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 det