Bacillus

Anticancer Drugs, 1997 Mar, 8(3), 217 - 24
Cellular cancer vaccine induces delayed-type hypersensitivity reaction and augments antibody response to tumor-associated carbohydrate antigens (sialyl Le(a), sialyl Le(x), GD3 and GM2) better than soluble lysate cancer vaccine; Ravindranath MH et al.; Allogenic whole cell and lysate cancer vaccines are associated with very different clinical outcome, which could be due to different immune responses to critical tumor-associated antigens . We used a guinea pig model to evaluate the immune responses to melanoma-associated carbohydrate antigens administered in whole cell and soluble lysate vaccines produced from the same cell lines and administered with or without Bacille Calmette-Guerin (BCG) . Animals immunized with whole cell vaccine developed a significantly higher delayed-type hypersensitivity (DTH) reaction . The IgG response to all tumor-associated carbohydrate antigens except GD2 was significantly higher in animals immunized with whole cell vaccine than lysate vaccine . This study indicates that whole cell vaccine is superior to soluble or lysate vaccine because it induces a better immune response against cell-surface antigens . The addition of BCG significantly increased the antibody response, suggesting that an exogenous adjuvant may immunopotentiate antigens better in the presence of an intact cell membrane.

Transgenic Res, 1997 Mar, 6(2), 169 - 76
Expression of a Bacillus thuringiensis cryIA(c) gene in transgenic peanut plants and its efficacy against lesser cornstalk borer; Singsit C et al.; The invasion of peanut (Arachis hypogaea L.) pods and seeds by aflatoxin-forming species of Aspergillus is linked to injury by the lesser cornstalk borer and frequently causes a severe reduction in crop quality . The lesser cornstalk borer is susceptible to the lepidopteran-active Bacillus thuringiensis insecticidal crystal protein . We have introduced a codon-modified Bacillus thuringiensis cryIA(c) gene into peanut using microprojectile bombardment . The toxin-coding region of a Bt cryIA(c) gene was reconstructed for expression in plants and the resulting 3.4 kb gene cassette (promoter: 1.8 kb coding: 3') was directly cloned into the BglII site of plant transformation vectors . The vectors contained the hph gene, conferring resistance to the antibiotic hygromycin . Somatic embryos initiated from immature peanut cotyledons of two cultivars were used as the target for bombardment . DNA from hygromycin-resistant embryogenic cell lines, regenerated plants, and a progeny plant showed the presence and integration of hph and Bt genes by PCR and/or Southern blot analyses . ELISA immunoassay of the CryIA(c) protein from the hygromycin-selected plants showed the expression of CryIA(c) protein up to 0.18% of total soluble protein . Insect feeding bioassay of transformed plants indicated various levels of resistance to the lesser cornstalk borer, from complete larval mortality to a 66% reduction in larval weight . A negative correlation between percent survival or larval weight and the amount of Bt CryIA(c) protein was recorded indicating in general that the higher the protein level the lower the survival or larval weight of the insect . Based on leaf bioassay, transformation of peanut with vectors containing the Bt cryIA(c) gene may be effective in protecting the peanut plants from damage by lepidopteran insect larvae of lesser cornstalk borer.

Acta Neuropathol (Berl), 1997 Mar, 93(3), 301 - 5
Necrotizing Bacillus cereus infection of the meninges without inflammatory reaction in a patient with acute myelogenous leukemia: a case report; Motoi N et al.; A 64-year-old man in a severely immunocompromised state due to acute myelogenous leukemia died, respirator-unaided, about 10 h after the abrupt onset of coma . An earlier blood culture had yielded Bacillus cereus . The autopsy, performed 2 h after death, demonstrated diffuse subarachnoid hemorrhage without berry aneurysms, and the formalin-fixed brain was tinged with gray-brownish discoloration . The sections of the brain presented a whitish tint of the surface layer of all portion of the cerebral cortices, even those in the sulci . Histological examination of the brain revealed leptomeningeal B . cereus dissemination, and widespread necrosis of the leptomeninges and arachnoid vessels without inflammatory cell reaction . The grossly recognizable whitish surface layer of the cerebral cortex showed overt hyperchromatism, and contained neurons more degenerative than those located in the deeper cortical layer . The total absence of inflammatory reaction may be explained by a combination of the immunocompromised state of the patient and the character of B . cereus infection, which in itself induces little inflammatory reaction . The prominent lesions were confined to the cerebral surface layer and leptomeningeal tissue including the arachnoid vessels, which were all bathed in the cerebrospinal fluid, suggesting that some necrotizing toxins had been secreted into the fluid by the B . cereus . The necrosis of arachnoid vessels is thought to have in turn caused diffuse subarachnoid hemorrhage and marked disturbance of the cerebral blood flow, resulting in the terminal coma.

Mol Microbiol, 1997 Mar, 23(5), 1053 - 62
Regulation of expression, genetic organization and substrate specificity of xylose uptake in Bacillus megaterium; Schmiedel D et al.; Xylose uptake in Bacillus megaterium depends on expression of a putative H+/xylose symporter encoded by xylT, the last gene in the xyl operon . Insertional inactivation of xylT leads to an apparent uptake deficiency determined with whole cells and severely slower growth on xylose as sole carbon source . Expression of xylT is xylose inducible and subject to carbon catabolite repression mediated by CcpA and cre . Northern analysis of the xyl mRNA reveals that a potential stem-loop structure located in the non-translated region between xylA and xylB presumably acts as a transcriptional terminator, as it leads to different amounts of the respective mRNA sections: the 5'-xylA portion is very abundant, while the 3'-xylBT portion constitutes only a fraction of it . XylT has an apparent Michaelis constant (KM) of approx . 100 microM and is competitively inhibited by glucose with an inhibitor constant KI of 16 mM.

Mol Microbiol, 1997 Mar, 23(5), 935 - 44
The penicillin sensory transducer, BlaR, involved in the inducibility of beta-lactamase synthesis in Bacillus licheniformis is embedded in the plasma membrane via a four-alpha-helix bundle; Hardt K et al.; Prediction studies, conformational analyses and membrane-topology mapping lead to the conclusion that the penicillin sensory transducer, BlaR, involved in the inducibility of beta-lactamase synthesis in Bacillus licheniformis, is embedded in the plasma membrane bilayer via four transmembrane segments TM1-TM4 that form a four-alpha-helix bundle . The extracellular 262-amino-acid-residue polypeptide, S340-R601, that is fused at the carboxy end of TM4, possesses the amino acid sequence signature of a penicilloyl serine transferase . It probably functions as penicillin sensor . As an independent entity, this polypeptide behaves as a high-affinity penicillin-binding protein . As a component of the full-size BlaR, it adopts a different conformation presumably because of interactions with the extracellular 63-amino-acid-residue P53-S115 loop that connects TM2 and TM3 . Reception of the penicillin-induced signal requires a precise conformation of the sensor but it does not involve penicilloylation of the serine residue S402 of motif STYK . Signal transmission through the plasma membrane by the four-alpha-helix bundle may proceed in a way comparable to that of the aspartate receptor, Tar . Signal emission in the cytosol by the intracellular 189-amino-acid-residue Y134-K322 loop that connects TM3 and TM4, may proceed via the activation of a putative metallopeptidase.

