluble enzyme, and the immobilized alpha-amylases were well suited to the continuous hydrolysis of starch conducted at elevated temperatures . Although the specific activities of the enzymes immobilized on PVA-FEP were lower than for that immobilized to Sepharose 4B, these novel supports showed far superior strength . The enzymes immobilized on PVA-FEP were able to be readily recovered from stirred batch bioreactors for repeated reuse, whereas the enzymes immobilized to Sepharose were fractured and fragmented under similar conditions of stirring . A conventional fixed-bed bioreactor was found to be unsuitable for continuous starch hydrolysis owing to an unacceptable build-up of pressure drop across the bed . However, an expanded bed reactor containing alpha-amylase immobilized on solid PVA-coated perfluorocarbon showed great potential for the continuous hydrolysis of starch . Only 20% of the enzyme activity was lost after use for 3 weeks at 72 degrees C . It is concluded that PVA-coated solid perfluorocarbon is a highly promising support for use in immobilized enzyme engineering.

Biotechnol Appl Biochem, 1998 Oct, 28 ( Pt 2), 95 - 8
Development of Bacillus thuringiensis fermentation and process control from a practical perspective; Yang XM et al.; Bacillus thuringiensis (Bt) is the most widely used biopesticide producer in the biological control market . It is very critical for the Bt pesticide industry to be able to achieve a high yield in the Bt fermentation process in order to reduce its cost and compete with chemical pesticides in the market . We review the overall development of Bt fermentation process research and provide our point of view for the future research opportunities and potential improvements . This minireview covers the areas of fermentation physiology, growth dynamics and high-yield process control . It is pointed out that many studies aimed to improve spore count and process research focusing on toxin protein yield is lacking . In addition, significant development opportunities reside in the process development for the genetically engineered Bt strains expressing multiple toxin proteins.

Int J Immunopharmacol, 1998 Jul, 20(7), 359 - 68
Stimulation of macrophages by Bacillus firmus: production of nitric oxide and cytokines; Zidek Z et al.; Immunostimulatory properties of gram-positive Bacillus firmus were investigated under in vitro conditions using murine peritoneal macrophages . B . firmus stimulated in a concentration and time dependent manner the secretion of tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10), but it had no influence upon interferon-gamma (IFN-gamma) and interleukin-2 (IL-2) production . It also substantially augmented production of nitric oxide (NO) induced by exogenous IFN-gamma . Inhibitory experiments using neutralizing antibodies against TNF-alpha and/or IL-10 have demonstrated that these cytokines are responsible for triggering the underlying mechanism(s) leading to enhanced NO production . The cytokine-stimulatory and NO-costimulatory properties could participate in the antiinfectious and anticancer effects of B . firmus, detected previously in the in vivo experiments.

Plant J, 1998 Aug, 15(4), 553 - 61
Co- and/or post-translational modifications are critical for TCH4 XET activity; Campbell P et al.; TCH4 encodes a xyloglucan endotransglycosylase (XET) of Arabidopsis thaliana . XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall . Therefore, XET function may affect cell shape and plant morphogenesis . To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity . Recombinant baculoviruses were designed to produce distinct forms of TCH4 . TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co- and post-translational modifications is virtually inactive . TCH4 proteins, with and without a polyhistidine tag, that harbor an intact N-terminus are directed to the secretory pathway . Thus, as predicted, the N-terminal region of TCH4 functions as a signal peptide . TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS-PAGE in the presence of dithiothreitol (DTT) . This disulfide bond(s) is essential for full XET activity . TCH4 is glycosylated in vivo; glycosidases that remove N-linked glycosylation eliminated 98% of the XET activity . Thus, co- and/or post-translational modifications are critical for optimal TCH4 XET activity . Furthermore, using site-specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL motif (E97) is essential for activity . A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization . These data support the hypothesis that, in analogy to Bacillus beta-glucanases, this region may be the active site of XET enzymes.

Biochemistry, 1998 Sep 29, 37(39), 13446 - 52
The 0.78 A structure of a serine protease: Bacillus lentus subtilisin; Kuhn P et al.; Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B . lentus subtilisin crystals has been collected to a resolution of 0.78 A . The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution . Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain . Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated . The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement . Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp) . Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad . The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond . This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution . The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad . While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.

Prog Nucleic Acid Res Mol Biol, 1998, 61, 23 - 64
Regulation of cytochrome P450 gene transcription by phenobarbital; Kemper B; The ability of phenobarbital to induce levels of drug metabolism in mammals has been known for over 40 years . However, the molecular mechanisms underlying increased expression of the genes of the key enzyme in drug metabolism, cytochrome P450, have not been elucidated, primarily because in vitro model systems in which the induction could be studied were not available . Transfected primary cultured hepatocytes, transfection of liver in situ, and transgenic mice now provide suitable models for phenobarbital induction . In this review, progress toward understanding the mechanism of phenobarbital induction of gene expression is discussed with an emphasis on the mammalian genes, CYP2B1, CYP2B2, and Cyp2b10, which are most highly inducible by phenobarbital . Barbiturate induction of P450s in Bacillus megaterium, which is the system best understood, and its relevance to mammalian mechanisms of induction are also discussed . In B . megaterium, the binding of a repressor to several motifs is reversed by direct effects of barbiturates and by induction of positively acting factors . One of the repressor binding sites, the barbie box, is present in many mammalian phenobarbital-inducible genes, including the promimal promoter regions of CYP2B1, CYP2B2, and Cyp2B10 . In the mammalian P450 genes, evidence has been proposed for phenobarbital-regulated elements both in the proximal promoter region and in a distal enhancer region . The role of the proximal region is controversial . A positively acting element that overlaps the barbie box sequence and a negative element have been proposed to mediate induction of CYP2B1/2, based primarily on protein binding and cell-free transcription assays . In contrast, other investigators have not found differences in phenobarbital-dependent protein binding in the proximal promoter region nor mediation of phenobarbital induction by this region . A distal gene fragment, at about -2000 kb in CYP2B1, CYP2B2, and Cyp2b10, has been shown to be a phenobarbital-responsive enhancer independent of proximal promoter elements . This fragment contains several binding sites for proteins and several functional elements, including an NF-1 site, and, therefore, has been designated as a phenobarbital-responsive unit . Possible models are presented in which phenobarbital treatment induces altered chromatin structure, which allows the binding of positively acting factors, or activates factors already bound, to the distal enhancer and the proximal promoter.

Lett Appl Microbiol, 1998 Sep, 27(3), 168 - 72
A RAPD-PCR method for large-scale typing of Bacillus cereus; Nilsson J et al.; A robust RAPD-PCR procedure for large-scale typing of Bacillus cereus was developed . It is based on a simple DNA preparation, involving only freezing and boiling of cells in water with active carbon . By using a computerized system for data collection and processing, an efficient system for handling RAPD patterns for large-scale investigations was achieved . The procedure was highly discriminatory for Bacillus cereus strains and was found to give reproducible classification of RAPD fingerprints for five reference strains.

J Appl Microbiol, 1998 Aug, 85(2), 211 - 8
Bactericidal activity of carvacrol towards the food-borne pathogen Bacillus cereus; Ultee A et al.; Carvacrol, a natural plant constituent occurring in oregano and thyme, was investigated for its bactericidal effect towards the food-borne pathogen Bacillus cereus . Carvacrol showed a dose-related growth inhibition of B . cereus . At concentration of 0.75 mmol l-1 and above, total inhibition of the growth was observed . Below this concentration, carvacrol extended the lag-phase, reduced the specific growth rate and reduced the maximum population density . Incubation for 40 min in the presence of 0.75-3 mmol l-1 carvacrol decreased the number of viable cells of B . cereus exponentially . Spores were found to be approximately 2-3 fold less sensitive to carvacrol than vegetative cells . Bacillus cereus cells showed reduced susceptibility towards carvacrol at pH 7.0 compared with different values between pH 4.5 and 8.5 . The culture and exposure temperatures had a significant influence on the survival of vegetative cells . The highest death rate of cells was observed at an exposure temperature of 30 degrees C . Membrane fluidity was found to be an important factor influencing the bactericidal activity of carvacrol.

Mol Cells, 1998 Aug 31, 8(4), 466 - 70
Cloning and characterization of plastid ribosomal protein S16 gene from potato (Solanum tuberosum L . cv Désirée); Bae JM et al.; The plastid ribosomal protein s16 (rps16) gene was cloned from potato (Solanum tuberosum L . ssp . tuberosum cv Desiree) by PCR amplification to obtain a new homologous recombination site of plastid transformation . The potato rps16 genomic clone was 1627 bp in size and the coding region was interrupted by an 859 bp intron . Exon I was 40 bp, encoding 13 amino acids and exon II was 227 bp, encoding a 76 amino acid polypeptide . The nucleotide sequence of the rps16 gene from the "Desiree" potato shared perfect identity with the sequence from the "Superior" potato in the coding region . Three nucleotide substitutions, two nucleotide insertions, and one nucleotide deletion were found between the intron sequence of both "Desiree" and "Superior" cultivars . The amino acid sequence of the potato rps16 gene showed a high level of identity with rice, maize, tobacco, and mustard (84-94%) and a relatively low level compared with Bacillus stearothermophilus and E . coli (27-28%) . Expression of the rps16 gene was strong in chloroplasts and transcripts were detectable in amyloplasts, suggesting that the rps16 gene is active in nonphotosynthetic plastids as well as in photosynthetic plastids . These results indicate that the potato rps16 gene can be used as a new homologous recombination site of plastid transformation for potato cultivars.

Genetika, 1998 Jul, 34(7), 992 - 5
{Variability of pericentromeric chromatin in chromosome 2 of ovarian nurse cells during inbreeding in Anopheles atroparvus V.Tiel}; Burlak VA et al.; We investigated the variability of pericentromeric chromatin of chromosome 2 in ovarian nurse cells (trophocytes) in two laboratory lines of malaria mosquito Anopheles atroparvus V . Tiel and in their hybrids . One line had been raised by means of sib inbreeding, the other kept at constantly high population density . The inbreeding was shown to result in an increased percentage of chromosomes bearing an achromatinic zone in the centromeric region, which resulted in chromosome breakage . Toxicological tests demonstrated an increase in the sensitivity of the progeny of females with abnormal morphotypes of chromosome 2 to the entomopathogenic bacterium Bacillus thuringiensis israelensis . The appearance of the achromatinic zone is attributed to local chromatin underreplication accompanying chromosome polytenization . Possible reasons for this phenomenon and its implication for adaptation are discussed.

Cancer Radiother, 1998 Apr, 2 Suppl 1, 19s - 26s
{Treatment of superficial tumors of the bladder}; Patard JJ et al.; Superficial bladder cancer includes T1, Ta, TIS transitional cell carcinomas of the TNM classification . These tumors represent a spectrum disease with different biological behavior . Some tumors may recur on the same mode for years, others have a definite metastatic risk in the short range . The main difficulty in the management of these tumors is to predict their potential aggressiveness based on clinicopathological parameters . Their management is based on the initial endoscopy and histopathological analysis . Molecular markers will reach the clinical level in order to better predict the prognosis and improved the non invasive tools for follow-up . For low risk tumors, transurethral resection (TUR) and surveillance allow an adequate tumor control . For high risk tumors, conservative treatment is based on a complete transurethral resection which need confirmation by a second look TUR and prophylactic endovesical instillations of bacille Calmette Guerin (BCG) . The response to the first BCG cycle represent a crucial indicator of tumor behavior . For intermediate risk lesions, the advantage of prophylactic endovesical instillations have been finally established either using BCG or chemotherapy (Mitomycine C) . However in this setting, various therapeutic modalities (maintenance therapy, dose, repeat cycles) are proposed and their relative efficacy remain to be established . For this later group of tumor it is more likely that the use of maintenance therapy or repeated courses increase the chance of conservative treatment . Other therapeutic modalities exist (oral interferon inductors, photochemotherapy) but remain second line or investigative therapies . In conclusion, progress in the understanding of the natural history of bladder cancer allow a better management of these tumor in order to optimize the ratio between the oncologic efficacy and quality of life.

J Bacteriol, 1998 Oct, 180(19), 5077 - 84
Structure and mechanism of action of the protease that degrades small, acid-soluble spore proteins during germination of spores of Bacillus species; Nessi C et al.; The germination protease (GPR) of Bacillus megaterium initiates the degradation of small, acid-soluble proteins during spore germination . Trypsin treatment of the 46-kDa GPR zymogen (termed P46) removes an approximately 15-kDa C-terminal domain generating a 30-kDa species (P30) which is stable against further digestion . While P30 is not active, it does autoprocess to a smaller form by cleavage of the same bond cleaved in conversion of P46 to the active 41-kDa form of GPR (P41) . Trypsin treatment of P41 cleaves the same bond in the C-terminal part of the protein as is cleaved in the P46-->P30 conversion . While the approximately 29-kDa species generated by trypsin treatment of P41 is active, it is rapidly degraded further by trypsin to small inactive fragments . These results, as well as a thermal melting temperature for P41 which is 13 degreesC lower than that for P46 and the unfolding of P41 at significantly lower concentrations of guanidine hydrochloride than for P46, are further evidence for a difference in tertiary structure between P46 and P41, with P46 presumably having a more compact stable structure . However, circular dichroism spectroscopy revealed no significant difference in the secondary structure content of P46 and P41 . The removal of approximately 30% of P46 or P41 without significant loss in enzyme activity localized GPR's catalytic residues to the N-terminal two-thirds of the molecule . This finding, as well as comparison of the amino acid sequences of GPR from three different species, analysis of several site-directed GPR mutants, determination of the metal ion content of purified GPR, and lack of inhibition of P41 by a number of protease inhibitors, suggests that GPR is not a member of a previously described class of protease.

Clin Sci (Lond), 1998 Oct, 95(4), 397 - 407
Importance of glutamine metabolism in murine macrophages and human monocytes to L-arginine biosynthesis and rates of nitrite or urea production; Murphy C et al.; 1.The intermediates of biochemical cycles are commonly utilized for biosynthetic processes; thus at least one intermediate must be replenished de novo to provide constant flux through the cycle . The utilization of L-arginine for NO synthesis in macrophages may thus reduce the concentration of intermediates of the urea cycle . It is possible that a glutamine-utilizing pathway exists in mononuclear phagocytes that may connect with the urea cycle.2.In this paper we report that mouse peritoneal resident and Bacillus Calmette-Guerin (BCG)-activated macrophages and human monocytes are capable of utilizing glutamine at high rates, contain sufficient activity of the enzymes required to convert glutamine to citrulline (and subsequently citrulline to arginine) to account for observed rates of nitrite synthesis in the absence of extracellular L-arginine, and will release nitrite when exposed to intermediates of the proposed glutamine-->arginine pathway.3.The rate of nitrite production (in the absence of extracellular arginine) was reduced by culturing macrophages or monocytes in the presence of the glutaminase inhibitor 6-diazo 5-oxo norleucine.4.The rate and extent of arginase secretion, glutamine utilization, nitrite production (basal and lipopolysaccharide-stimulated) and phosphate-dependent glutaminase activity from BCG-activated macrophages was increased compared with resident cells.5.We suggest that the elevated arginase secretion rates in activated macrophages would effectively increase the intracellular concentration of arginine available for conversion to NO via inducible nitric oxide synthase, the expression of which is known to increase on activation of macrophages or monocytes . Additionally, the rate of L-arginine biosynthesis from glutamine may be increased on immunostimulation of the macrophage.