Eur Respir J, 1997 Mar, 10(3), 619 - 23
Neonatal BCG vaccination in Ireland: evidence of its efficacy in the prevention of childhood tuberculosis; Kelly P et al.; Data from the 1986 and 1991 National Tuberculosis Survey were used, together with the Census of Population for those years, to try and determine whether bacille Calmette-Guerin (BCG) vaccination policy had any influence on the reported incidence of tuberculosis in the Republic of Ireland . The age-specific incidence of tuberculosis for the country as a whole and for local areas, was determined with respect to neonatal BCG vaccination . Based on these data, the reported incidence of tuberculosis in people aged 15 yrs or younger in areas without a policy of neonatal BCG was shown to be significantly higher compared to areas that use neonatal BCG vaccination (p = 1.5x10(-5) in 1986; and p = 1.0x10(-7) for 1991) . It was estimated that some 646 vaccinations needed to be given to prevent one case of tuberculosis in 1986, and that the figure for 1991 was 551 vaccinations . This evidence supports a policy of continued neonatal bacille Calmette-Guerin vaccination in the population of the Republic of Ireland at present.

Clin Chem, 1997 Mar, 43(3), 533 - 8
Enzymatic assay of D-mannose in serum; Etchison JR et al.; We describe a new and improved enzymatic assay for determining the concentration of D-mannose in sera . Serum D-glucose is selectively converted to glucose-6 phosphate with the highly specific thermostable glucokinase (EC 2.7.1.2) from Bacillus stearothermophilus . The anionic reaction products and excess substrates are removed by a rapid and simple anion-exchange chromatography step in microcentrifuge spin columns . D-Mannose in the glucose-depleted sample is then assayed spectrophotometrically by using coupled enzymatic reactions . The quantitative elimination of glucose from the serum samples allowed the accurate and reproducible assay of serum mannose in the 0-200 mumol/L range . Recovery of mannose added to serum (5-200 mumol/L) was 94% +/- 4.4% . The intraassay CV was 6.7% at 40 mumol/L mannose (n = 5; 39.6 +/- 1.6 mumol/L) and 4.4% at 80 mumol/L (n = 11; 75.0 +/- 1.8 mumol/L); the interassay CV at these concentrations was 12.2% (n = 7; 36.9 +/- 2.1 mumol/L) and 9.8% (n = 7; 74.2 +/- 2.7 mumol/L), respectively . Sera from 11 healthy human volunteers contained an average of 54.1 +/- 11.9 mumol/L mannose (range 36-81 mumol/L).

Vet Surg, 1997 Mar-Apr, 26(2), 121 - 5
Evaluation of skin bacterial flora before and after aseptic preparation of clipped and nonclipped arthrocentesis sites in horses; Hague BA et al.; OBJECTIVE: This study evaluates skin bacterial flora before and after aseptic preparation of clipped and nonclipped arthrocentesis sites in horses . STUDY DESIGN: The hair over one midcarpal joint and one distal interphalangeal joint on each horse was clipped . The contralateral joint served as the nonclipped comparison . ANIMALS OR SAMPLE POPULATION: Twelve adult horses . METHODS: A prescrub sample for microbial culture was taken from the dorsal surface of all four joints for each horse . Each site was aseptically prepared with povidone iodine and 70% alcohol, followed by postscrub sampling for microbial culture . Colony forming units (CFUs) were determined for each sample, 24 hours after inoculation of blood agar plates . RESULTS: There was no significant difference (P > .05) in number of postscrub CFUs between clipped and nonclipped skin over the midcarpal or distal interphalangeal joints . Percent bacterial reduction (mean +/- SD%) after aseptic preparation differed significantly (P = .02) between clipped (99.8 +/- .003%) and nonclipped (96.2 +/- .05%) skin at the midcarpal joint, but not at the distal interphalangeal joint (clipped, 98.5 +/- .03% and nonclipped, 97.8 +/- 0.21%) . There was a significant difference (P = .009) in number of prescrub CFUs obtained from clipped and nonclipped skin for the midcarpal joint . There was no significant difference in number of prescrub CFUs between clipped and nonclipped skin at the distal interphalangeal joint . Bacteria isolated from both clipped and nonclipped skin sampled postscrub included Bacillus sp, nonhemolytic Staphylococcus sp, and Micrococcus sp . CONCLUSIONS: The presence of hair over the midcarpal and distal interphalangeal joints does not appear to inhibit the ability of antiseptics to effectively reduce bacterial flora to an acceptable level for arthrocentesis . CLINICAL RELEVANCE: Aseptic preparation of the skin over the midcarpal and distal interphalangeal joints can be accomplished without hair removal in horses.

Clin Exp Immunol, 1997 Mar, 107(3), 462 - 7
Immune response to Plasmodium falciparum antigens in Cameroonian primigravidae: evolution after delivery and during second pregnancy; Fievet N et al.; Mechanisms responsible for the increase in malaria susceptibility during pregnancy, and in particular during the first pregnancy, have not been elucidated . T and B cell responses to leucoagglutinin, bacille Calmette-Guerin (BCG) and to six Plasmodium falciparum antigens were longitudinally investigated in 33 pregnant women during their first pregnancy, after delivery, and during second pregnancy . Parasitological data obtained from the same women during and after the first pregnancy demonstrated the higher risk of P . falciparum infection during this pregnancy . Plasma levels of antibodies to Pf155/ RESA were lower during pregnancy than after delivery . Conversely, antibodies to P . falciparum asexual blood stages were higher during pregnancy than after delivery, suggesting that during pregnancy the regulation of antibody production may be variously impaired depending upon the antigens . The most striking finding of the present study is the impairment of the IL-2 cellular response during the first pregnancy . Conversely, proliferative responses, as well as IL-4 and interferon-gamma (IFN-gamma) responses, were either unaffected or moderately enhanced . No difference in humoral and cellular responses was observed between first and second pregnancy . The impairment of the IL-2 responses involved the response to malaria peptides and proteins, as well as the response to non-malarial antigens and to the mitogen leucoagglutinin . Thus, the alteration of malaria immunity might rather fall into the general frame of the depression of cellular immunity during pregnancy than involve a specific malaria phenomenon.

J Invertebr Pathol, 1997 Mar, 69(2), 92 - 104
A new tissue technique for evaluating effects of Bacillus thuringiensis toxins on insect midgut epithelium; Braun L et al.; Epithelial tissue wholemounts were produced after enzymatic removal of basal lamina and connective tissue from midguts of Trichoplusia ni larvae . Wholemounts were nourished in artificial hemolymph and tissue viability was assessed for up to 24 hr using the vital dyes trypan blue, acridine orange (AO), propidium iodide (PI), and 4', 6-diamidino-2-phenylindole (DAPI) . Peritrophic membrane synthesis and modification of Bacillus thuringiensis Cry1Ac protoxin to active toxin confirmed some normal epithelial function . Vital staining using the combination of AO and PI, or DAPI revealed altered membrane permeability in columnar epithelial and regenerative cells of tissues treated with activated Cry1Ac toxin while feeding and oral inoculation bioassays verified Cry1Ac toxicity . DAPI was selected to identify target cells in a rapid and highly sensitive assay.

Arch Biochem Biophys, 1997 Mar 1, 339(1), 17 - 23
Microbial system for polysaccharide depolymerization: enzymatic route for gellan depolymerization by Bacillus sp . GL1; Hashimoto W et al.; A bacterium-producing polysaccharide lyase (gellan lyase) was isolated from soil samples and identified to be Bacillus sp . The lyase was purified from the culture fluid of the bacterium (designated Bacillus sp . GL1) grown in the presence of gellan as a carbon source . The purified gellan lyase depolymerized deacetylated gellan and gave a single oligosaccharide product with a molecular weight of 646, which was determined by fast atom bombardment mass spectrometry . The structure of the product was determined by the combination of mass spectrometry, HPLC analysis, and high-resolution proton nuclear magnetic resonance spectroscopy to be a tetrasaccharide of glucuronyl-glucosyl-rhamnosyl-glucose with unsaturated glucuronic acid at the nonreducing terminal . When incubated in cell extracts of Bacillus sp . GL1, the tetrasaccharide was first converted to trisaccharide without unsaturated glucuronyl residue, and the trisaccharide was then converted to hydrolyzed monosaccharides glucose and rhamnose . These results show that, in the bacterium Bacillus sp . GL1, gellan is first depolymerized to give a tetrasaccharide, a repeating unit in gellan molecule, by an extracellular gellan lyase and then the tetrasaccharide is hydrolyzed to monosaccharides by successive actions of intracellular exoglycosidases.

Appl Environ Microbiol, 1997 Mar, 63(3), 1182 - 4
Maltodextrin stimulates growth of Bacillus cereus and synthesis of diarrheal enterotoxin in infant milk formulae; Rowan NJ et al.; One hundred reconstituted milk-based infant formulae (IMF) representative of 10 leading brands available in many European Economic Community countries were examined for Bacillus cereus and for the presence of diarrheal enterotoxin . Sixty-three reconstituted IMF supported growth of the organism after 14 h at 25 degrees C, and in 4 of these, which contained maltodextrin, enterotoxin was detected . Reconstituted IMF (and basal synthetic media) supplemented with > or = 0.1% maltodextrin supported both growth of B . cereus and diarrheal toxin production when incubated for 14 h or more at 25 degrees C.

Appl Environ Microbiol, 1997 Mar, 63(3), 1054 - 7
Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis; Asano SI et al.; A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined . Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K . Shinagawa, p . 181-193, in A . E . Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B . cereus strain, but no report of cloning of the enterotoxin gene has been published . In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B . cereus FM1 made with the lambda gt11 vector . An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions . The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues . In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B . cereus gene . These primers were designed to amplify the full protein-coding region . PCR conducted with DNA preparations from the B . thuringiensis subsp . sotto and B . thuringiensis subsp . israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B . cereus enterotoxin . Nucleotide sequences of the amplified DNA segments showed that these B . thuringiensis strains contain an enterotoxin gene very similar to that of B . cereus . Further PCR screening of additional B . thuringiensis strains with four primer pairs in one reaction revealed that some additional B . thuringiensis strains contain enterotoxin-like genes.

Appl Environ Microbiol, 1997 Mar, 63(3), 1006 - 10
Comparison of the cellular fatty acid composition of a bacterium isolated from a human and alleged to be Bacillus sphaericus with that of Bacillus sphaericus isolated from a mosquito larvicide; Siegel JP et al.; The cellular fatty acid (CFA) composition of the cytoplasmic membrane of a bacillus isolated from a human lung and deposited in the National Collection of Type Cultures as Bacillus sphaericus NCTC 11025 was determined by gas-liquid chromatography . The CFA composition of B . sphaericus 2362, isolated from a microbial larvicide, and those of B . sphaericus reference strains obtained from public collections were also determined . Samples were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages . Samples that linked at a Euclidean distance of < or = 2.0 U were considered to belong to the same strain . NCTC 11025 and the type strain of B . sphaericus, ATCC 14577, were mixed; all other isolates were monotypic . The predominant fatty acid in NCTC 11025 was 12-methyltetradecanoic acid, while the predominant fatty acid in the remaining isolates was 13-methyltetradecanoic acid . NCTC 11025 linked to the other isolates at a Euclidean distance of 83.8 U, and we concluded that it belongs to a different species that we could not identify . We could distinguish among six DNA homology groups of B . sphaericus by using fatty acids . Within DNA homology group IIA, strain 2362 could be distinguished from other strains belonging to serotype H5a, 5b . We concluded that CFA analysis is a useful technique to determine if future human isolates identified as B . sphaericus in fact belong to other species of bacteria or whether the isolates originated from commercial products.

Appl Environ Microbiol, 1997 Mar, 63(3), 956 - 61
Identification of ATP-dependent phosphofructokinase as a regulatory step in the glycolytic pathway of the actinomycete Streptomyces coelicolor A3(2); Alves AM et al.; The ATP-dependent phosphofructokinase (ATP-PFK) of Streptomyces coelicolor A3(2) was purified to homogeneity (1,600-fold) and characterized (110 kDa, with a single type of subunit of 40 kDa); it is allosterically inhibited by phosphoenolpyruvate . Cloning of the pfk gene of S . coelicolor A3(2) and analysis of the deduced amino acid sequence (343 amino acids; 36,667 Da) revealed high similarities to the PPi-PFK enzyme from Amycolatopsis methanolica (tetramer, nonallosteric; 70%) and to the allosteric ATP-PFK enzymes from other bacteria, e.g., Escherichia coli (tetramer; 37%) and Bacillus stearothermophilus (tetramer, 41%) . Further structural and functional analysis of the two actinomycete PFK enzymes should elucidate the features of these proteins that determine substrate specificity (ATP versus PPi) and allosteric (in)sensitivity.