Plant Mol Biol, 1998 Oct, 38(3), 347 - 56
The sugarcane bacilliform badnavirus promoter is active in both monocots and dicots; Tzafrir I et al.; Regions of the sugarcane bacilliform badnavirus genome were tested for promoter activity . The genomic region spanning nucleotides 5999-7420 was shown to possess promoter activity as exemplified by its ability to drive the expression of the coding region of the uidA gene of Escherichia coli, in both Avena sativa and Arabidopsis thaliana . In A . sativa, the promoter was active in all organs examined and, with the exception of the anthers where the expression was localized, this activity was constitutive . In A . thaliana, the promoter activity was constitutive in the rosette leaf, stem, stamen, and root and limited primarily to vascular tissue in the sepal and the silique . The transgene was inherited and active in progeny plants of both A . sativa and A . thaliana.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1994 Nov, 27(4), 196 - 205
{Screening and rapid identification of Bacillus thuringiensis mutants}; Su YC et al.; Mutants of Bacillus thuringiensis subsp . kurstaki NTU 9 and Bt 158, which were isolated previously for using the diamondback moth as a target insect in Taiwan, were screening by either protein electrophoresis of intracellular proteins or enzyme-linked immunosorbent assay (ELISA) . The optimal conditions of effective protein electrophoresis were (1) 24-hour cells harvested from nutrient broth were crashed by petite glass beads followed by centrifugation . And (2) the supernatant pretreated by heating at 60 degrees C for 2 minutes was electrophoresed with 7.5% native PAGE at 110 voltages . On ELISA, the antiserum used was obtained from rabbits immunized with Bt 158 crystal protein . Optimal antigen coating concentration of ELISA, attained by chequer-board titration method, was 10 micrograms/ml . Antigens (crystal protein) in samples were detected by competitive inhibition method with antiserum diluted to 10(4) fold . By using protein electrophoresis and ELISA methods, two isolates A 71 and BN 11, were denoted respectively as qualitative and quantitative mutants of Bacillus thuringiensis.

Infect Immun, 1998 Oct, 66(10), 4895 - 902
Surface structure, hydrophobicity, phagocytosis, and adherence to matrix proteins of Bacillus cereus cells with and without the crystalline surface protein layer; Kotiranta A et al.; Nonopsonic phagocytosis of Bacillus cereus by human polymorphonuclear leukocytes (PMNs) with particular attention to bacterial surface properties and structure was studied . Two reference strains (ATCC 14579(T) and ATCC 4342) and two clinical isolates (OH599 and OH600) from periodontal and endodontic infections were assessed for adherence to matrix proteins, such as type I collagen, fibronectin, laminin, and fibrinogen . One-day-old cultures of strains OH599 and OH600 were readily ingested by PMNs in the absence of opsonins, while cells from 6-day-old cultures were resistant . Both young and old cultures of the reference strains of B . cereus were resistant to PMN ingestion . Preincubation of PMNs with the phagocytosis-resistant strains of B . cereus did not affect the phagocytosis of the sensitive strain . Negatively stained cells of OH599 and OH600 studied by electron microscopy had a crystalline protein layer on the cell surface . In thin-sectioned cells of older cultures (3 to 6 days old), the S-layer was observed to peel off from the cells . No S-layer was detected on the reference strains . Extraction of cells with detergent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major 97-kDa protein from the strains OH599 and OH600 but only a weak 97-kDa band from the reference strain ATCC 4342 . One-day-old cultures of the clinical strains (hydrophobicity, 5.9 to 6.0%) showed strong binding to type I collagen, laminin, and fibronectin . In contrast, reference strains (hydrophobicity, -1.0 to 4.2%) as well as 6-day-old cultures of clinical strains (hydrophobicity, 19.0 to 53.0%) bound in only low numbers to the proteins . Gold-labelled biotinylated fibronectin was localized on the S-layer on the cell surface as well as on fragments of S-layer peeling off the cells of a 6-day-old culture of B . cereus OH599 . Lactose, fibronectin, laminin, and antibodies against the S-protein reduced binding to laminin but not to fibronectin . Heating the cells at 84 degreesC totally abolished binding to both proteins . Benzamidine, a noncompetitive serine protease inhibitor, strongly inhibited binding to fibronectin whereas binding to laminin was increased . Overall, the results indicate that changes in the surface structure, evidently involving the S-layer, during growth of the clinical strains of B . cereus cause a shift from susceptibility to PMN ingestion and strong binding to matrix and basement membrane proteins . Furthermore, it seems that binding to laminin is mediated by the S-protein while binding to fibronectin is dependent on active protease evidently attached to the S-layer.

CA Cancer J Clin, 1998 Sep-Oct, 48(5), 263 - 8
Bladder cancer: twenty years of progress and the challenges that remain; Lamm DL; The most obvious improvements in the care of patients with bladder cancer over the past 20 years are bacille Calmette-Guerin immunotherapy for superficial bladder cancer and cisplatin and methotrexate-based combination chemotherapy for advanced disease . The challenges that remain are prevention, early detection, and improved treatment of both superficial and advanced disease . Meeting these challenges requires research, financial investment, and public education.

Can J Microbiol, 1998 May, 44(5), 465 - 70
Inactivation of enzymes within spores of Bacillus megaterium ATCC 19213 by hydroperoxides; Palop A et al.; The organic hydroperoxides t-butyl hydroperoxide, cumene hydroperoxide, and peracetic acid were found to act similarly to hydrogen peroxide in causing inactivation of enzymes within intact spores of bacillus megaterium ATCC 19213 concomitant with mortality . Spores treated with lethal levels of the agents were germinated and permeabilized for enzyme assays . The hierarchy of sensitivities among enolase, glucose-6-phosphate dehydrogenase (G6Pdh), and pyruvate kinase to inactivation varied somewhat with the specific hydroperoxide used, possibly because of the differences in the types of radicals generated . However, each agent inactivated each of the enzymes, albeit at different rates . Comparative assessments of enzyme inactivation by lethal levels of H2O2 or by moist heat showed that some enzymes, such as G6Pdh, are highly sensitive to inactivation, while others, such as ATPases, are much more resistant . The enzymes G6Pdh and aldolase were highly sensitive to hydroperoxide inactivation and also to moist heat, while pyruvate kinase was much more sensitive to hydroperoxides than to moist heat . Our overall interpretation of the findings is that hydroperoxides and moist heat can produce cumulative damage to sensitive enzymes within spores, which progressively diminishes the capacities of the cells to undergo the outgrowth required for return to vegetative life.

Syst Appl Microbiol, 1998 Mar, 21(1), 162 - 70
Serotype H5a5b is a major clone within mosquito-pathogenic strains of Bacillus sphaericus; Zahner V et al.; Seventy six mosquito pathogenic strains of Bacillus sphaericus and 10 non-pathogens were examined by pulsed field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA . Non-pathogenic strains were clearly distinguished from the entomopathogenic types which were assigned to 21 groups (SmaI restriction patterns; SRPs) . Some agreement between SRP based on PFGE and serotyping was noted, in particular all 39 strains of serotype 5a5b examined revealed identical SRPs indicating total conservation of the SmaI restriction site in these bacteria . Serotype 5a5b (SRP 12) strains comprise a widely distributed and abundant clonal lineage . Most serotypes, however, were divided into several SRPs . Seven strains from serotype 2a2b were covered in five SRPs in which toxin synthesis was correlated with chromosomal structure . Similarly, toxicity correlated with SRP in strains from serotypes 3 and 6.

Syst Appl Microbiol, 1998 Mar, 21(1), 144 - 50
A novel isolate of Bacillus thuringiensis serovar leesis that specifically exhibits larvicidal activity against the moth-fly, Telmatoscopus albipunctatus; Higuchi K et al.; A soil isolate designated 88-KO-14-45, belonging to Bacillus thuringiensis serovar leesis (H33), exhibited larvicidal activity against the moth-fly, Telmatoscopus albipunctatus (Diptera: Psychodidae), but not for larvae of the culicine and aedine mosquitoes and Lepidoptera . Purified parasporal inclusions had an LC50 value of 5.78 micrograms/ml for the larval moth-fly, but gave no mortality against larvae of Culex pipiens molestus (Diptera: Culicidae) at protein concentrations up to 10 mg/ml . Electron microscopic observations revealed that the parasporal inclusions are homogeneous round-shaped bodies enclosed with thick, electron dense envelopes . Haemolytic activity against sheep erythrocytes was not detected in the solubilized inclusions . SDS-PAGE showed that the inclusions are composed of 72, 68, 56 and 30 kDa proteins . Immunologically, these proteins were unrelated to the inclusion proteins of B . thuringiensis serovar israelensis, while a 70 kDa protein of the strain 73-E-10-2 (B . thuringiensis serovar darmstadiensis) was seroactive to antibodies against proteins of 88-KO-14-45.

Syst Appl Microbiol, 1998 Mar, 21(1), 97 - 106
Environmental distribution and diversity of Bacillus thuringiensis in Spain; Iriarte J et al.; Bacillus thuringiensis was isolated from 301 out of 1,005 samples collected in Spain from agricultural and non-cultivated soils, dust from stored products, and dead insects . Based on the production of parasporal crystals, 1,401 isolates were identified as B . thuringiensis after examining 11,982 B . thuringiensis-like colonies . We found a greater presence of B . thuringiensis in dust from grain storages than in other habitats . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the spore-crystal mixtures revealed diverse populations of B . thuringiensis which were differentiated in at least 92 distinct protein profiles . Serological identification also showed great diversity among the Spanish isolates which were distributed among 38 of the 58 known serovars . The most frequently found serovars were aizawai, kurstaki, konkukian, morrisoni, and thuringiensis, which together represented more than 50% of the serotyped isolates . In preliminary toxicity assays, a number of isolates were found to show significant insecticidal activity against the lepidopterans Heliothis armigera (76.1% of the assayed isolates), Spodoptera exigua (50.5%), and Plutella xylostella (19.7%) . Thirty five isolates were toxic to both H . armigera and S . exigua, and eight were toxic to S . exigua and P . xylostella . Four and one isolates were toxic to the coleopterans Leptinotarsa decemlineata and Colaspidema atrum, respectively, and three to the dipteran Tipula oleracea . The electrophoretic pattern and serovar of most of the isolates with toxic activity were consistent with those reported in the literature, although other isolates revealed unusual protein profiles, were assigned to new H serovars, or were included in H serovars not previously reported within such pathotypes.

Biochem Mol Biol Int, 1998 Aug, 45(5), 1011 - 20
Biochemical characterization of the third domain from Bacillus thuringiensis Cry1A toxins; Vazquez-Padron RI et al.; Cry proteins from Bacillus thuringiensis have insecticidal properties . The function of domains I and II has been described but domain III has so far eluded understanding . Domain III from Cry1Ab and Cry1Ac has been cloned, expressed in E . coli and injected to rabbits with the aid of characterizing them immunologically . Interestingly, polyclonal antibodies against Cry1Ab fragment did not recognize either the native Cry1Ab toxin or the Cry1Ac fragment while those against the latter did recognize either the native Cry1Ac toxin or the Cry1Ab protein fragment . A combination of information from sequence comparison and hydrophobicity profile indicates that these protein fragments possibly adopt different spatial dispositions within the respective toxins.

FEBS Lett, 1998 Aug 14, 433(1-2), 41 - 3
C-terminal domain of beta-1,3-glucanase H in Bacillus circulans IAM1165 has a role in binding to insoluble beta-1,3-glucan; Yamamoto M et al.; The deduced amino acid sequences of 72-kDa beta-1,3-glucanase from Bacillus circulans WL-12 (GIcA) and 91-kDa enzyme from B . circulans IAM1165 (BglH) are highly homologous, except that the latter has an additional long C-terminal region composed of 192 amino acid residues . Two mutant enzymes (BgIH deprived of the C-terminal region and GIcA with the C-terminal region added) were constructed . The enzymes possessing the C-terminal region bound more abundantly to pachyman (insoluble beta-1,3-glucan) and A.spergillus oryzae cell wall than those not possessing the region . This indicates that the C-terminal region participated in binding of the enzymes to insoluble beta-1,3-glucan.

Eur J Biochem, 1998 Aug 1, 255(3), 710 - 7
Substrate binding to a cyclodextrin glycosyltransferase and mutations increasing the gamma-cyclodextrin production; Parsiegla G et al.; Bacterial cyclodextrin glycosyltransferases use starch to produce cyclic maltooligosaccharides (cyclodextrins) which are of interest in various applications . The cyclization reaction gives rise to a spectrum of ring sizes consisting of predominantly six to eight glucosyl units . Using the enzyme from Bacillus circulans strain no . 8, binding studies have been performed with several substrates and analogues . The observed binding modes differ in detail, but agree in general with data on homologous enzymes . Based on these binding studies, two mutations were designed that changed the production spectrum from the predominant product beta-cyclodextrin of the wild-type enzyme towards gamma-cyclodextrin, which is of practical interest because it is rare and can encapsulate larger nonpolar compounds.

J Clin Oncol, 1998 Sep, 16(9), 2913 - 20
Correlation of specific immune responses with survival in melanoma patients with distant metastases receiving polyvalent melanoma cell vaccine; Hsueh EC et al.; PURPOSE: The mechanisms that underlie the clinical efficacy of melanoma vaccines are not well understood . We hypothesized that the type and strength of the immune response generated by CancerVax (John Wayne Cancer Institute, Santa Monica, CA), a polyvalent melanoma cell vaccine (PMCV), might be correlated with its effect on overall survival (OS) . PATIENTS AND METHODS: Seventy-seven patients began PMCV therapy after complete surgical resection of distant metastatic melanoma . During the first two treatments, PMCV was administered with bacille Calmette-Guerin (BCG) . Blood was drawn at 0, 2, 4, 8, and 12 weeks to measure serum titers of immunoglobulin G (IgG) and IgM antibodies against a tumor-associated 90-kd glycoprotein antigen (TA90) expressed on most melanoma cells, including those of PMCV . Cellular immune response to PMCV was assessed by delayed-type hypersensitivity (DTH) . General immune competence was assessed by skin tests to purified protein derivative (PPD), mumps, and candida . RESULTS: Median follow-up time was 31.5 months . Within the first 12 weeks of PMCV immunotherapy, there was a significant increase in the anti-TA90 IgM (P=.0001) and IgG titers (P=.0001), and in the DTH response to PMCV (P=.0001) . Univariate analysis showed that high anti-TA90 IgM titer and strong PMCV-DTH were associated with improved survival (P=.051 and .0173, respectively), whereas elevated anti-TA90 IgG was correlated with decreased survival (P=.0119) . Multivariate analysis considering clinical variables and PMCV immune responses identified anti-TA90 IgM, anti-TA90 IgG, and PMCV-DTH as significant independent variables influencing survival following PMCV immunotherapy (P=.0342, .0105, and .0082, respectively) . These responses to PMCV were not correlated with immune responses to BCG and therefore were not a manifestation of general immune competence for responses to unrelated antigens . The median survival time and 5-year survival rate were more than 76 months and 75%, respectively, if both anti-TA90 IgM and PMCV-DTH responses were strong (> or = 800 and > or = 7 mm, respectively; n=29); 32 months and 36%, respectively, if only one response was strong (n=35); and 19 months and 8%, respectively, if neither was strong (n=13) (P < .0001) . CONCLUSION: PMCV induces both humoral and cell-mediated immune responses to melanoma-associated tumor antigens, the type and strength of which appear to be directly related to its therapeutic efficacy.

J Gastroenterol Hepatol, 1998 Aug, 13(8), 833 - 9
Hepatobiliary tuberculosis; Alvarez SZ; Tuberculosis is known to involve the liver in different ways . The term hepatobiliary tuberculosis refers to the localized form of hepatic tuberculosis as a distinct clinical entity, with signs and symptoms related to the hepatobiliary tract . Its clinical features and the different diagnostic aids used in its diagnosis are reviewed . Plain abdominal radiographs showing diffuse hepatic calcifications seen in approximately 50% of cases are almost diagnostic for hepatobiliary tuberculosis . Liver biopsies obtained either by ultrasound, computed tomography or laparoscopy, showing caseating granuloma usually establish the diagnosis . In the absence of caseation necrosis, a positive acid-fast bacillus (AFB) or culture for Mycobacterium tuberculosis is needed to establish the diagnosis . A polymerase chain reaction assay for the identification of Mycobacterium tuberculosis in liver biopsy specimens is a new development . Treatment is similar to that used for pulmonary tuberculosis . Quadruple therapy (using four anti-tuberculosis drugs) is recommended, generally for 1 year . For patients with obstructive jaundice, in addition to anti-tuberculous treatment, biliary decompression should be performed either by stent insertion during endoscopic retrograde cholangiopancreatology, by percutaneous transhepatic biliary drainage or by surgical decompression whenever feasible.

Mycopathologia, 1997-98, 140(3), 163 - 9
Inhibition of aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus; Munimbazi C et al.; Six isolates of Bacillus pumilus were tested for their ability to inhibit aflatoxin production of Aspergillus parasiticus NRRL 2999 in yeast extract sucrose (YES) broth . Aflatoxin production was inhibited in both simultaneous and deferred antagonism assays, suggesting that the inhibitory activity was due to extracellular metabolites(s) produced in cell-free supernatant fluids of cultured broth . The inhibition was not due to organic acids or hydrogen peroxide produced by B . pumilus since the inhibitory activity was not lost after pH adjustment or treatment of supernatant fluids with catalase . A range of media tested for the production of inhibitory metabolite(s) in supernatant fluids showed that all media supported bacterial growth and production of the metabolite(s) . The metabolite(s) were produced over a wide range of temperature (25 to 37 degrees C) and pH (4 to 9) of growth of B . pumilus . They were stable over a wide range of pH (4 to 10) and were not inactivated after autoclaving at 121 degrees C for 30 minutes.

Int J Syst Bacteriol, 1998 Jul, 48 Pt 3, 1031 - 5
Bacillus pseudomycoides sp . nov; Nakamura LK; Previous DNA relatedness studies showed that strains identified as Bacillus mycoides segregated into two genetically distinct yet phenotypically similar groups, one being B . mycoides sensu stricto and the other, an unclassified taxon . In the present study, the taxonomic position of this second group was assessed by measuring DNA relatedness and determining phenotypic characteristics of an increased number of B . mycoides strains . Also determined was the second group's 16S RNA gene sequence . The 36 B . mycoides strains studied segregated into two genetically distinct groups showing DNA relatedness of about 30%; 18 strains represented the species proper and 18 the second group with intragroup DNA relatedness for both groups ranging from 70 to 100% . DNA relatedness to the type strains of presently recognized species with G+C contents of approximately 35 mol% (Bacillus alcalophilus, Bacillus cereus, Bacillus circulans, Bacillus lentus, Bacillus megaterium and Bacillus sphaericus) ranged from 22 to 37% . Although shown to be genetically distinct taxa, the two B . mycoides groups exhibited highly similar (98%) 16S RNA sequences . Phylogenetic analyses showed that both B . mycoides and the second group clustered closely with B . cereus . Although not distinguishable by physiological and morphological characteristics, the two B . mycoides groups and B . cereus were clearly separable based on fatty acid composition . The data established that the second B . mycoides group merits recognition as a new species for which the name Bacillus pseudomycoides is proposed . The type stain is NRRL B-617(T).

Curr Microbiol, 1998 Oct, 37(4), 245 - 50
Localization of putative virulence genes on a physical map of the bacillus thuringiensis subsp . gelechiae chromosome
Lovgren A, Carlson CR, Eskils K, Kolsto AB.
The insect pathogen Bacillus thuringiensis (Bt) has earlier been shown to possess virulence factors in addition to the crystal toxins . Bt subsp . gelechiae strain Bt13 lacks crystals but is still virulent to lepidopteran insects . Among the virulence co-expressed genes are two phospholipases; phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-degrading phospholipase C (PC-PLC), flagellin, and beta-lactamase I . In addition to these putative virulence factors the toxic neutral metalloprotease immune inhibitor A (InA) has been identified . In this paper we report a circular 5.9 Mb combined physical and genetic map of the of the Bt subsp . gelechiae chromosome . The genes encoding PI-PLC, PC-PLC, InA, flagellin, and beta-lactamase I are shown to be scattered over the chromosome . The PLC-encoding genes have been cloned from Bt13, and DNA sequencing showed that the Bt subsp . gelechiae PLC genes are >90% identical to their previously cloned equivalents from Bt or B . cereus . An HD-1 crystal toxin (cryIA) gene probe was found to hybridize to the Bt13 chromosome, but not to extrachromosomal elements.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 565 - 71
Bacillus horti sp . nov., a new gram-negative alkaliphilic bacillus; Yumoto I et al.; Novel Gram-negative alkaliphilic strains were isolated from soil obtained from Atsuma, Hokkaido, Japan . The isolates were strictly aerobic rods that produced subterminally located ellipsoidal spores . Chemotaxonomic characteristics of the isolates included the presence of meso-diaminopimelic acid in the cell wall and a DNA G + C content of 40.2-40.9 mol% . The major isoprenoid quinone was menaquinone-7 and the cellular fatty acid profile consisted of a significant amount of 15-C branched-chain acids, iso-C15:0 and anteiso-C15:0 . The growth rate was higher at pH 8-10 than at pH 7 . Comparative sequence analysis of 16S rDNA of 14 alkaliphilic Bacillus strains indicates that the isolated strain has an equidistant relationship to three already defined rRNA groups of alkaliphilic Bacillus species . Based on the morphological and physiological characteristics, as well as phylogenetic position as determined by 16S rDNA analysis and DNA-DNA relatedness data, it is concluded that these isolates should be designated as a new species, for which the name Bacillus horti is proposed . The type strain is K13T (= JCM 9943T).

J Exp Med, 1998 Sep 7, 188(5), 845 - 54
Recombinant Mycobacterium bovis bacillus Calmette-Guérin secreting merozoite surface protein 1 (MSP1) induces protection against rodent malaria parasite infection depending on MSP1-stimulated interferon gamma and parasite-specific antibodies; Matsumoto S et al.; The merozoite surface protein 1 (MSP1) has emerged as a leading malaria vaccine candidate at the erythrocytic stage . Recombinant bacillus Calmette-Guerin (rBCG), which expressed a COOH-terminal 15-kD fragment of MSP1 of Plasmodium yoelii (MSP1-15) as a fusion protein with a secretory protein of Mycobacterium kansasii, was constructed . Immunization of mice with this rBCG induced a higher degree of protection against blood-stage parasite infection than with recombinant MSP1-15 in the RIBI adjuvant (RIBI ImmunoChem Research, Inc., Hamilton, MT) or incomplete Freund's adjuvant systems . We studied the mechanism of protection induced by MSP1-15, and found that interferon (IFN)-gamma had a major role in protection in all adjuvant systems we examined . Mice that produced low amounts of MSP1-15 stimulated IFN-gamma and could not control parasite infection . The antibody against MSP1-15 did not play a major role in protection in this system . After parasite infection, immunoglobulin G2a antibodies, which had been produced by IFN-gamma stimulation, were induced and subsequently played an important role in eradicating parasites . Thus, both cellular and humoral immune responses were essential for protection from malaria disease . These data revealed that BCG is a powerful adjuvant to induce such a protective immune response against malaria parasites.

Biochemistry, 1998 Sep 8, 37(36), 12404 - 11
Crystal structure of the zinc-dependent beta-lactamase from Bacillus cereus at 1.9 A resolution: binuclear active site with features of a mononuclear enzyme; Fabiane SM et al.; The structure of the zinc-dependent beta-lactamase II from Bacillus cereus has been determined at 1.9 A resolution in a crystal form with two molecules in the asymmetric unit and 400 waters (space group P3121; Rcryst = 20.8%) . The active site contains two zinc ions: Zn1 is tightly coordinated by His86, His88, and His149, while Zn2 is loosely coordinated by Asp90, Cys168, and His210 . A water molecule (W1) lies between the two zinc ions but is significantly closer to Zn1 and at a distance of only 1.9 A is effectively a hydroxide moiety and a potential, preactivated nucleophile . In fact, Asp90 bridges W1 to Zn2, and its location is thus distinct from that of the bridging water molecules in the binuclear zinc peptidases or other binuclear zinc hydrolases . Modeling of penicillin, cephalosporin, and carbapenem binding shows that all are readily accommodated within the shallow active site cleft of the enzyme, and the Zn1-bound hydroxide is ideally located for nucleophilic attack at the beta-lactam carbonyl . This enzyme also functions with only one zinc ion present . The Zn1-Zn2 distances differ in the two independent molecules in the crystal (3.9 and 4.4 A), yet the Zn1-W1 distances are both 1.9 A, arguing against involvement of Zn2 in W1 activation . The role of Zn2 is unclear, but the B . cereus enzyme may be an evolutionary intermediate between the mono- and bizinc metallo-beta-lactamases . The broad specificity of this enzyme, together with the increasing prevalence of zinc-dependent metallo-beta-lactamases, poses a real clinical threat, and this structure provides a basis for understanding its mechanism and designing inhibitors.

Microbiol Mol Biol Rev, 1998 Sep, 62(3), 807 - 13
Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins; Crickmore N et al.; The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production . The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989 . Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion . Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent . Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups . A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed . Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences . In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.

Microbiol Mol Biol Rev, 1998 Sep, 62(3), 775 - 806
Bacillus thuringiensis and its pesticidal crystal proteins; Schnepf E et al.; During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research . These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism's pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge . Other studies have focused on the ecological role of the B . thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests . Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Gene, 1998 Jul 3, 214(1-2), 177 - 85
Molecular cloning of a GPI-anchored aminopeptidase N from Bombyx mori midgut: a putative receptor for Bacillus thuringiensis CryIA toxin; Hua G et al.; An aminopeptidase N (APN) with a molecular weight of 110kDa was released from the midgut membrane of Bombyx mori by phosphatidylinositol-specific phospholipase C (PI-PLC), and purified to a homogeneous state . This 110-kDa APN was different from the 100-kDa APN that we previously reported, in chromatographic behaviors, substrate specificity, and N-terminal and internal amino acid sequences . However, the N-terminal sequence of 110-kDa APN, DPAFRLPTTTRPRHYQVTLT, was highly homologous with those of Manduca sexta and Heliothis virescens APNs, which were identified as a receptor for an insecticidal toxin of Bacillus thuringiensis . From a B . mori midgut cDNA library, we cloned the 110-kDa APN cDNA that possessed a 2958-bp open reading frame encoding a 111573-Da polypeptide of 986 residues . The sequence of the eicosa-peptide Asp42Thr61 deduced from the cDNA was completely matched with the N-terminal sequence of the mature 110-kDa APN . One potential N-glycosylation site, HEXXHXW zinc-binding motif and characteristic proline-rich repeats were observed in the ORF . Moreover, the primary sequence contained two hydrophobic peptides on N- and C-termini . The N-terminal peptide sequence showed characteristics of leader peptide for secretion and the C-terminal peptide contained a possible glycosylphosphatidylinositol (GPI) anchoring site . Taken together, the deduced amino acid sequence suggests that the 110-kDa APN is a GPI-anchored protein and a specific receptor protein for B . thuringiensis CryIA delta-endotoxin.

Int J Food Microbiol, 1998 Jul 21, 42(3), 139 - 45
Isolation and partial characterization of a thermostable extracellular protease of Bacillus polymyxa B-17; Matta H et al.; Bacillus polymyxa B-17, a sporeforming psychrotroph produced a thermostable protease . The protease was purified to homogeneity from cell free broth culture by precipitation with ammonium sulfate and gel filtration through Sephadex G-100 . The enzyme had a temperature optimum at 50 degrees C and shared significant activity at 70 degrees C . The protease was also active over a wide range of pH, 5.5 to 10.0, and had optimum activity at pH 7.5 . It was inhibited by metal chelating agents and has a molecular weight of 30 kDa.

J Infect Dis, 1998 Sep, 178(3), 760 - 8
Peptide-specific T cell response to Mycobacterium tuberculosis: clinical spectrum, compartmentalization, and effect of chemotherapy; Wilkinson RJ et al.; The T cell repertoire of 59 patients with untreated tuberculosis was compared with that of 46 bacille Calmette-Guerin-vaccinated controls by assaying the proliferative responses to six permissively recognized peptides from the 16-, 19-, and 38-kDa molecules of Mycobacterium tuberculosis . A trend from higher to lower reactivity following this order: vaccinated controls > lymph node disease > localized extrapulmonary > pulmonary > pleural was seen for 4 of the peptides (P < .03) . The decreased response of blood lymphocytes from patients with pleural tuberculosis was partially accounted for by sequestration of peptide-responsive cells within the pleural fluid . Chemotherapy "reversed" the depressed proliferative responses of patients with pulmonary and pleural tuberculosis depending on the peptide origin, being greatest for peptides of 16 kDa, transient for those of 19 kDa, and least for those of 38 kDa . These data demonstrate antigen specificity in the decreased responsiveness of patients with tuberculosis.