J Bacteriol, 1997 Mar, 179(5), 1824 - 7
Most of the propeptide is dispensable for stability and autoprocessing of the zymogen of the germination protease of spores of Bacillus species; Pedersen LB et al.; Loss of 3, 7, or 10 of the amino-terminal 15 residues removed upon autoactivation of the zymogen of the germination protease (GPR), which initiates protein degradation during germination of spores of Bacillus species, did not result in significant changes in (i) the lack of enzymatic activity of the zymogen, (ii) the rate of zymogen autoactivation, or (iii) the unreactivity of the zymogen's single SH group . Removal of 13 amino-terminal residues resulted in a partially active enzyme whose SH group was as reactive as the fully active enzyme . These findings suggest that at least a part of the propeptide blocks access to the enzyme's active site . However, the free propeptide did not inhibit the enzyme.

J Bacteriol, 1997 Mar, 179(5), 1664 - 70
Molecular characterization of the Bacillus stearothermophilus PV72 S-layer gene sbsB induced by oxidative stress; Kuen B et al.; S-layer protein variation from a hexagonally ordered (SbsA; 130 kDa) to a obliquely ordered (SbsB; 98 kDa) protein in Bacillus stearothermophilus PV72 is mediated by an increased oxygen supply . To elucidate the molecular basis of S-layer protein variation in B . stearothermophilus PV72, the sbsB gene, coding for the 98-kDa protein, was cloned by means of inverse PCR technology and sequenced . The sbsB coding region cloned in pUC18 was expressed in Escherichia coli, without its own regulatory upstream sequences but with its putative transcriptional terminator . The reading frame of sbsB (2,760 nucleotides) is predicted to encode a protein of 920 amino acids, including the signal sequence . Amino acid sequence comparison of SbsA and SbsB did not reveal any significant homology . The expression of sbsB in E . coli resulted in an accumulation of SbsB self-assembly products in the cytoplasm.

J Clin Microbiol, 1997 Mar, 35(3), 566 - 9
PCR identification of Mycobacterium bovis BCG; Talbot EA et al.; The attenuated bacillus Calmette-Guerin (BCG) vaccine strain is derived from a virulent strain of Mycobacterium bovis . BCG is difficult to differentiate from other strains of M . bovis and other members of the M . tuberculosis complex by conventional methods . Recently, a genomic region designated RD1 was found to be present in all virulent M . bovis and M . tuberculosis strains tested but deleted from all BCG strains tested . With this information, a multiplex PCR method was developed to detect the RD1 deletion . A large collection of BCG and other M . tuberculosis complex strains from diverse host and geographic origins was tested . RD1 was deleted in 23 of 23 BCG strains . RD1 was present in 129 of 129 other M . tuberculosis complex strains . This multiplex PCR method can be used as a tool for the rapid and specific identification of BCG.

J Clin Microbiol, 1997 Mar, 35(3), 544 - 7
Isolation of Bartonella (Rochalimaea) henselae: effects of methods of blood collection and handling; Brenner SA et al.; Bartonella (Rochalimaea) henselae causes cat-scratch disease, bacillary angiomatosis, peliosis hepatis, and fever in humans . B . henselae can be difficult to culture axenically, and as many as 5 weeks may be required before colonies are visible . We compared how different methods of blood collection and handling affect isolation of this pathogen . Blood specimens from B . henselae-infected cats were collected in both EDTA and Isolator blood-lysis tubes and were subsequently plated onto rabbit blood-brain heart infusion agar by using three different schedules: plating immediately, plating after 24 h at 25 degrees C, and plating after 26 days at -65 degrees C . Colonies were counted 14 and 35 days after plating . Blood collected in tubes containing EDTA, frozen at -65 degrees C, and then plated on blood agar yielded a median of 60,000 CFU/ml, compared with 25,333 CFU/ml after collection in the Isolator tubes (P < 0.01) . Frozen blood yielded the largest number of B . henselae colonies for any of the schedules tested . These results support previous observations that the Isolator system is more sensitive than tubes containing EDTA for isolation of B . henselae and suggest that, for cat blood, collection in tubes containing EDTA and subsequent freezing may further improve the sensitivity of detection of B . henselae.

Curr Microbiol, 1997 Mar, 34(3), 144 - 8
Bacillus sphaericus penicillin V acylase: purification, substrate specificity, and active-site characterization; Pundle A et al.; Penicillin V acylase from Bacillus sphaericus was purified to homogeneity with an overall yield of 15% . The enzyme exhibited comparatively high specificity for penicillin V, penicillin G, and other related compounds being hydrolyzed at less than 10% of the rate of penicillin V . Moreover, the high rate of hydrolysis was observed when the side chain of the substrate molecule was unsubstituted . Lysine-modifying reagents inactivated the enzyme rapidly . Kinetics and titration studies indicated the involvement of lysine in the catalytic activity of the enzyme.

Biochemistry, 1997 Feb 25, 36(8), 2257 - 65
Positioning the acid/base catalyst in a glycosidase: studies with Bacillus circulans xylanase; Lawson SL et al.; The mechanism of action employed by a glycosidase is dictated, in part, by the distance between the two catalytic carboxylic acids . In the retaining endo-beta-1,4-xylanase from Bacillus circulans, this critical distance (approximately 5.5 A) has been altered by mutagenesis of the putative acid/base catalyst Glu172 . An increase in the separation (Glu172Asp) resulted in a 400-fold decrease in the k(cat) value for xylan hydrolysis . By contrast, a decrease in the separation, achieved by the selective carboxymethylation of the Glu172Cys mutant, caused only a 25-fold reduction in the rate of xylan hydrolysis . Altering the length of the acid/base catalyst had a less detrimental effect on the hydrolysis of aryl xylobiosides, with k(cat)/Km values being reduced only 3-23-fold relative to the wild-type enzyme . Complete removal of the carboxyl group had a more dramatic effect . The Glu172Cys and Glu172Gln mutants exhibited no measurable activity on xylan or phenyl xylobioside, substrates which require acid catalysis . However, these mutants were capable of hydrolyzing aryl xylobiosides with relatively good leaving groups (pKa < 5.5), which need little protonic assistance . The addition of sodium azide caused significant rate increases for the hydrolysis of 2,5-dinitrophenyl beta-xylobioside (pKa = 5.15) by Glu172Cys and Glu172Gln . Thus, the absence of an acid/base catalyst can be partially compensated for by the addition of an anionic nucleophile . These results are consistent with Glu172 functioning as the acid/base catalyst in B . circulans xylanase and emphasize the functional importance of the carboxyl group found at this position.