Steroids, 1998 Sep, 63(9), 484 - 95
Microbial hydroxylation of acetylaminosteroids; Holland HL et al.; An investigation of the microbial biotransformation of a range of 3 beta-, 17 beta-, and 20-acetylamino C18 to C21 steroids by microorganisms known to hydroxylate conventional steroids has been undertaken, using Aspergillus ochraceus, Bacillus megaterium, Curvularia lunata, and Rhizoputus arrhizus . A . ochraceus and B . megaterium gave products of 11 alpha- and 15 beta-hydroxylation, respectively . In the case of C . lunata, the products were predominantly those of this organism's normal C-11 beta- and C-14 alpha-hydroxylating pathways, but in one instance, 3 beta-acetylamino-7 alpha-hydroxy-5 alpha-androstan-17-one, appeared to results from direction of the site of hydroxylation by the substitution pattern of the substrate . The products from R . arrhizus generally corresponded to those previously obtained from normal steroids of similar skeleton, with 6 beta- 11 alpha-hydroxylation predominating, but again the sites of hydroxylation and the range of hydroxylated products were found to depend on the substitution pattern of the substrate.

Appl Environ Microbiol, 1998 Sep, 64(9), 3533 - 5
Purification and properties of two thermostable alkaline xylanases from an alkaliphilic bacillus sp
Gessesse A.
Two xylanases, designated XylA and XylB, were purified from the culture supernatant of the alkaliphilic Bacillus sp . strain AR-009 . The molecular masses of the two enzymes were estimated to be 23 kDa (XylA) and 48 kDa (XylB) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The optimum pHs for activity were 9 for XylA and 9 to 10 for XylB . The temperature optima for the activity of XylA were 60 degreesC at pH 9 and 70 degreesC at pH 8 . XylB was optimally active at 75 degreesC at pH 9 and 70 degreesC at pH 8 . Both enzymes were stable in a broad pH range and showed good stability when incubated at 60 and 65 degreesC in pH 8 and 9 buffers.

Appl Environ Microbiol, 1998 Sep, 64(9), 3525 - 9
Discrimination of psychrotrophic and mesophilic strains of the Bacillus cereus group by PCR targeting of major cold shock protein genes; Francis KP et al.; Detection of psychrotrophic strains (those able to grow at or below 7 degreesC) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology . This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7 degreesC) by means other than growth at low temperature, which takes 5 to 10 days for detection . Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B . cereus group and discriminating between psychrotrophic and mesophilic strains . It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B . cereus group.

Appl Environ Microbiol, 1998 Sep, 64(9), 3282 - 9
Enzymatic properties of a novel liquefying alpha-amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequences; Igarashi K et al.; A novel liquefying alpha-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378 . The specific activity of purified LAMY was approximately 5,000 U mg of protein-1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme . The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55 degreesC . The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9 . This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction . Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme . The structural gene for LAMY contained a single open reading frame 1, 548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids . The calculated molecular mass of the extracellular mature enzyme was 55,391 Da . LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B . licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%) . The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY . Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (alpha/beta)8 barrel motif in domain A.

Am Ind Hyg Assoc J, 1998 Aug, 59(8), 540 - 6
Sampling and analytical method development for qualitative assessment of airborne mycobacterial species of the Mycobacterium tuberculosis complex; Schafer MP et al.; This article presents a novel, qualitative approach for detecting airborne M . tuberculosis . Culturing or sample purification is not required . A DNA diagnostic method involving the polymerase chain reaction (PCR) coupled to an enzymatically generated color reaction was used for direct detection of M . bovis BCG (Bacillus of Calmette-Guerin), a surrogate for pathogenic M . tuberculosis . Fewer than 10 mycobacteria were detected with no culturing using this bioanalytical method . Analysis was completed in 1 to 1.5 days, in contrast to traditional culturing methods requiring a minimum of 2-3 weeks . To evaluate an air sampling method coupled to a PCR bioanalytical method, liquid cultures of the surrogate were aerosolized and collected for PCR analyses using 37-mm filter cassettes containing polytetrafluoroethylene filters . An Andersen six-stage (viable) particle sizing sampler was employed as a reference sampler . Aerosolized BCG impacted onto Andersen agar plates required incubation periods of 6-8 weeks before small colony forming units could be detected and enumerated . Although the BCG mean length of the rod-shaped particles was 8.3 microns, the airborne BCG particles were collected predominantly on the Andersen 4-6 stages, representing aerodynamic diameters 0.7 to 3.3 microns . Approximately 25 mycobacteria were detected without culturing using the PCR-filter cassette method . This approach could be used to detect airborne mycobacterial species of the M . tuberculosis complex and could permit the early detection of contaminated indoor air . Also, the efficacy of environmental controls could be evaluated and monitored . This approach could also be used to study the expulsion of infectious particles from patients and may permit risk assessment in regard to personal respiratory protection.

J Immunol, 1998 Sep 1, 161(5), 2356 - 64
Molecular cloning and immunologic reactivity of a novel low molecular mass antigen of Mycobacterium tuberculosis; Coler RN et al.; Polypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors . One such Ag, which elicited strong proliferation and IFN-gamma production, was further characterized . The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M . tuberculosis genomic DNA library . The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli . The predicted m.w . of the recombinant protein without its signal peptide was 8.4 kDa . By Southern analysis, the DNA encoding this mycobacterial protein was found in the M . tuberculosis substrains H37Rv, H37Ra, Erdman, and "C" strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guerin, Pasteur) . The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum . Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-gamma, IL-10, and TNF-alpha, but not IL-5, from human PBMC isolated from PPD-positive healthy donors . Mtb 8.4 did not stimulate PBMC from PPD-negative donors . Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis . Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M . tuberculosis.

J Econ Entomol, 1998 Aug, 91(4), 864 - 8
Isolation of aerobic microbes from Ixodes scapularis (Acari: Ixodidae), the vector of Lyme disease in the eastern United States; Martin PA et al.; The spirochete Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Benner is transmitted by Ixodes scapularis Say, a vector of Lyme disease . As a 1st step into investigating the possibility of biocontrol of the tick, we identified the microbiota associated with the ticks . We collected, identified, and determined the sex of ticks from foliage and deer . Seventy-three initial bacterial isolates were recovered from 43 ticks (27 adults and 16 nymphs) . The bacteria isolated from nymphs were qualitatively different (mainly gram-negative cocci) from the bacteria isolated from adult ticks (gram-negative and gram-positive rods) . To determine long-term viability, these isolates were stored for 6 mo under laboratory conditions . After storage, 63 surviving bacterial isolates were characterized using the Biology System of identification by substrate utilization . Forty-four isolates were identified to the species level . Our characterization efforts focused on the 40 spore-forming bacteria, which could prove useful in the biocontrol of ticks . Eleven species of Bacillus were identified . Bacillus thuringiensis-B . cereus was the predominant species group isolated . Six isolates from this group formed crystals.

Mol Membr Biol, 1998 Apr-Jun, 15(2), 69 - 74
Microcalorimetric study on the phase behaviour of S-layer coated liposomes; Kupcu S et al.; Isolated S-layer subunits from Bacillus coagulans E38-66/v1 were recrystallized on positively charged, unilamellar liposomes composed of dipalmitoylphosphatidylcholine, cholesterol and hexadecylamine . The thermotropic phase behaviour of S-layer coated and uncoated liposomes was characterized by differential scanning microcalorimetry indicating for both preparations a broad transition around 50 degrees C due to the chain-melting from a liquid-ordered gel-like to a liquid-ordered fluid phase as described for phosphatidylcholine/cholesterol mixtures . The slightly higher phase transition temperature for the S-layer coated liposomes were explained by increased intermolecular order . Cross-linking the S-layer subunits covalently to hexadecylamine with glutaraldehyde induced phase separation within the liposomes . Based on deconvolution of the normalized excess heat capacity functions it was proposed that the different lipid domains arise from phospholipids representing different degrees of mobility.

Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10797 - 802
Identification of HLA class II-restricted determinants of Mycobacterium tuberculosis-derived proteins by using HLA-transgenic, class II-deficient mice; Geluk A et al.; T helper 1 cells play a major role in protective immunity against mycobacterial pathogens . Since the antigen (Ag) specificity of CD4(+) human T cells is strongly controlled by HLA class II polymorphism, the immunogenic potential of candidate Ags needs to be defined in the context of HLA polymorphism . We have taken advantage of class II-deficient (Ab0) mice, transgenic for either HLA-DRA/B1*0301 (DR3) or HLA-DQB1*0302/DQA*0301 (DQ8) alleles . In these animals, all CD4(+) T cells are restricted by the HLA molecule . We reported previously that human DR3-restricted T cells frequently recognize heat shock protein (hsp)65 of Mycobacterium tuberculosis, and only a single hsp65 epitope, p1-20 . DR3.Ab0 mice, immunized with bacillus Calmette-Guerin or hsp65, developed T cell responses to M . tuberculosis, and recognized the same hsp65 epitope, p1-20 . Hsp65-immunized DQ8.Ab0 mice mounted a strong response to bacillus Calmette-Guerin but not to p1-20 . Instead, we identified three new DQ8-restricted T cell epitopes in the regions 171-200, 311-340, and 411-440 . DR3.Ab0 mice immunized with a second major M . tuberculosis protein, Ag85 (composed of 85A, 85B, and 85C), also developed T cell responses against only one determinant, 85B p51-70, that was identified in this study . Importantly, subsequent analysis of human T cell responses revealed that HLA-DR3+, Ag85-reactive individuals recognize exactly the same peptide epitope as DR3.Ab0 mice . Strikingly, both DR3-restricted T cell epitopes represent the best DR3-binding sequences in hsp65 and 85B, revealing a strong association between peptide-immunodominance and HLA binding affinity . Immunization of DR3.Ab0 with the immunodominant peptides p1-20 and p51-70 induced T cell reactivity to M . tuberculosis . Thus, for two different Ags, T cells from DR3.Ab0 mice and HLA-DR3+ humans recognize the same immunodominant determinants . Our data support the use of HLA-transgenic mice in identifying human T cell determinants for the design of new vaccines.

Immunol Cell Biol, 1998 Aug, 76(4), 363 - 8
Antigen-specific lymphocytes enhance nitric oxide production in Mycobacterium bovis BCG-infected bovine macrophages; Carpenter E et al.; Nitric oxide (NO) production was evaluated in macrophages isolated from Mycobacterium bovis bacille Calmette-Guerin (BCG)-immunized, and control non-immunized, cattle . Incubation of M . bovis BCG-infected macrophages with recombinant bovine IFN-gamma led to increased nitrite levels in culture supernatants . It was also demonstrated that NO production by autologous M . bovis BCG-infected macrophages increased in a linear relationship with the number of antigen-specific lymphocytes added to cultures . The elevated NO levels were also associated with increased IFN-gamma secretion . Treatment of cultures with the NO inhibitor, N-monomethyl L-arginine (L-NMMA), reduced the levels of NO without affecting the metabolic activity of internalized M . bovis BCG . Our results suggest that synthesis of NO may constitute an integral part of the cell-mediated antigen-specific response against M . bovis BCG . However, although the presence of lymphocytes does partially inhibit multiplication of M . bovis BCG in macrophages, it appears that the activity of NO, or the levels produced in monocyte-derived macrophages, may be insufficient to influence the growth of the intracellular mycobacteria.

Prog Neurobiol, 1998 Oct, 56(1), 19 - 35
Inflammation in the CNS: balance between immunological privilege and immune responses; Matyszak MK; Inflammatory components play an important part in many diseases of the central nervous system (CNS) . Recent evidence suggests that this may also be true of diseases which were previously considered as purely neuro-degenerative . However, it is also clear that inflammatory responses in the CNS differ in many ways from responses in non-CNS tissues . Some of these differences have been demonstrated by the use of animal models . For example, when bacteria are injected into the brain parenchyma, they induce a typical acute inflammatory response . However, unlike in other tissues, bacteria which are not cleared from the brain parenchyma remain undetected by the immune system . Some bacteria, such as bacillus Calmette-Guerin, can persist in the brain parenchyma for months sequestered in microglia and perivascular macrophages . When an animal with an intraparenchymal bacteria deposit is later sensitised peripherally, an immune response is evoked at the site of the deposits . The lesions induced in the CNS parenchyma are T-cell mediated and show characteristics typical of a delayed-type hypersensitivity response . The lesions produce a breakdown of the blood-brain barrier and demyelination . These immune responses are similar to those described for multiple sclerosis lesions . The responses to bacteria are unique to the brain parenchyma . Pathogens injected into the ventricles induce inflammatory responses similar to those in other non-CNS tissues: there is an acute inflammatory response which develops spontaneously into an immune mediated response within the first week.

Curr Opin Immunol, 1998 Aug, 10(4), 413 - 7
Mendelian susceptibility to mycobacterial infection in man; Altare F et al.; Selective susceptibility to poorly pathogenic mycobacteria, such as bacille Calmette-Guerin vaccine and environmental non-tuberculous mycobacteria, has long been suspected to be a mendelian disorder but its molecular basis has remained elusive . Recently, recessive mutations in the interferon-gamma-receptor receptor ligand-binding chain, interferon-gamma-receptor signalling chain, IL-12 p40 subunit and IL-12-receptor beta 1 chain genes have been identified in a number of patients with disseminated mycobacterial infection . Although genetically distinct, these conditions are immunologically related and highlight the essential role of interferon-gamma-mediated immunity in the control of mycobacteria in man.

Br J Urol, 1998 Aug, 82(2), 213 - 23
Bacille Calmette-Guérin in superficial transitional cell carcinoma; Mungan NA et al.; The mechanisms by which BCG exerts its antitumour activity remain unclear . Attachment of BCG to the bladder via FN has been shown to be an important step in initiating its antitumorigenic activity . The mechanism(s) by which BCG operates requires LAK cells, BCG-activated killer cells, T lymphocytes (CD4) helper cells and CD8 suppressor/cytotoxic cells) and monocytes . The optimal route of administration is intravesical . The efficacy of a BCG vaccine depends on the viability, dose and strain . Differences in efficacy and side-effects have not been shown between different strains . Low-dose regimens successfully protect from recurrences, with fewer side-effects . The initial schedule of BCG is a course of six instillations in 6 weeks; when the patient fails this course, two possibilities arise . The first is maintenance therapy; response rates improve but there is more local and systemic toxicity . The second is a further 6-week course, and this seems most useful in those with a sustained response to the initial treatment . The clinical response to BCG therapy can be monitored using cytokine measurements or p53 determinations . Toxicity remains a major problem in BCG treatment and triple antituberculosis combination therapy should be given for 3 months in those with severe systemic side-effects . The use of prophylactic isoniazid is not recommend to decrease side-effects . The clinical results of BCG have been good, with success rates of 58-100%, with a minimal follow-up of one year in prophylaxis . BCG seems superior to intravesical therapy, but at the cost of inducing more adverse effects . BCG is not indicated for low- and intermediate-risk patients, in whom chemotherapy is the first choice . BCG can also be used to eliminate tumour after an incomplete TUR, or in patients who are unfit for surgery, with a 60-70% success rate . The primary and best treatment for CIS is intravesical BCG; encouraging results have been reported, with success rate of 42-83% after a minimal follow-up of one year . Although currently BCG seems to be the choice for high-risk superficial TCC, many questions remain unanswered, especially about the mechanism(s) of action, the optimal dose and clinical schedule.

J Biochem (Tokyo), 1998 Sep, 124(3), 642 - 7
Substrate specificity of pepstatin-insensitive carboxyl proteinase from Bacillus coagulans J-4; Shibata M et al.; Bacillus coagulans J-4 carboxyl proteinase, designated as J-4, is characterized as alcohol-resistant and insensitive to aspartic proteinase inhibitors such as pepstatin, diazoacetyl-DL-norleucinemetylester, and 1,2-epoxy-3-(p-nitrophenoxy)propane . Here, its substrate specificity was elucidated by using two series of chromogenic substrates, Lys-Pro-Ala-Lys-Phe*Nph (p-nitrophenylalanine:* is cleavage site)-Arg-Leu (XVI) and Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu (RS6), in which the amino acid residues at positions P5-P2, P2', and P3' were systematically substituted . Kinetic parameters were determined for both sets of peptides . J-4 was shown to hydrolyze Lys-Pro-Ala-Ala-Phe-Nph-Arg-Leu most effectively among the XVI series . The kinetic parameters of this peptide were Km = 20.0 +/- 3.24 microM, kcat = 15.4 +/- 0.71 s-1, and kcat/Km = 0.769 +/- 0.128 microM-1.s-1 . Among the RS6 series, Lys-Pro-Ile-Pro-Phe-Nph-Arg-Leu was hydrolyzed most effectively . The kinetic parameters of this peptide were Km = 13.7 +/- 1.30 microM, kcat = 9.65 +/- 0.38 s-1, and kcat/Km = 0.704 +/- 0.072 microM-1.s-1 . These systematic analyses revealed that J-4 had a unique preference for the P2 position: J-4 preferentially hydrolyzed peptides having an Ala or Pro residue in the P2 position . Other carboxyl proteinases preferred peptides having hydrophobic and bulky amino acid residue such as Leu in the P2 position . Thus, J-4 was found to differ considerably in substrate specificity from the other carboxyl proteinases reported so far.

J Appl Microbiol, 1998 Jul, 85(1), 17 - 24
Sporulation temperature affects initiation of germination and inactivation by high hydrostatic pressure of Bacillus cereus; Raso J et al.; The influence of sporulation temperature (20, 30 and 37 degrees C) on the heat resistance and initiation of germination and inactivation by high pressure on Bacillus cereus ATCC 14579 spores was investigated . Spores sporulated at 37 degrees C were the most heat-resistant . However, spores sporulated at 20 degrees C were more resistant to the initiation of germination and inactivation by high pressure . Spores were more sensitive to pressure at higher treatment temperatures . At 25 degrees C, there was an optimum pressure (250 MPa) for the initiation of germination for the three suspensions; at higher temperatures an increase of pressure up to 690 MPa caused progressively more germination . Resistance to the germinability and inactivation by high pressure of the spore population was distributed heterogeneously . Semilogarithmic curves of the ungerminated and survival fraction of B . cereus spores were concave . The resistant fraction of the spore population was lower at higher treatment temperatures . At 60 degrees C after 30 s of treatment at 690 MPa almost 5 log cycles of the population of B . cereus sporulated at 20 degrees C was germinated, and more than 7 log cycles of the population of B . cereus sporulated at 30 and 37 degrees C . The same treatment inactivated 4, 6 and 7 log cycles of the population of B . cereus sporulated at 20, 30 and 37 degrees C, respectively.

Am J Infect Control, 1998 Aug, 26(4), 393 - 8
Comparative evaluation of the sporicidal activity of new low-temperature sterilization technologies: ethylene oxide, 2 plasma sterilization systems, and liquid peracetic acid; Rutala WA et al.; OBJECTIVE: This study was undertaken to evaluate the efficacy of 4 new low-temperature sterilization technologies: ethylene oxide with hydrochlorofluorocarbons, a liquid peracetic acid immersion system (Steris System 1 Processor), and 2 plasma sterilization processes that use vaporized hydrogen peroxide (Sterrad 100 and the Sterrad 100S) . The Sterrad 100S system potentially improves sterilizer efficacy by using 2 cycles of a diffusion stage and a plasma stage per sterilization cycle . METHODS: Flat stainless steel carriers were inoculated with approximately 10(6) Bacillus stearothermophilus spores . These carriers were aseptically placed in the middle of 40 cm long stainless steel lumens (hollow tubes) . Two types of lumen were used:(1) a lumen test unit with a removable 5 cm center piece (1.2 cm diameter) of stainless steel sealed to the narrower steel tubing by hard rubber septums and (2) a straight lumen . Three different diameters of the lumen test unit (1, 2, and 3 mm) and a single diameter of the straight lumen (3 mm) were studied . At least 40 replicates were performed for each type of lumen and sterilization method . After inoculation, the test unit was evaluated in 1 of the low-temperature sterilization technologies . After sterilization, the carriers were cultured in trypticase soy broth for 14 days at 55 degrees C and assessed for growth of B stearothermophilus spores . RESULTS: Our results demonstrated that ethylene oxide with hydrochlorofluorocarbons, the Sterrad 100s, and the Sterrad 100S half cycle were highly effective in killing approximately 10(6) B stearothermophilus spores present in the center of narrow-lumen stainless steel tubes . As the lumen diameter decreased with the lumen test unit, the Sterrad 100 demonstrated reduced ability to kill B stearothermophilus spores present on the carrier . At the smallest diameter tested (1 mm), the Sterrad 100 system failed 74% of the time . The Steris System 1 was not effective in completely eliminating the 10(6) inoculum under test conditions . CONCLUSION: The Sterrad 100S was significantly superior to the Sterrad 100 system and equivalent to ethylene oxide with hydrochlorofluorocarbons . Introduction of this new Sterrad 100S system should improve the margin of safety and reduce processing costs by its use of a shorter cycle time . The Steris System 1 is limited by diffusion of the chemical sterilant into the interior of the lumen test unit.

J Bacteriol, 1998 Sep, 180(17), 4734 - 8
Cloning, expression, and catabolite repression of a gene encoding beta-galactosidase of Bacillus megaterium ATCC 14581; Shaw GC et al.; A gene encoding beta-galactosidase, designated mbgA, was isolated from Bacillus megaterium ATCC 14581 . Chromosomal beta-galactosidase production could be dramatically induced by lactose but not by isopropyl-beta-D-thiogalactopyranoside (IPTG) and was subject to catabolite repression by glucose . Disruption of mbgA in the B . megaterium chromosome resulted in loss of lactose-inducible beta-galactosidase production . A 27-bp inverted repeat was found to overlap the mbgA promoter sequence . Two partially overlapping catabolite-responsive elements (CREs) were identified within the inverted repeat . Base substitutions within CRE-I and/or CRE-II caused partial relief from catabolite repression . The results suggest that the 27-bp inverted repeat may serve as a target for a catabolite repressor(s).

Environ Health Perspect, 1998 Sep, 106(9), A432 - 7
Natural born killers; Schmidt CW; This year, 30 million acres of the corn, cotton, and potatoes planted in the United States will have been genetically engineered to produce an endotoxin normally found in the microbe Bacillus thuringiensis (Bt), a self-contained pesticide that will be toxic only to target insects . Transgenic pest-resistant crops are a cost-effective alternative to chemical pesticides, and may offer a way to help feed the world's growing population with minimal environmental impact.

FEBS Lett, 1998 Aug 7, 432(3), 150 - 4
Phospholipase cleavage of glycosylphosphatidylinositol reconstituted in liposomal membranes; Villar AV et al.; Glycosylphosphatidylinositol (GPI) purified from rat liver lipids was incorporated into lipid bilayers of defined compositions, in the form of large unilamellar vesicles . The GPI concentration in the bilayers was kept constant at 25 mole%, whereas the remaining lipids being phosphatidylcholine, phosphastidylethanolamine, sphingomyelin and/or cholesterol were varied . The resulting liposomes consisted of spherical vesicles, approximately 100 nm in diameter, that could keep their aqueous contents separated from the extravesicular medium . When these liposomes were treated with either Bacillus cereus phosphatidylinositol-phospholipase C, Trypanosoma brucei GPI-phospholipase C, or bovine serum GPI-phospholipase D, GPI was hydrolyzed at different rates, depending on the enzyme and the bilayer lipid composition . These observations open the way to biophysical and biochemical studies of enzymic GPI cleavage under defined conditions . Extensive GPI hydrolysis was observed in certain cases that could allow the use of these systems for the preparation of inositol phosphoglycans, proposed second messengers of a wide variety of hormones, cytokines and growth factors.

Soc Sci Med, 1998 Jul, 47(2), 195 - 202
The meanings of tuberculosis for Mexican migrant farmworkers in the United States; Poss JE; The timely diagnosis and treatment of tuberculosis is an important public health problem in both developed and developing nations . In the United States, migrant farmworkers are estimated to be about six times more likely than other employed adults to develop tuberculosis . The purpose of this study was to investigate explanatory models of tuberculosis among Mexican migrant farmworkers working in western New York state . In-depth interviews were conducted with 26 farmworkers using an open-ended question format . All interviews were conducted in migrant camps and were audio-taped, translated and transcribed by the researcher . Data analysis was performed using Glaser and Strauss' grounded theory method of analysis which involves continuous and simultaneous data collection, coding, and analysis . Study participants included 21 males and 5 females ranging in age from 18 to 65 . Respondents had worked as migrant farmworkers an average of 10 years and had an average of five years of schooling . Two-thirds of the participants had previously attended a tuberculosis education program, and four had received treatment for tuberculosis infection in the past . Farmworkers' explanations of tuberculosis etiology, severity, symptoms, prevention, treatment, and social significance are described as well as their beliefs about tuberculosis skin testing and the bacillus Calmette-Guerin (BCG) vaccine . Migrant farmworkers' explanatory models were similar in many aspects to the medical model of tuberculosis, although farmworkers had numerous misconceptions about BCG vaccination . Health care workers should be aware that Mexican migrant farmworkers may have beliefs about tuberculosis that are very compatible with participation in testing and treatment programs if such programs are made accessible to them.

Appl Microbiol Biotechnol, 1998 Jul, 50(1), 48 - 54
Cloning of a new endoglucanase gene from Bacillus sp . BP-23 and characterisation of the enzyme . Performance in paper manufacture from cereal straw; Blanco A et al.; The gene ce1A, encoding an endoglucanase from the strain Bacillus sp . BP-23, was cloned and expressed in Escherichia coli . The nucleotide sequence of a 1867-bp DNA fragment containing the ce1A gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44,803 Da . The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases . The ce1A gene product synthesized in E . coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel . Activity was enhanced in the presence of 10 mM Mg2+ and Ca2+ and showed its maximum at 40 degrees C and pH 4.0 . Study of the performance of Ce1A on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper . Ce1A treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished . Electron-microscope analysis showed that the surface of straw fibres was modified by Ce1A.

Zentralbl Veterinarmed B, 1998 Aug, 45(6), 363 - 71
Cilia-associated respiratory (CAR) bacillus infection in conventionally reared rabbits; Caniatti M et al.; This study was designed to investigate the prevalence of Cilia-associated respiratory (CAR) bacillus infection in rabbits reared for meat production in Italy and to correlate the presence of CAR bacillus with inflammatory lesions of the respiratory tract . Seventy health, 3-month-old, New Zealand White rabbits, raised in 10 different rabbitries in Northern Italy were randomly selected at slaughter . No gross lesions were found at necropsy in any rabbit . In each animal, the trachea and lungs were sampled, fixed in 10% formalin, embedded in paraffin and stained with the Warthin-Starry method to evaluate the presence of CAR bacillus, and with haematoxylin and eosin to evaluate the presence of inflammatory lesions . CAR bacillus was present in 50 out of 70 rabbits (71.4%) with a prevalence of the infection that varied from 30% to 100% in the seven rabbitries . CAR bacillus was present both in the trachea and bronchi in 23 cases (32.8%), only in the trachea in 24 cases (34.3%) and only in the bronchi in three cases (4.3%) . Inflammatory lesions were found in the trachea (22 cases, 31.4%) and the bronchi (58 cases, 82.8) . There was a strong, statically significant correlation between the presence of CAR bacillus in the bronchi and bronchial inflammatory lesions (P < 0.0001) . This study indicates that CAR bacillus infection is widespread in conventionally reared rabbits in Italy and that a possible correlation exists between the presence of CAR bacillus and bronchial inflammatory lesions.

Haematologica, 1998 Jul, 83(7), 670 - 2
Bacteremia caused by CDC group IV c-2 in a patient with acute leukemia; Salar A et al.; Human infection due to CDC group IV c-2, a gram negative bacillous, are rare . We describe a case of nosocomial bacteremia caused by this organism in a neutropenic patient with acute lymphoblastic leukemia and include a literature review of CDC group IV c-2 infection in patients with hematologic malignancies.

J Protein Chem, 1998 Jul, 17(5), 463 - 71
Limited proteolysis of Bacillus thuringiensis CryIG and CryIVB delta-endotoxins leads to formation of active fragments that do not coincide with the structural domains; Zalunin IA et al.; Bacillus thuringiensis "true" toxins consist of three domains: the N-terminal, alpha-helical domain followed by two beta-structural domains . Their limited proteolysis does not proceed at the domain boundaries, but is directed to the loops within the domains . There are at least two patterns of the limited proteolysis of "true" toxins . The first pattern, observed for CryIA and CryIVD delta-endotoxins, results in the proteolysis of the loops connecting beta-strands of the second domain . The second pattern, detected for CryIG and CryIVB proteins, consists in the cleavage of the loop connecting the fifth and the sixth alpha-helices of the first domain . The choice between the routes depends on the size, sequence, and dynamics of the loop that define its accessibility to a proteinase . Bioassay of CryIG and CryIVB delta-endotoxin fragments indicates that only two alpha-helices, the sixth and the seventh within the first domain, followed by the two beta-structural domains are sufficient for the insecticidal activity.