Biochemistry, 1997 Feb 18, 36(7), 1567 - 72
A single mutation in cytochrome P450 BM3 changes substrate orientation in a catalytic intermediate and the regiospecificity of hydroxylation; Oliver CF et al.; Phenylalanine 87 of Bacillus megaterium cytochrome P450 BM3, a residue close to the heme in the substrate binding pocket, has been replaced by alanine by site-directed mutagenesis . The substitution had no effect on the rate of hydroxylation of laurate and increased the affinity for laurate of both the intact enzyme and its heme domain by 2.6-6-fold in the ferric state . NMR paramagnetic relaxation measurements showed that in the initial ferric enzyme-substrate complex, where the substrate binds relatively far from the heme, the substitution had no effect on the position or orientation of the bound substrate . However, in the next intermediate in the catalytic cycle, the reduced enzyme, the position of the bound substrate was altered so that the terminal methyl group was 3.1 A from the iron in the mutant, compared to 5.1 A in the wild-type enzyme . Analysis of the products of the action of the enzyme on laurate and myristate showed that the mutant catalyzed hydroxylation almost exclusively at the omega position, in marked contrast to the wild-type enzyme, with which no hydroxylation at this position was observed.

Rev Prat, 1997 Feb 15, 47(4), 382 - 7
{Treatment of superficial bladder tumors}; Chopin DK; Superficial bladder cancer, T1, Ta, TIS transitional cell carcinomas of the TNM classification, represent a spectrum disease with different biological behavior . The main difficulty in the management of these tumors is to predict their potential aggressiveness based on clinicopathological parameters . Their management is based on endoscopy findings and histopathological analysis . For low risk tumors, transurethral resection and surveillance allow an adequate tumor control . For high risk tumors, conservative treatment is based on a complete transurethral resection and prophylactic endovesical instillations of bacillus Calmette-Guerin . For intermediate risk lesions, the advantage of prophylactic endovesical instillations have been finally established either using BCG or chemotherapy (mitomycine C) . However in this setting, various therapeutic modalities (maintenance therapy, dose, repeat cycles) are proposed and their relative efficacy remains to be established.

J Immunol, 1997 Feb 15, 158(4), 1949 - 55
Human T cell responses induced by vaccination with Mycobacterium bovis bacillus Calmette-Guérin; Ravn P et al.; Many aspects of the widely used bacillus Calmette-Guerin (BCG) vaccine against tuberculosis are still the subject of controversy . There is a huge variation in efficacy from one clinical trial to another and no relationship between vaccine-induced skin test conversion and subsequent protection . We have studied in vitro cell-mediated immune responses primed by BCG vaccination in 22 healthy Danish donors with different levels of in vitro purified protein derivative (PPD) reactivity before vaccination . The study demonstrated a markedly different development of reactivity to mycobacterial Ags depending on the prevaccination sensitivity to PPD . Previously sensitized donors mounted a potent and highly accelerated recall response within the first week of BCG vaccination . Nonsensitized donors, in contrast, exhibited a gradually increasing responsiveness to mycobacterial Ags, reaching maximal levels between day 56 and 365 postvaccination . The recognition of different classes of Ags were induced in a stepwise manner: culture filtrate Ags were recognized 1 wk postvaccination followed by cell wall, membrane, and the cytosolic Ag fraction . The T cell response primed by BCG vaccination was characterized as a CD4 response with a Th1-like cytokine pattern and substantial levels of Ag-specific cytotoxicity . The specificity of the T cell response generated was broad and directed to a range of culture filtrate Ag fractions . The study shows that BCG vaccination of previously nonsensitized donors can provide important data on potentially protective immune responses in humans and suggest a careful evaluation of prevaccination sensitivity when investigating vaccine-induced immunity.

Biochemistry, 1997 Feb 11, 36(6), 1329 - 42
Determination of the structure of alanine racemase from Bacillus stearothermophilus at 1.9-A resolution; Shaw JP et al.; The molecular structure of alanine racemase from Bacillus stearothermophilus was determined by X-ray crystallography to a resolution of 1.9 A . The alanine racemase monomer is composed of two domains, an eight-stranded alpha/beta barrel at the N-terminus, which includes residues 1-240, and a C-terminal domain essentially composed of beta-strand (residues 241-388) . In the structure of the dimer the mouth of the alpha/beta barrel of one monomer faces the second domain of the other monomer . The pyridoxal 5'-phosphate (PLP) cofactor lies in and above the mouth of the alpha/beta barrel and is covalently linked via an aldimine linkage to Lys39, which is at the C-terminus of the first beta-strand of the alpha/beta barrel . This is the first example of a PLP cofactor binding in the active site of a alpha/beta barrel . A number of other residues are involved in maintaining the position of the PLP in the protein . Of these, Arg219 is the most interesting, as it forms a hydrogen bond with the pyridine nitrogen of the cofactor . This is the first known occurrence of such an interaction with PLP and is expected to influence the electron delocalization in the PLP-alanine intermediates . A second arginine residue, Arg136, donates a hydrogen bond to the phenolic oxygen of PLP and may be involved in the binding of substrate as well as stabilization of intermediates . Finally, Tyr265', from the second monomer, is postulated to be 2 proton donor to the carbanion intermediate.

Gene, 1997 Feb 7, 185(2), 251 - 5
Identification of a second transcriptional start site for the insecticidal protein gene cryIVA of Bacillus thuringiensis subsp . israelensis; Yoshisue H et al.; Expression of cryIVA, one of the insecticidal protein genes of B . thuringiensis subsp . israelensis, is regulated at the transcriptional level . The cryIVA gene is specifically transcribed during the stationary phase of this bacterium . As shown in our previous report {Yoshisue et al . (1993a)}, the transcription from the -364 position of the cryIVA gene is conducted by the major promoter P1 that is functional during middle stages of the stationary phase of B . thuringiensis . In the present study, we have identified a second transcriptional start point P2 for the cryIVA gene in addition to P1, the major transcriptional start point . The transcription from P2 of the cryIVA gene occurred later than that from P1, during later stages of stationary phase of B . thuringiensis subsp . israelensis . The -10 and -35 nt sequences upstream from P2 of cryIVA are similar to those of the omega 28-specific promoters of B . thuringiensis genes and of the omega K-specific promoters of B . subtilis genes . It is most likely that the region upstream from P2 of cryIVA contains the nt sequences that determine the omega 28-specific promoter, the second one, for the cryIVA gene.

J Public Health Manag Pract, 1996 Summer, 2(3), 32 - 40
School-based tuberculosis testing and treatment program: comparing directly observed preventive therapy with traditional preventive therapy; Sass P et al.; A high-school-based health center instituted a tuberculosis testing and treatment program . Compliance of purified protein derivative (PPD) positive students with isonicotine hydrazine (INH) preventive therapy was compared for two groups of students: those in directly observed preventive therapy (DOPT) and those in traditional therapy . Four hundred thirty six students were tested and 429 returned for a reading: 209 (49 percent) students had a positive PPD test . The rate of positivity did not vary by age, sex, or years in the United States . There was no relationship between bacille Calmette-Guerin (BCG) vaccination and rate of positivity or mean size of induration . The compliance rate for all courses of DOPT was significantly higher (54 percent) than that for traditional therapy (26 percent) . The compliance rates for the first and second attempts to complete a course of DOPT was 71 percent . Completion of therapy did not differ by age, sex, or years in the United States . Haitian students completed therapy more often than students from Jamaica . We believe it is effective and efficient to base tuberculosis testing and treatment with DOPT in the school setting.