Proteins, 1998 Aug 15, 32(3), 276 - 88
Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A, Mg2+ Ap5A, and Mn2+ Ap5A reveal an intermediate lid position and six coordinate octahedral geometry for bound Mg2+ and Mn2+; Berry MB et al.; Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A, Mn2+ Ap5A, and Mg2+ Ap5A have been determined by X-ray crystallography to resolutions of 1.6 A, 1.85 A, and 1.96 A, respectively . The protein's lid domain is partially open, being both rotated and translated away from bound Ap5A . The flexibility of the lid domain in the ternary state and its ability to transfer force directly to the the active site is discussed in light of our proposed entropic mechanism for catalytic turnover . The bound Zn2+ atom is demonstrably structural in nature, with no contacts other than its ligating cysteine residues within 5 A . The B . stearothermophilus adenylate kinase lid appears to be a truncated zinc finger domain, lacking the DNA binding finger, which we have termed a zinc knuckle domain . In the Mg2+ Ap5A and Mn2+ Ap5A structures, Mg2+ and Mn2+ demonstrate six coordinate octahedral geometry . The interactions of the Mg2+-coordinated water molecules with the protein and Ap5A phosphate chain demonstrate their involvement in catalyzing phosphate transfer . The protein selects for beta-y (preferred by Mg2+) rather than alpha-gamma (preferred by Mn2+) metal ion coordination by forcing the ATP phosphate chain to have an extended conformation.

Biochemistry, 1998 Aug 25, 37(34), 11707 - 13
Snapshots along an enzymatic reaction coordinate: analysis of a retaining beta-glycoside hydrolase; Davies GJ et al.; The enzymatic hydrolysis of O-glycosidic linkages is one of the most diverse and widespread reactions in nature and involves a classic "textbook" enzyme mechanism . A multidisciplinary analysis of a beta-glycoside hydrolase, the Cel5A from Bacillus agaradhaerens, is presented in which the structures of each of the native, substrate, covalent-intermediate, and product complexes have been determined and their interconversions analyzed kinetically, providing unprecedented insights into the mechanism of this enzyme class . Substrate is bound in a distorted 1S3 skew-boat conformation, thereby presenting the anomeric carbon appropriately for nucleophilic attack as well as satisfying the stereoelectronic requirements for an incipient oxocarbenium ion . Leaving group departure results in the trapping of a covalent alpha-glycosyl-enzyme intermediate in which the sugar adopts an undistorted 4C1 conformation . Finally, hydrolysis of this intermediate yields a product complex in which the sugar is bound in a partially disordered mode, consistent with unfavorable interactions and low product affinity.

J Appl Microbiol, 1998 Jun, 84(6), 959 - 68
Isolation and partial characterization of antifungal metabolites of Bacillus pumilus; Munimbazi C et al.; Antifungal metabolites produced by Bacillus pumilus in Potato Dextrose Broth (PDB) were isolated from culture supernatant fluid by precipitation with ammonium sulphate . The antifungal metabolites inhibited mycelial growth of many species of Aspergillus, Penicillium and Fusarium . They also inhibited production of aflatoxins, cyclopiazonic acid, ochratoxin A and patulin . The metabolites were heat-stable and remained active after sterilization at 121 degrees C for 15 min . Their activity was stable over a wide range of pH (2-10) . The metabolites were resistant to hydrolysis by various proteases, peptidases and other enzymes . They were also resistant to denaturation by many protein-denaturing detergents except Nonidet P-40 . The metabolites were soluble in water and relatively polar organic solvents . Chromatographic bioassay revealed that a crude precipitate of the metabolites contained only one compound with antifungal activity . The active compound did not form a fluorescent derivative with fluorescamine suggesting that the compound is either a cyclic polypeptide or a non-peptidic compound.

Lepr Rev, 1998 Jun, 69(2), 145 - 50
Fine-needle aspiration cytology of lepromatous leprosy; Singh N et al.; A prospective study correlating cytopathology with clinical morphology and histopathology in 22 patients with lepromatous leprosy was performed . Aspirates were taken from skin lesions in all patients . Lymph node aspirates were also performed in four patients with lymphadenopathy . Fine-needle aspirates yielded sufficient cellular material with excellent preservation of morphological detail . Diagnosis and correlation with bacillary index, clinical and histopathological findings was possible in all patients . In addition, the two patterns, partial and diffuse, of lymph node involvement could be recognized . Fine-needle aspiration cytology is a simple method for the laboratory assessment of leprosy.

Biochem Mol Biol Int, 1998 Jul, 45(4), 769 - 74
Analysis of 66 kDa toxin from Bacillus thuringiensis subsp . kurstaki reveals differential amino terminal processing of protoxin by endogenous protease(s); Kumar NS et al.; The endogenous protease(s) activated crystal toxin from Bacillus thuringiensis subsp . kurstaki was purified and examined . The purified toxin was homogenous, as demonstrated by two-dimensional polyacrylamide gel electrophoresis and contained 1.38 mumoles neutral sugar and 9 nmoles sialic acid per mg protein amino terminal amino acid sequence data revealed that the toxin is a cleavage product of 132 kDa protoxin with glutamic acid-30 of the deduced amino acid sequence of the crystal protein (Schnepf, H.E., Wong, H.C . and Whiteley, H.R . (1985) J . Biol . Chem . 260: 6264-6272) at the amino terminus.

Tuber Lung Dis, 1997, 78(3-4), 195 - 203
rhuIL-2 adjunctive therapy in multidrug resistant tuberculosis: a comparison of two treatment regimens and placebo; Johnson BJ et al.; SETTING: Low-dose recombinant human interleukin 2 (rhuIL-2) adjunctive immunotherapy in multidrug resistant tuberculosis (MDR-TB) patients . OBJECTIVE: Evaluation of the effects of daily versus pulse-administered rhuIL-2 compared to placebo . DESIGN: MDR-TB patients on best available antituberculous chemotherapy received rhuIL-2 for 30 consecutive days (daily therapy), or for 5 days followed by a 9-day 'rest', for three cycles (pulse therapy) . Placebo control patients received diluent . The cumulative total dose of rhuIL-2 given to each patient in either rhuIL-2 treatment group was the same . Patient immunologic, microbiologic, and radiologic responses were compared . RESULTS: The three treatment schedules induced different results . Immune activation was documented in patients receiving daily rhuIL-2 therapy . Numbers of CD25+ and CD56+ cells in the peripheral blood were increased in these patients, but not in patients receiving pulse rhuIL-2 or placebo . In addition, 5/8 (62%) patients receiving daily rhuIL-2 demonstrated reduced or cleared sputum bacterial load while only 2/7 (28%) pulse rhuIL-2 treated and 2/8 (25%) controls showed bacillary clearance . Chest radiographs of 7/12 (58%) patients receiving daily rhuIL-2 indicated significant improvement over 6 weeks . Only 2/9 (22%) pulse rhuIL-2-treated patients and 5/12(42%) placebo controls showed radiologic improvement . CONCLUSION: Daily low dose rhuIL-2 adjunctive treatment stimulates immune activation and may enhance the antimicrobial response in MDR-TB.

Tuber Lung Dis, 1997, 78(3-4), 185 - 93
Cellular and humoral immune responses to mycobacterial stress proteins in experimental pulmonary tuberculosis; Bartow RA et al.; OBJECTIVE: Immunity to mycobacterial stress protein antigens was studied in response to vaccination and/or virulent infection . DESIGN: Guinea pigs, either vaccinated with Mycobacterium bovis bacille Calmette-Guerin (BCG), infected by the pulmonary route with virulent M . tuberculosis, or vaccinated then infected, were studied for the development of cellular and humoral immunity to two recombinant mycobacterial stress proteins, hsp 65 and hsp 70 . RESULTS: Recombinant hsp 70 stimulated good proliferation in blood lymphocytes and, to a lesser extent, spleen and bronchotracheal lymph node lymphocytes from BCG-vaccinated guinea pigs . The proliferative responses to hsp 70 were diminished in both the spleen and lymph node cells following subsequent pulmonary challenge alone, but were boosted significantly by prior vaccination . Recombinant hsp 65 was much less active at inducing the proliferation of spleen and lymph node cells, with lowest responses observed in blood lymphocytes occurring in the cells from BCG-vaccinated, aerosol-challenged guinea pigs . Using a semi-quantitative dot blot procedure, serum antibodies to both hsp 65 and hsp 70 developed gradually following BCG vaccination, with all guinea pigs studied exhibiting significant seroreactivity after 15 weeks post-vaccination . In guinea pigs exposed to virulent M . tuberculosis by aerosol, serologic reactivity to hsp 70 was consistently stronger 6 weeks post-challenge in both vaccinated and non-vaccinated guinea pigs . In fact, 6 weeks following pulmonary exposure to M . tuberculosis in previously naive guinea pigs, 3 out of 6 animals had no detectable serum antibodies to hsp 65 . Somewhat surprisingly, antibody levels to both hsp 65 and hsp 70 were only slightly increased by prior BCG vaccination in guinea pigs exposed to virulent M . tuberculosis by the respiratory route . CONCLUSION: These results demonstrate that both hsp 65 and hsp 70 stimulate detectable humoral and cell-mediated immunity in guinea pigs vaccinated and/or infected under highly relevant conditions . There is little evidence that vaccination with BCG primes the guinea pig to make an anamnestic response to hsp 65 following virulent pulmonary challenge . The precise contribution of immunity to mycobacterial stress proteins to the pathogenesis of tuberculosis in this model remains to be elucidated.

Int J Tuberc Lung Dis, 1998 Aug, 2(8), 679 - 82
Bacille Calmette Guérin vaccination in pre-term infants; Sedaghatian MR et al.; OBJECTIVE: To evaluate the efficacy of bacillus Calmette Guerin (BCG) vaccine in pre-term infants . DESIGN: BCG vaccine was given to three groups of neonates: 1) 36 pre-terms born at different gestational ages (GA) vaccinated at birth, 2) 16 pre-terms vaccinated at 40 weeks post conceptional age, and 3) 20 full-terms vaccinated at birth . All of the infants were tested by purified protein derivative (PPD) 2-4 months post vaccination . RESULT: The mean sizes of BCG scarring and PPD tuberculin induration were largest in full-term and smallest in pre-term infants vaccinated at birth . The pre-term infants of lower GA (27-33 weeks) had non-significantly smaller BCG scar and PPD induration than the pre-terms of higher GA (34-36 weeks) . The results of logistic regression analyses revealed that female infants were more likely to have a BCG scar, and both birth weight and female sex were significantly associated with a reactive PPD induration . CONCLUSION: This study indicates that male pre-term infants of lower GA (<33 weeks) are less likely to develop BCG scar and a reactive PPD tuberculin test after BCG vaccination . These findings do not support routine BCG vaccination at birth of pre-term neonates of GA <33 weeks . Studies including larger groups of infants are needed to confirm these findings.

FEMS Microbiol Lett, 1998 Aug 1, 165(1), 35 - 41
Differential effects of helper proteins encoded by the cry2A and cry11A operons on the formation of Cry2A inclusions in Bacillus thuringiensis; Ge B et al.; To compare the differential effects of cry2A operon orf2 (29-kDa protein gene) and Cry11A operon orf3 (20-kDa protein gene) on Cry2A synthesis and inclusion formation, we expressed the cry2A gene along with either the 29-kDa gene, 20-kDa gene, or both genes . Constructs containing 20-kDa, in the presence or absence of 29-kDa, produced more Cry2A than constructs which lacked this gene . Cry2A synthesis was also higher when the 29-kDa gene was included with 20-kDa in the construct . However, even in the presence of increased Cry2A synthesis facilitated by the 20-kDa gene, typical Cry2A crystals did not form if the 29-kDa gene was not included in the construct . These results suggest that the 29-kDa and 20-kDa proteins have different functions, with the 20-kDa protein acting like a molecular chaperone to enhance net Cry2A synthesis, and the 29-kDa protein likely serving as a template for the stabilization of Cry2A molecules and their organization into the rectangular inclusion characteristic of wild-type Cry2A crystals.

Mem Inst Oswaldo Cruz, 1998 Jul-Aug, 93(4), 441 - 4
Studies on the Bacillus sphaericus larvicidal activity against malarial vector species in Amazonia; Rodrigues IB et al.; In this work, bioassays were carried out in laboratory conditions (average temperature 26 +/- 2 degrees C) to test ten strains of Bacillus sphaericus, isolated from Brazilian soils against third instar larvae from anopheline species recorded as malaria vectors in Amazonian-Anopheles nuneztovari and An . darlingi . With the former mosquito, three strains--S2, S20 and S46 showed relative activity, in 24 and 48 hr exposure to the B . spahericus strains . With the latter only the S2 and S20 were effective in the 48 hr reading . The studied strains that showed the most adequate response in the Amazonian region were S2 and S20 showing broader and more efficient results . Therefore, S2 was the most effective when the 24 and 48 hr readings were considered, because it showed the greatest relative activity values.

Prep Biochem Biotechnol, 1998 Aug, 28(3), 261 - 9
Enalapril microbial biosensor; Fleschin S et al.; Enalapril maleate (EMa) belongs to a new class of antihypertensive agents known as angiotensin converting enzyme (ACE) inhibitors . This paper describes the development of a microbial biosensor for EMa using induced Bacillus subitilis cells . This biosensor measures the acceleration of respiration during specific metabolic pathways of this drug . It has been applied, with good results, for determination of the active ingredient in the pharmaceutical tablet formulations.

J Food Prot, 1998 May, 61(5), 563 - 70
Combined effects of pH, nisin, and temperature on growth and survival of psychrotrophic Bacillus cereus; Jaquette CB et al.; Growth of vegetative cells and outgrowth of spores of enterotoxigenic psychrotrophic Bacillus cereus in refrigerated minimally processed food products is a public health concern . A study was undertaken to determine the combined effects of pH, nisin, and temperature on growth and survival of 20 strains of B . cereus . The minimum growth temperatures in tryptic soy broth (pH 7.3) and brain heart infusion broth (BHI broth, pH 7.4) were 5 degrees C for two strains and 8 degrees C for five other strains . Vegetative cells of four of eight strains grew at 8 degrees C in BHI broth (pH 6.01 and 6.57) containing 10 micrograms of nisin per ml . At 15 degrees C, all strains grew at pH 5.53 to 6.57; three strains tolerated nisin at 50 micrograms/ml (pH 6.57), whereas two other strains had a maximum tolerance of 10 micrograms of nisin per ml . Tolerance of vegetative cells of B . cereus to nisin increased as the pH of the broth was increased from 5.53 to 6.01 and again to pH 6.57 . Outgrowth of spores (six of six strains tested) was inhibited by 5 and 50 micrograms of nisin per ml at 8 and 15 degrees C, respectively . At 15 degrees C, outgrowth of spores of two strains occurred at pH 6.52 in BHI broth containing 10 micrograms of nisin per ml . The effectiveness of nisin in controlling the growth of psychrotrophic strains of B . cereus capable of causing human illness was more pronounced at 8 degrees C than at 15 degrees C and as the pH was decreased from 6.57 to 5.53 . Studies to determine the effectiveness of nisin in controlling growth of psychrotrophic B . cereus in nonpasteurized foods held at refrigeration temperatures are warranted.

Biochemistry, 1998 Aug 18, 37(33), 11621 - 8
Phospholipase C hydrolysis of phospholipids in bilayers of mixed lipid compositions; Ruiz-Arguello MB et al.; Phosphatidylcholine phospholipase C (EC 3.1.4.3) from Bacillus cereus has been assayed with substrates in the form of large unilamellar vesicles . Phosphatidylcholine, phosphatidylethanolamine (also a substrate for the enzyme), sphingomyelin, and cholesterol have been mixed in various proportions, in binary, ternary, and quaternary mixtures . A lag period, followed by a burst of enzyme activity, has been found in all cases . The activity burst was always accompanied by an increase in turbidity of the vesicle suspension . Varying lipid compositions while keeping constant all the other parameters leads to a range of lag times extending over 2 orders of magnitude (from 0.13 to 38.0 min), and a similar variability is found in maximal enzyme rates (from 0.40 to 55.9 min-1) . Meanwhile, the proportion of substrate that is hydrolyzed during the lag period remains relatively constant at 0.10% moles of total lipid, in agreement with the idea that enzyme activation is linked to vesicle aggregation through diacylglycerol-rich patches . Phosphatidylethanolamine and cholesterol enhance the enzyme activity in a dose-dependent way: they reduce the lag times and increase the maximal rates . The opposite is true of sphingomyelin . These lipids exert each its own peculiar effect, positive or negative, either alone or in combination, so that the susceptibility of a given mixture to the enzyme activity can be to some extent predicted from its composition . Phospholipase C activity is not directly influenced by the formation of nonlamellar structures . However, the presence of lipids with a tendency to form nonlamellar phases, such as phosphatidylethanolamine or cholesterol, stimulates the enzyme even under conditions at which purely lamellar phases exist . Conversely sphingomyelin, a well-known stabilizer of the lamellar phase, inhibits the enzyme . Thus phospholipase C appears to be regulated by the overall geometry and composition of the bilayer.

J Food Prot, 1998 Mar, 61(3), 344 - 9
Validation of antibiotic residue tests for dairy goats; Zeng SS et al.; The SNAP test, LacTek test (B-L and CEF), Charm Bacillus sterothermophilus var . calidolactis disk assay (BsDA), and Charm II Tablet Beta-lactam sequential test were validated using antibiotic-fortified and -incurred goat milk following the protocol for test kit validations of the U.S . Food and Drug Administration Center for Veterinary Medicine . SNAP, Charm BsDA, and Charm II Tablet Sequential tests were sensitive and reliable in detecting antibiotic residues in goat milk . All three assays showed greater than 90% sensitivity and specificity at tolerance and detection levels . However, caution should be taken in interpreting test results at detection levels . Because of the high sensitivity of these three tests, false-violative results could be obtained in goat milk containing antibiotic residues below the tolerance level . Goat milk testing positive by these tests must be confirmed using a more sophisticated methodology, such as high-performance liquid chromatography, before the milk is condemned . LacTek B-L test did not detect several antibiotics, including penicillin G, in goat milk at tolerance levels . However, LacTek CEF was excellent in detecting ceftiofur residue in goat milk.

J Food Prot, 1998 Feb, 61(2), 196 - 200
Effectiveness of cleaning and disinfection procedures on the removal of enterotoxigenic bacillus cereus from infant feeding bottles; Rowan NJ et al.; Reconstituted infant milk formulas are considered a food class of high risk because of the susceptibility of the infant population to enteric bacterial pathogens, severe response to enterotoxins, and increased mortality . Twenty infant feeding bottles, contaminated with different levels of enterotoxigenic Bacillus cereus, were subjected in triplicate to a variety of commonly used cleaning and disinfection procedures Although thorough cleaning reduced microbial numbers, it did not remove all B . cereus present . Three commercially available disinfection procedures (i.e., one chemical and two thermal) successfully eliminated this organism when the level of contamination was <10(5) organisms ml(-1) . However, the chemical disinfection method failed to eliminate enterotoxigenic B . cereus totally at potentially hazardous contamination levels (i.e., greater than or equal to 10(5) organisms ml(-1)) that may be encountered under storage abuse conditions in the home.

J Egypt Soc Parasitol, 1998 Aug, 28(2), 461 - 79
Autoclaved cercarial vaccine: a new hope against schistosomiasis parasitologic, histopathologic and immunologic studies; Eissa MM et al.; The efficacy of autoclaved cercarial vaccine (ACV) in eliciting protective immunity against Schistosoma mansoni infection was investigated in Swiss strain Albino mice . Two main groups of animals were used . One served as control group and the second was vaccinated with ACV mixed with Bacille Clamette-Guerin (BCG) as an adjuvant, in a single, double and triple doses 2 weeks apart . Six weeks after the first vaccination, all animals were challenged with freshly liberated cercariae of Schistosoma mansoni then sacrificed 7 weeks later . Parasitological, histopathological and immunological studies showed promising results which gave hope in evolution of anti-schistosomal vaccine.

EMBO J, 1998 Aug 17, 17(16), 4545 - 58
The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif; Davies C et al.; We report the 1.7 A crystal structure of ribosomal protein S4 from Bacillus stearothermophilus . To facilitate the crystallization, 41 apparently flexible residues at the N-terminus of the protein have been deleted (S4Delta41) . S4Delta41 has two domains; domain 1 is completely alpha-helical and domain 2 comprises a five-stranded antiparallel beta-sheet with three alpha-helices packed on one side . Domain 2 is an insertion within domain 1, and it shows significant structural homology to the ETS domain of eukaryotic transcription factors . A phylogenetic analysis of the S4 primary structure shows that the likely RNA interaction surface is predominantly on one side of the protein . The surface is extensive and highly positively charged, and is centered on a distinctive canyon at the domain interface . The latter feature contains two arginines that are totally conserved in all known species of S4 including eukaryotes, and are probably crucial in binding RNA . As has been shown for other ribosomal proteins, mutations within S4 that affect ribosome function appear to disrupt the RNA-binding sites . The structure provides a framework with which to probe the RNA-binding properties of S4 by site-directed mutagenesis.

Int J Food Microbiol, 1998 Jun 30, 42(1-2), 1 - 7
Current microbiological status of 'health foods' sold in Canada; Warburton DW et al.; A follow-up survey was conducted across Canada to evaluate the current status of 'health foods' sold in Canada . A total of 1239 sample units of 'health foods' were analysed for a variety of bacteria, including aerobic colony counts (ACC), coliforms, aerobic and anaerobic sporeformers, Escherichia coli and Bacillus cereus . Results presented indicate that 16.8-18.4% of the 'health foods' exceed ACC guidelines, 16.0-17.8% exceeded coliform guidelines, 35.7% exceeded aerobic sporeformer guidelines, 81.4% exceeded anaerobic sporeformer guidelines, and 9% exceeded B . cereus guidelines . Some ACC were further identified and found to be opportunistic pathogens, including the genera Bacillus, Enterococcus, and Staphylococcus . It was concluded that more extensive surveillance of this industry by health officials is needed.

Biochem Biophys Res Commun, 1998 Aug 10, 249(1), 207 - 12
Structural and functional role of tryptophan in xylanase from an extremophilic Bacillus: assessment of the active site; Nath D et al.; Microenvironment and conformation of the active site of xylanase from an extremophilic Bacillus was deciphered for the first time using fluorescence spectroscopy . NBS modified enzyme showed complete inactivation and the kinetic analysis implicated the presence of an essential tryptophan at the active site of xylanase . Xylan (0.5%) protected the enzyme completely from inactivation with NBS, whereas it afforded 35% protection against the loss of fluorescence, suggesting that not all the tryptophans are involved at the substrate binding site . Quenching studies revealed that acrylamide was more efficient than KI and CsCl as indicated by the higher Stern-Volmer quenching constants (Ksv) . The steric factor represented by the percentage accessibility of the tryptophan residues of XylII was higher with the positively charged Cs+ (80) than with the negatively charged I- (10), suggesting that the tryptophan residues are located in a relatively electronegative environment . In the presence of 6 M Gdn HCl the fluorescence shifted to 350 nm with increased accessibility of the fluorophore to the quenchers . The proximity of the essential carboxyl groups with a high pKa value of 6.9 {Chauthaiwale and Rao (1994) Biochim . Biophys . Acta} probably contributes to the electronegative environment of the tryptophan residue . Our results on sequence analysis of the gene encoding for XylII (Accession Number U83602 in the GenBank database) have shown that Trp 61 is highly conserved and may play a role in the structure-function relationship of the enzyme.

Nucleic Acids Res, 1998 Sep 1, 26(17), 4012 - 8
The role of supercoiling in mycobacteriophage L5 integrative recombination; Pena CE et al.; The genome of temperate mycobacteriophage L5 integrates into the chromosomes of its hosts, including Mycobacterium smegmatis , Mycobacterium tuberculosis and bacille Calmette-Guerin . This integrase-mediated site-specific recombination reaction occurs between the phage attP site and the mycobacterial attB site and requires the mycobacterial integration host factor . Here we examine the role of supercoiling in this reaction and show that integration is stimulated by DNA supercoiling but that supercoiling of either the attP or the attB substrate enhances recombination . Supercoiling thus facilitates a post-synaptic recombination event . We also show that, while supercoiling is not required for the production of a recombinagenic intasome, a mutant attP DNA deficient in binding of the host factor acquires a dependence on supercoiling for intasome formation and recombination.

Syst Appl Microbiol, 1998 Jun, 21(2), 179 - 84
A novel class of mosquitocidal delta-endotoxin, Cry19B, encoded by a Bacillus thuringiensis serovar higo gene; Hwang SH et al.; Partially digested HincII fragments of DNA from a mosquitocidal strain of Bacillus thuringiensis serovar higo were cloned into pBluescript II SK(+) and propagated in Escherichia coli . Recombinant cells were screened immunologically for the production of parasporal inclusion antigens . One E . coli clone harboring a recombinant plasmid exhibited larvicidal activity to Culex pipiens molestus, but not to Anopheles stephensi . Hybridization experiments revealed that the gene of the toxin protein is located on a 110 kb plasmid of B . thuringiensis serovar higo . Sequence analysis detected an open reading frame of 2046 nucleotides encoding a polypeptide of 682 amino acid residues with a predicted molecular weight of 78,467 . The gene encoded five block regions commonly conserved in the insecticidal protein genes of B . thuringiensis . Amino acid sequence of the 78 kDa protein shared 49% identity and 56% similarity with that of the Cry19A protein from B . thuringiensis serovar jegathesan . A new class of delta-endotoxin protein, designated Cry19B, was established on the basis of this protein.

Parazitologiia, 1998 May-Jun, 32(3), 264 - 7
{The effect of infection by the entomopathogenic bacterium Bacillus thuringiensis on the spread of microsporidia in an inversion-polymorphic population of the malarial mosquito Anopheles messeae (Diptera: Culicidae)}; Burlak VA et al.; A specificity of distribution of the microsporidia Parathelohania sp . in a natural population of Anopheles messeae and in those, which survived after the treatment by subspecies Bacillus thuringiensis fukuokaensis, B . t . darmstadiensis and B . t . kyushuensis (pathotype B), were observed . The microsporidia Parathelohania sp . affected males and it did not show a genotypic specificity in control, but it had a genotypic specificity after the B . thuringiensis subspecies treatment.

Arch Kriminol, 1998 May-Jun, 201(5-6), 172 - 81
{Morphologic detection of Bacillus cereus in blank cartridges}; Rothschild MA et al.; Wound infections after gunshot wounds from live ammunition can produce serious complications . It is well known that projectiles per se are neither sterile nor does their firing cause sterilization . The germs on the surface of a projectile enter the body together with the projectile and are thus introduced into the wound together with skin bacteria . However it is less known that wound infections can occur in wounds caused by the gas jet from blank ammunition (mainly from shots at very close range) . In such ammunition without a projectile, the propellant particles are usually contaminated with bacteria which find their way into the wound together with skin germs . In previous investigations, we have microbiologically detected the species Bacillus cereus in the propellant of blank cartridges . In the present study, we have applied scanning electron microscopic methods to find out which areas of the blank cartridges are colonized by these bacteria . For this purpose 20 blank cartridges, each from 4 different manufacturers, were electronmicroscopically examined . B . cereus only found on the surface of intact nitrocellulose particles but not in the interior of broken prepared propellant particles . Bacterial structures were not morphologically identified on black powder particles.

Plant Physiol, 1998 Aug, 117(4), 1445 - 61
Direct evidence for rapid degradation of Bacillus thuringiensis toxin mRNA as a cause of poor expression in plants; De Rocher EJ et al.; It is well established that the expression of Bacillus thuringiensis (B.t.) toxin genes in higher plants is severely limited at the mRNA level, but the cause remains controversial . Elucidating whether mRNA accumulation is limited transcriptionally or posttranscriptionally could contribute to effective gene design as well as provide insights about endogenous plant gene-expression mechanisms . To resolve this controversy, we compared the expression of an A/U-rich wild-type cryIA(c) gene and a G/C-rich synthetic cryIA(c) B.t.-toxin gene under the control of identical 5' and 3' flanking sequences . Transcriptional activities of the genes were equal as determined by nuclear run-on transcription assays . In contrast, mRNA half-life measurements demonstrated directly that the wild-type transcript was markedly less stable than that encoded by the synthetic gene . Sequences that limit mRNA accumulation were located at more than one site within the coding region, and some appeared to be recognized in Arabidopsis but not in tobacco (Nicotiana tabacum) . These results support previous observations that some A/U-rich sequences can contribute to mRNA instability in plants . Our studies further indicate that some of these sequences may be differentially recognized in tobacco cells and Arabidopsis.

Plant Physiol, 1998 Aug, 117(4), 1433 - 43
Premature polyadenylation at multiple sites within a Bacillus thuringiensis toxin gene-coding region; Diehn SH et al.; Some foreign genes introduced into plants are poorly expressed, even when transcription is controlled by a strong promoter . Perhaps the best examples of this problem are the cry genes of Bacillus thuringiensis (B.t.), which encode the insecticidal proteins commonly referred to as B.t . toxins . As a step toward overcoming such problems most effectively, we sought to elucidate the mechanisms limiting the expression of a typical B.t.-toxin gene, cryIA(c), which accumulates very little mRNA in tobacco (Nicotiana tabacum) cells . Most cell lines transformed with the cryIA(c) B.t.-toxin gene accumulate short, polyadenylated transcripts . The abundance of these transcripts can be increased by treating the cells with cycloheximide, a translation inhibitor that can stabilize many unstable transcripts . Using a series of hybridizations, reverse-transcriptase polymerase chain reactions, and RNase-H-digestion experiments, poly(A+) addition sites were identified in the B.t.-toxin-coding region corresponding to the short transcripts . A fourth polyadenylation site was identified using a chimeric gene . These results demonstrate for the first time to our knowledge that premature polyadenylation can limit the expression of a foreign gene in plants . Moreover, this work emphasizes that further study of the fundamental principles governing polyadenylation in plants will have basic as well as applied significance.