J Infect Dis, 1997 Feb, 175 Suppl 1, S268 - 71
Strengthening routine immunization services in the Western Pacific through the eradication of poliomyelitis; Aylward RB et al.; Infant immunization coverage in the Western Pacific Region of the World Health Organization was reviewed to evaluate the impact of polio eradication activities on routine immunization services . The trend in bacille Calmette-Guerin (one dose; BCG), diphtheria-tetanus toxoids-pertussis (three doses; DTP3), and measles (one dose) vaccination rates was analyzed from the beginning of eradication activities in 1990 to 1994 in the five polio-endemic countries that conducted supplementary oral polio vaccine immunization . In China and the Philippines, coverage for each antigen remained at or above 90% and 85%, respectively, while in Vietnam, coverage for all three antigens rose from 85% to 95% . BCG, DTP3, and measles vaccine coverage more than doubled in the People's Democratic Republic of Lao and increased by >30% in the Kingdom of Cambodia during the same period.

Anasthesiol Intensivmed Notfallmed Schmerzther, 1997 Feb, 32(2), 124 - 9
{Bacillus cereus pneumonia after thoracic trauma . Case report and review of the literature}; Bastian L et al.; We report on the case of a pneumonia caused by Bacillus cereus in a patient who sustained severe thoracic trauma, fractures of ribs 3-12, left lung contusion and haematopneumothorax . Bronchoscopic alveolar lavage (BAL) performed on ICU day four helped identifying the underlying cause of pneumonia . Inspite of early diagnosis and antibiotic treatment with clindamycin, cefotaxim and tobramycin, which rapidly eliminated the bacteria from the patient's lungs, the patient eventually died from the severe underlying injuries . However, this case emphasises that early performed BAL and rapid microbiological staining techniques can help diagnose pneumonia in critically ill patients . We also review the literature on pulmonary infections caused by Bacillus cereus.

Appl Biochem Biotechnol, 1997 Feb-Mar, 62(2-3), 219 - 31
Purification and characterization of the D-hydantoinase from Bacillus circulans; Luksa V et al.; A D-hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from Bacillus circulans . Purification of two hundred forty-three-fold was achieved with an overall yield of 12% . The relative molecular mass of the native enzyme is 212,000 and that of the subunit is 53,000 . This enzyme is an acidic protein with an isoelectric point of 4.55 . The enzyme is sensitive to thiol reagent and requires metal ions for its activity . The optimal conditions for the hydantoinase activity are pH 8.0-10.0 and a temperature of 75 degrees C . The enzyme is the most stable in a pH range of 8.5-9.5 and up to 60 degrees C . The enzyme is significantly stable not only at high temperatures but also on treatment with protein denaturant SDS . These remarkable properties are used for the purification procedure.

Appl Biochem Biotechnol, 1997 Feb-Mar, 62(2-3), 191 - 200
Characterization of a monoclonal antibody that specifically inhibits pullulanase activity of Bacillus circulans amylase-pullulanase enzyme; Kim CH et al.; A monoclonal antibody (MAb) against amylase pullulanase enzyme from Bacillus circulans, which hydrolyzes not only the alpha-1,6-glycosidic linkage but also the alpha-1,4-glycosidic linkage to the same extent, has been produced by the fusion of BALB/c mouse spleen cells immunized with the native enzyme and P3x63Ag8U1 myeloma cells, and examined for inhibition of pullulanase activity in order to characterize the catalytic site of the pullulanase . The MAb recognizes active enzyme, but not the SDS-denatured or heat-inactivated protein, indicating that the antibody is highly conformational-dependent, specific for active enzyme . The antibody inhibited the pullulanase activity, but not amylase activity . The monoclonal antibody immunoblotted the enzyme and immunoprecipitated the enzyme . The immunoprecipitation was inhibited in the presence of substrate, pullulan, and the MAb competitively inhibited the binding of pullulan to the enzyme . The MAb, therefore, recognizes the pullulan-binding site of the enzyme . Kinetic analysis showed that the MAb inhibited pullulanase activity with inhibition constant (Ki) of 0.77 microgram/mL, providing evidence that the antibody decreases the catalytic rate of enzyme activity and has an effect on substrate binding . These results strongly confirm the previous observations that APE may have two different active sites responsible for the expression of amylase and pullulanase activities (Kim, C.H . and Kim, Y.S . Eur . J . Biochem . 1995, 227, 687-693).

Pediatr Surg Int, 1997 Feb, 12(2-3), 220 - 3
Is BCG vaccine innocent?
Karnak I, Senocak ME, Buyukpamukcu N, Gocmen A.
Four patients admitted to the Hacettepe University Department of Pediatric Surgery between 1987 and 1995, two with Bacille Calmette-Guerin (BCG) lymphadenitis and two with multisystem postvaccination tuberculosis (MPT), are presented . The hospital records and records of the Ministery of Health Tuberculosis Control Department were evaluated to determine the complications of BCG vaccine . The most common complication was lymphadenitis with or without suppuration (0.3 per thousand - 3 per thousand) . Surgical intervention was required in two BCG lymphadenitis cases and two cases of MPT . Involved lymph nodes were excised in two lymphadenitis cases . Colostomy and percutaneous nephrostomy was performed in the first case of MPT in addition to triple antituberculous drug therapy . Although BCG lymphadenitis is self limited, chronically discharging nodes and tumor-like lymphadenopathy masses need to be excised . On the other hand, MPT has a silent nature with resistance to antituberculous drug therapy . Surgical intervention may be required, directed to the involved systems.

J Biochem (Tokyo), 1997 Feb, 121(2), 244 - 50
Lysyl-tRNA synthetase from Bacillus stearothermophilus . Formation and isolation of an enzyme-lysyladenylate complex and its analogue; Takita T et al.; The formation of an enzyme.lysyladenylate complex was studied with a highly purified lysyl-tRNA synthetase {L-lysine:tRNALYS ligase (AMP-forming); EC 6.1.1.6} from Bacillus stearothermophilus . The apparent dissociation equilibrium constants of the enzyme with L-lysine and ATP in the process of the complex formation were estimated to be 50.9 and 15.5 microM, respectively, at pH 8.0, 30 degrees C, by fluorometric measurement . The isolated enzyme.lysyladenylate complex was relatively stable with a rate constant of decomposition of 1.7 x 10(-5) s-1 at pH 8.5 and 0 degree C . The rate constant of transfer of L-lysine from the complex to Escherichia coli tRNA was 1.2 x 10(-2) S-1 at pH 8.5 and 0 degree C . The effects of replacing L-lysine by several analogues on the complex formation were examined . L-Lysine hydroxamate, a strong inhibitor of the L-lysine dependent ATP-PPi exchange reaction, produced a stable complex with the enzyme and ATP, enzyme.lysinehydroxamate-AMP probably being formed . The binding stoichiometry of the assumed L-lysinehydroxamate-AMP per mol of the dimer enzyme was 1:1.

Toxicon, 1997 Feb, 35(2), 305 - 9
Hemolytic activity in extracts of the diatom Nitzschia; Rangel M et al.; The few reports about diatom toxins are related to central nervous system toxicity, induced by domoic acid . In the present work Nitzschia sp . (Bacillariophyceae) was studied . The cells were cultured in f/2 medium, under 4000 lux and 14/10 hr light/dark cycle . After massive growth (5 x 10(6) cells/ml) the diatom cells were filtered, and an extract was prepared and partitioned in two fractions (polar and apolar) . After cell harvesting by filtration, the diatom cells were shaken in artificial sea water to extract the water-soluble extracellular matrix (mucilage) . An extract was prepared with the washed cells (free of mucilage), and polar and apolar fractions were obtained . Hemolytic assays were performed using 4.0 and 0.5% erythrocyte suspensions . Both the diatom polar and apolar fractions showed hemolytic activity . The membrane phospholipid sphingomyelin was tested as an acceptor for the hemolysins in the polar and apolar fractions . The mucilage did not exhibit hemolytic activity.

Appl Microbiol Biotechnol, 1997 Feb, 47(2), 120 - 6
Extracellular production of a hybrid beta-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors; Miksch G et al.; Cultivation conditions for the extracellular production of a hybrid beta-glucanase from Bacillus were established by using Escherichia coli JM 109 carrying the plasmid pLF3 . This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid beta-glucanase gene of Bacillus . When controlled by the fic promoter, the kil gene led to a higher total production of beta-glucanase and a higher protein secretion than when it was under control of the bolA promoter . When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion . With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed . In defined media the secretion of beta-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation . The strain with the highest production and secretion yield of beta-glucanase {E . coli JM 109(pLF3)} was tested on the fermenter scale.

J Econ Entomol, 1997 Feb, 90(1), 130 - 4
Toxicity of an isolate of Bacillus thuringiensis subspecies darmstadiensis to adults of the Mexican fruit fly (diptera: (Diptera:Tephritidae) in the laboratory; Martinez AJ et al.; Centrifugation pellets obtained from an isolate of Bacillus thuringiensis subspecies darmstadiensis (Guat 1) cultured from a Guatemalan soil sample were found to be toxic to Anastrepha ludens (Loew) adults in the laboratory . We developed a bioassay diet that consisted of a mixture of the bacterium, a protein source, and sugar . A pH of 4.1 of the mixture was needed to obtain maximum adult mortality . One meal of the diet, which lasted from 30 s to 4 min, was enough to cause > 70% mortality of both fed or starved adults . Mortality of fed adults was 70-75% following a feeding period of 60 min and mortality of starved adults was 80-90% following a feeding period of 30 min . The isolate was toxic to adults from 1 to 21 d old.

Biochem Mol Biol Int, 1997 Feb, 41(2), 243 - 55
The growth kinetics of intracellular bacille Calmette-Guerin (BCG) within human monocyte-derived macrophages of different ethnic populations; Fazal N; In this paper we have examined intracellular mycobacterial growth in human monocyte-derived macrophage cell cultures . Cells from three population groups were selected to compare and contrast the kinetics of intracellular BCG growth both by determining changes in metabolic activity of dividing bacteria by radio-isotope incorporation and growth/survival by estimating colony forming unit by Fazal's Microcolony Counting Method . These data indicate that BCG infects and multiplies intracellularly in human macrophages and may serve as an in vitro model for studying inherent capability of mycobacteria to grow and infect macrophage populations . There is however, no inhibition or reduction of intracellular mycobacterial growth from a Caucasian male and female, and an Afrocarribean female donor macrophages . On the contrary, there is a ten-fold increase in the replication of mycobacteria over a 7-day infection period . Patterns of BCG growth within macrophage cultures from different donors were determined and variation calculated at different days post-infection . Experiment to experiment coefficient of variation of intracellular BCG growth estimates for a single donor is between 4-12% . In contrast, the coefficient of variation of intracellular BCG growth between three different macrophage donors studied is < 4%.

Biochem Mol Biol Int, 1997 Feb, 41(2), 227 - 34
Effect on product specificity of cyclodextrin glycosyltransferase by site-directed mutagenesis; Kim YH et al.; Cyclodextrin glycosyltransferase (CGTase; EC 2.4.1.19) catalyzes the degradation of starch into cyclodextrins through an intramolecular transglycosylation reaction . Tyr-89, Asn-94, and Tyr-100 are located near the putative active center . To analyze their roles in product specificity, Tyr-89, Asn-94, and Tyr-100 of CGTase from alkalophilic Bacillus sp . I-5 were replaced with different amino acids . Among the mutants, the N94S mutant protein produced about two times more alpha-cyclodextrin than the wild-type at all incubation times . The Y89F and Y100F mutant proteins were changed to more beta-specific enzymes . From these results it is suggested that the changing of the residues located at the near active site can change the product specificity of CGTase.

Biosci Biotechnol Biochem, 1997 Feb, 61(2), 362 - 4
A gene encoding endo-1,4-beta-glucanase from Bacillus sp . 22-28; Miyatake M et al.; Clones of a gene encoding an endo-1,4-beta-glucanase (EC 3.2.1.4) were obtained from Bacillus sp . 22-28, and the nucleotides were sequenced . A recombinant plasmid, pMK5, included a complete ORF of 2352 bp that encoded 783 amino acid residues . Another plasmid, pM3, which showed enzymatic activity in E . coli JM109, was also obtained, and it included an incomplete ORF of 1873 bp, which lacked the original stop codon and 479 bp of the C-terminal region . The enzymes purified from both of the two types of transformants have shown almost the same properties in comparison with that of the wild type Bacillus sp . 22-28.