Pathol Int, 1998 Jul, 48(7), 507 - 11
Gastrospirillum hominis and Helicobacter pylori infection in Thai individuals: comparison of histopathological changes of gastric mucosa; Yali Z et al.; The presence of Helicobacter pylori (H . pylori) in the stomach is closely associated with histological signs of chronic active gastritis and peptic ulcer . Another spiral organism named Gastrospirillum hominis (G . hominis) has led to further interest in the bacterial pathogenesis of gastritis . Due to the low prevalence of G . hominis, it is difficult to evaluate its biological behavior . Recently 16 cases of G . hominis-associated gastritis were found in 257 Thai individuals, which made it possible to study the biological characteristics of G . hominis and its relationship with gastric mucosal inflammation . The results showed that H . pylori and G . hominis could be easily observed in the lower third of the mucous layer and in the mucosa of the gastric pits by means of toluidine blue staining . Both bacteria immunostained positive . Helicobacter pylori were usually in the shape of curved bacillary while G . hominis often appeared in spiral configuration . In 257 cases of Thai subjects, 169 cases were found to be H . pylori positive, the detection rate was 65.7%, and 16 cases were G . hominis positive, with a 6.2% detection rate . In G . hominis infection, 43.6% of cases had normal gastric mucosa . Superficial, erosive and atrophic gastritis cases were 13.2, 10.9 and 12.5%, respectively . Mucosal inflammation was usually severe in H . pylori, but neutrophil polymorph infiltration was often mild and focal in G . hominis infection . Although no G . hominis infection with carcinoma was shown in our cases, the occurrence of mucosal atrophy, metaplasia and dysplasia was higher in both bacterial infections compared with H . pylori- and G . hominis-negative cases . It is suggested that G . hominis may be partly responsible for the mucosal inflammation and some malignant-associated lesions.

Braz J Med Biol Res, 1998 Jun, 31(6), 763 - 9
Construction of occluded recombinant baculoviruses containing the full-length cry1Ab and cry1Ac genes from Bacillus thuringiensis; Ribeiro BM et al.; The administration of baculoviruses to insects for bioassay purposes is carried out, in most cases, by contamination of food surfaces with a known amount of occlusion bodies (OBs) . Sinceper os infection is the natural route of infection, occluded recombinant viruses containing crystal protein genes (cry1Ab and cry1Ac) from Bacillus thuringiensis were constructed for comparison with the baculovirus prototype Autographa californica nucleopolyhedrovirus (AcNPV) . The transfer vector pAcUW2B was used for construction of occluded recombinant viruses . The transfer vector containing the crystal protein genes was cotransfected with linearized DNA from a non-occluded recombinant virus . The isolation of recombinant viruses was greatly facilitated by the reduction of background "wild type" virus and the increased proportion of recombinant viruses . Since the recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve the pathogenicity of the recombinant viruses when compared with the wild type AcNPV, and in order to compare expression levels of the full-length crystal proteins produced by non-occluded and occluded recombinant viruses the full-length cry1Ab and cry1Ac genes were chosen for construction of occluded recombinant viruses . The recombinant viruses containing full-length and truncated forms of the crystal protein genes did not seem to improve its pathogenicity but the size of the larvae infected with the recombinant viruses was significantly smaller than that of larvae infected with the wild type virus.

J Mol Biol, 1998 Aug 21, 281(3), 539 - 51
An investigation of the dynamics of ribosomal protein L9 using heteronuclear NMR relaxation measurements; Lillemoen J et al.; The dynamic properties of ribosomal protein L9 from Bacillus stearothermophilus were investigated in solution using an analysis of nitrogen-15 longitudinal and transverse relaxation rates and amide nitrogen-proton nuclear Overhauser effects . The relaxation rates of the amide nitrogen nuclei were found to be correlated with the angle between the amide nitrogen-proton bond vectors and the long axis of the protein . This directional dependence of the nuclear relaxation rates is consistent with the protein having a highly elongated shape in solution, consistent with that observed in previous X-ray crystallographic studies of the crystalline form . Analysis of the nuclear relaxation data shows that the solvent-exposed nine-turn alpha helix connecting the two domains has a relatively high degree of order, in contrast to the connecting helix in the similarly shaped, but functionally different, calmodulin protein . The rotational correlation times associated with the amide nitrogen atoms of the N-terminal domain are on average slightly shorter than those of the C-terminal domain and connecting helix, providing evidence that the N-terminal domain exhibits some degree of independence in tumbling, in addition to other fast internal motions . The putative RNA-binding surfaces in each of the protein domains are characterized by relatively low order parameters, indicating that these are the most flexible regions of the molecule . Overall, the picture of the internal dynamics provided by nuclear relaxation measurements is similar to that obtained from a detailed study of amide proton exchange rates, but differs markedly from the picture provided by crystallographic temperature factors . The present study describes a molecule with unusual and complex dynamic properties, and supports a model where the protein functions as a "molecular strut" within the ribosome .

Biochemistry, 1998 Aug 11, 37(32), 11332 - 42
Probing the mechanism of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases by chemical rescue of inactive mutants at catalytically essential residues; Viladot JL et al.; The role of the key catalytic residues Glu134 and Glu138 in the retaining 1,3-1,4-beta-glucanase from Bacillus licheniformis is probed by a chemical rescue methodology based on enzyme activation of inactive mutants by the action of added nucleophiles . While Glu134 was proposed as the catalytic nucleophile on the basis of affinity labeling experiments, no functional proof supported the assignment of Glu138 as the general acid-base catalyst . Alanine replacements are prepared by site-directed mutagenesis to produce the inactive E138A and E134A mutants . Addition of azide reactivates the mutants in a concentration-dependent manner using an activated 2, 4-dinitrophenyl glycoside substrate . The chemical rescue operates by a different mechanism depending on the mutant as deduced from 1H NMR monitoring and kinetic analysis of enzyme reactivation . E138A yields the beta-glycosyl azide product arising from nucleophilic attack of azide on the glycosyl-enzyme intermediate, thus proving that Glu138 is the general acid-base residue . Azide activates the deglycosylation step (increasing kcat), but it also has a large effect on a previous step (as seen by the large decrease in KM, the increase in kcat/KM, and the pH dependence of activation), probably increasing the rate of glycosylation through Bronsted acid catalysis by enzyme-bound HN3 . By contrast, azide reactivates the E134A mutant through a single inverting displacement to give the alpha-glycosyl azide product, consistent with Glu134 being the catalytic nucleophile . Formate as an exogenous nucleophile has no effect on the E138A mutant, whereas it is a better activator of E134A than azide . Although the reaction yields the normal hydrolysis product, a transient compound was detected by 1H NMR, tentatively assigned to the alpha-glycosyl formate adduct . This is the first case where a nonmodified sugar gives a long-lived covalent intermediate that mimics the proposed glycosyl-enzyme intermediate of retaining glycosidases.

J Infect Dis, 1998 Aug, 178(2), 584 - 8
DNA sequence resembling vanA and vanB in the vancomycin-resistant biopesticide Bacillus popilliae; Rippere K et al.; The origin of high-level vancomycin resistance in enterococci is unknown . Biopesticidal powders containing spores of Bacillus popilliae, which is vancomycin-resistant, have been used for >50 years in the United States for suppression of Japanese beetle populations . Using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci, an amplicon in B . popilliae was identified and sequenced . The putative ligase gene in B . popilliae had 76.8% and 68.4%-68.9% nucleotide identity to the sequences of the vanA and vanB genes, respectively . There was 75.3% and 69.3%-69.9% identity between the translation of the putative ligase gene in B . popilliae and the translation of the vanA and vanB genes, respectively . We have identified a gene resembling vanA and vanB in B . popilliae . The gene in B . popilliae may have been a precursor to or have had an ancestral gene in common with vancomycin resistance genes in enterococci.

J Virol, 1998 Sep, 72(9), 7407 - 19
A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit; Masterson PJ et al.; The human papillomavirus (HPV) E1 and E2 proteins bind cooperatively to the viral origin of replication (ori), forming an E1-E2-ori complex that is essential for initiation of DNA replication . All other replication proteins, including DNA polymerase alpha-primase (polalpha-primase), are derived from the host cell . We have carried out a detailed analysis of the interactions of HPV type 16 (HPV-16) E1 with E2, ori, and the four polalpha-primase subunits . Deletion analysis showed that a C-terminal region of E1 (amino acids {aa} 432 to 583 or 617) is required for E2 binding . HPV-16 E1 was unable to bind the ori in the absence of E2, but the same C-terminal domain of E1 was sufficient to tether E1 to the ori via E2 . Of the polalpha-primase subunits, only p68 bound E1, and binding was competitive with E2 . The E1 region required (aa 397 to 583) was the same as that required for E2 binding but additionally contained 34 N-terminal residues . In confirmation of these differences, we found that a monoclonal antibody, mapping adjacent to the N-terminal junction of the p68-binding region, blocked E1-p68 but not E1-E2 binding . Sequence alignments and secondary-structure prediction for HPV-16 E1 and other superfamily 3 (SF3) viral helicases closely parallel the mapping data in suggesting that aa 439 to 623 constitute a discrete helicase domain . Assuming a common nucleoside triphosphate-binding fold, we have generated a structural model of this domain based on the X-ray structures of the hepatitis C virus and Bacillus stearothermophilus (SF2) helicases . The modelling closely matches the deletion analysis in suggesting that this region of E1 is indeed a structural domain, and our results suggest that it is multifunctional and critical to several stages of HPV DNA replication.

J Bacteriol, 1998 Aug, 180(16), 4146 - 53
Influence of the secondary cell wall polymer on the reassembly, recrystallization, and stability properties of the S-layer protein from Bacillus stearothermophilus PV72/p2; Sara M et al.; The high-molecular-weight secondary cell wall polymer (SCWP) from Bacillus stearothermophilus PV72/p2 is mainly composed of N-acetylglucosamine (GlcNAc) and N-acetylmannosamine (ManNAc) and is involved in anchoring the S-layer protein via its N-terminal region to the rigid cell wall layer . In addition to this binding function, the SCWP was found to inhibit the formation of self-assembly products during dialysis of the guanidine hydrochloride (GHCl)-extracted S-layer protein . The degree of assembly (DA; percent assembled from total S-layer protein) that could be achieved strongly depended on the amount of SCWP added to the GHCl-extracted S-layer protein and decreased from 90 to 10% when the concentration of the SCWP was increased from 10 to 120 microg/mg of S-layer protein . The SCWP kept the S-layer protein in the water-soluble state and favored its recrystallization on solid supports such as poly-L-lysine-coated electron microscopy grids . Derived from the orientation of the base vectors of the oblique S-layer lattice, the subunits had bound with their charge-neutral outer face, leaving the N-terminal region with the polymer binding domain exposed to the ambient environment . From cell wall fragments about half of the S-layer protein could be extracted with 1 M GlcNAc, indicating that the linkage type between the S-layer protein and the SCWP could be related to that of the lectin-polysaccharide type . Interestingly, GlcNAc had an effect on the in vitro self-assembly and recrystallization properties of the S-layer protein that was similar to that of the isolated SCWP . The SCWP generally enhanced the stability of the S-layer protein against endoproteinase Glu-C attack and specifically protected a potential cleavage site in position 138 of the mature S-layer protein.

Kekkaku, 1998 Jun, 73(6), 387 - 94
{Tuberculosis statistics among the homeless population in Nagoya City from 1991 to 1995}; Yamanaka K et al.; A survey was conducted to clarify the tuberculosis (TB) situation among the homeless during the period from 1991 to 1995 in Nagoya city, using 5,222 registration cards of TB cases registered at one of Nagoya City's 16 Health Centers . Out of 5,222 TB cases, there were 269 homeless cases (267 male and 2 female) . Ninety-seven percent of them were pulmonary TB cases . The incidence and prevalence rates of TB per 100,000 among the homeless were estimated at around 1,500 and around 2,400, respectively, around 20 times higher than those of the non-homeless male over the 19 years of age . A decrease in the incidence rate of TB cases among the homeless was not seen, although the rate among the non-homeless decreased gradually . The percentage of infectious (bacillary and/or cavitary) tuberculous cases among the homeless was higher than in the non-homeless . In the infectious cases, the percentage of smear-positive bacillary cases or far advanced cavitary cases was 52.1% or 9.4% among the homeless compared to 48.1% or 2.6% among the non-homeless, respectively . The detection rate by chest X-ray examination of the homeless was 3.9%.

Urol Res, 1998, 26(3), 161 - 4
Immunohistochemical evaluation of p53, proliferating cell nuclear antigen (PCNA) and bcl-2 expression during bacillus Calmette-Guerin (BCG) intravesical instillation therapy for superficial bladder cancers; Okamura T et al.; Bacillus Calmette-Guerin (BCG) immunotherapy for superficial bladder cancer is now widespread, but non-effective cases are not uncommon and it has yet to be clarified why this is the case . In an attempt to cast light on this problem, we evaluated differences between effective and non-effective cases immunohistochemically using p53, proliferating cell nuclear antigen (PCNA), and bcl-2 antibodies . Between March 1988 and March 1996 a total of 79 superficial bladder cancer patients were treated with BCG intravesical instillation therapy after transurethral resection of bladder tumor (TUR-Bt) . Of these, 19 demonstrated recurrence after the initial treatment . From the 60 remaining patients without recurrence, we randomly chose 19 additional cases and evaluated both series for p53, PCNA and bcl-2 immunohistochemical staining using formalin-fixed, paraffin-embedded tissues . For the recurrent cases, material taken prior and subsequent to BCG therapy was available for 17 of the 19 patients . Positive staining for p53 was noted for 42.1% (8/19) of both recurrent and non-recurrent cases, without any difference between the two . The rates for PCNA and bcl-2 were 52.6% (10/19) and 47.4% (9/19) in recurrent, and 36.8% (7/19) and 78.9% (15/19) in non-recurrent cases, respectively . Thus, there was a significant difference for lower incidences of bcl-2 in recurrent cases (P = 0.044) . Values for p53 and bcl-2 were respectively 47.1% (8/17) and 41.2% (7/17) pre-treatment, and 52.9% (9/17) and 35.3% (6/17) post-treatment in the recurrence group . In contrast to the similarity in these results, PCNA positive cases were 52.9% (9/17) pre-treatment and 17.6% (3/17) post-treatment . These data suggest that there are differences between BCG-sensitive and BCG-resistant bladder cancers in terms of bcl-2 expression.

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