Biosci Biotechnol Biochem, 1997 Feb, 61(2), 276 - 80
Cloning of purine nucleoside phosphorylase II gene from Bacillus stearothermophilus TH 6-2 and characterization of its gene product; Hamamoto T et al.; Bacillus stearothermophilus TH 6-2 has two kinds of purine nucleoside phosphorylases (Pu-NPases) . The type I enzyme (Pu-NPase I) is a functional and structural homolog of eukaryotic purine nucleoside phosphorylases that catalyze the phosphorolysis of inosine, guanosine, and their derivatives . The type II enzyme (Pu-NPase II) is a minor enzyme that efficiently phosphorolyzes adenosine and its derivatives rather than other purine nucleosides like Escherichia coli Pu-NPase . The gene coding for Pu-NPase II (punB gene) has been cloned and sequenced from TH 6-2 strain . The deduced amino acid sequence of Pu-NPase II shared 54% identity with that of E . coli enzyme, while it had no significant homology to that of Pu-NPase I or eukaryotic enzymes . By producing the Pu-NPase II in E . coli cells, the Pu-NPase II has been purified and characterized.

Biosci Biotechnol Biochem, 1997 Feb, 61(2), 272 - 5
Cloning and expression of purine nucleoside phosphorylase I gene from Bacillus stearothermophilus TH 6-2; Hamamoto T et al.; Bacillus stearothermophilus TH 6-2 has two kinds of purine nucleoside phosphorylases (Pu-NPase I and Pu-NPase II) . The Pu-NPase I is a functional homolog of eukaryotic purine nucleoside phosphorylases that can catalyze the phosphorolysis of inosine and guanosine, but not adenosine, the primary substrate of Pu-NPase II . The Pu-NPase I gene of TH 6-2 has been cloned, sequenced, and expressed in E . coli . The gene corresponded to an open reading frame of 822 nucleotides that translates into a putative 274-amino acid protein with a molecular weight of 29,637 . The deduced amino terminus sequence completely coincided with that found for the purified enzyme . The cloned gene was overexpressed in E . coli by using the trc promoter to produce an active enzyme in large quantities . The amino acid sequence of Pu-NPase I shared 50% similarity with those of human and mouse purine nucleoside phosphorylases.

Biosci Biotechnol Biochem, 1997 Feb, 61(2), 251 - 5
Purification, properties, and N-terminal amino acid sequences of guar gum-degrading enzyme from Bacillus circulans K-1; Yosida S et al.; A guar gum-degrading enzyme of the newly isolated Bacillus circulans K-1 was purified to an electrophoretically homogeneous state . The molecular weight of the purified enzyme was 62,000 by SDS-PAGE . The purified enzyme was separated into a least six isozymes by isoelectric focusing and the pI of these isozymes were 5.4, 5.5, 5.6, 5.8, 6.0, and 6.2, respectively . The N-terminal amino acid sequences of the typical three of these proteins were all the same, Ala-Ser-Gly-Phe-Tyr-Val-Ser-Gly-Thr-Lys-Leu-Asp-Ala-Thr-Gly-Gln-Pro-Phe- Val- Met-Arg . The enzyme was most active at pH 6.9 and at 64 degrees C . The enzyme was activated slightly by Al3+ and inhibited strongly by Sn2+ and Zn2+, N-bromosuccinimide, 2-mercaptoethanol, and ethylenediamine-tetraacetic acid.

J Clin Oncol, 1997 Feb, 15(2), 796 - 807
Adoptive immunotherapy with vaccine-primed lymph node cells secondarily activated with anti-CD3 and interleukin-2; Chang AE et al.; PURPOSE: In preclinical studies, we have reported the ability to induce immune T cells in lymph nodes (LN) primed by in vivo vaccination with tumor cells admixed with a bacterial adjuvant . These LN cells can be activated and expanded ex vivo for the successful immunotherapy of established tumors . We have applied these methods to generate vaccine-primed LN in patients with advanced melanoma and renal cell cancer (RCC) for therapy . MATERIALS AND METHODS: Irradiated autologous tumor cells admixed with bacille Calmette-Guerin (BCG) were used to vaccinate patients . Seven days later, draining LN were removed for activation with anti-CD3 monoclonal antibody (mAb) followed by expansion in interleukin-2 (IL-2) . Activated LN cells were administered intravenously (IV) with the concomitant administration of IL-2 . RESULTS: A total of 23 patients were evaluated (11 melanoma and 12 RCC) . Vaccine-primed LN were expanded ex vivo with a mean of 8.4 x 10(10) cells administered per patient . Among 20 patients assessed, 15 demonstrated minimal cytotoxicity of autologous tumor cells by the activated LN cells, with the remaining mediating nonspecific cytotoxicity . By contrast, a majority of the activated LN cells showed highly specific release of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFN-gamma) to autologous but not allogeneic tumor stimulation . This tumor-specific cytokine release was found to be major histocompatibility complex (MHC) class I-restricted, which indicates the involvement of CD8+ cells . Among 11 melanoma patients, one had a partial tumor response . Among 12 RCC patients, two had complete and two partial responses . A trend (P = .066) between the enhancement of delayed-type hypersensitivity (DTH) reactivity to autologous tumor after therapy and tumor regression was observed . CONCLUSION: Tumor vaccines can be used to induce immunologically specific T-cell responses against melanoma and RCC in draining LN . Anti-CD3/IL-2 activation of primed LN cells can be reliably performed for clinical therapy and appears to have activity in patients with metastatic RCC.

Pediatr Infect Dis J, 1997 Feb, 16(2), 163 - 79
The expanding spectrum of Bartonella infections: II . Cat-scratch disease; Bass JW et al.; Recent advancements and developments in molecular biotechnology have allowed more precise reclassification of many microorganisms . With the use of these new taxonomy tools, several organisms previously thought to belong to other genera have been recently described as bartonellae . Of the 11 organisms now described as Bartonella spp., only four have been shown to be pathogenic for humans . Table 1 lists the four Bartonella human pathogens along with the their known epidemiology and the scope and range of disease associated with each . All are now considered to be bacteria and can be grown on blood-enriched agar although primary isolation in some may best be achieved in cell tissue culture . B . bacilliformis infection is limited to certain geographic regions in South America where the only human reservoir and the sandfly vector(s) that spreads the disease reside together . Specific antibiotic treatment is dramatically effective in treating the highly fatal, acute intraerythrocytic hemolytic form of the disease, but their effectiveness in treating the vascular proliferative forms (verruga peruana) or the chronic asymptomatic, bacteremic, carrier state of the disease has not been effective . This disease should remain confined to its present endemic geographic areas in South American unless asymptomatic bacteremic persons from these areas migrate to areas where sandflies and humans exist that are capable of establishing this infection in new endemic areas . B . quintana and B . henselae cause a wide range of clinical diseases in humans, the type and extent of which varies significantly with the immune status of the host . In immunocompetent hosts the pathologic response is granulomatous, suppurative, extracellular and intracellular, generally self-limited and usually unresponsive to antibiotic treatment, even to those drugs to which the organism is shown to be sensitive in vitro . In contrast, in immunocompromised hosts the pathologic response is vasculoproliferative, organisms may be seen intracellularly but they are often seen in abundance in extracellular clumps and infection is usually progressive and fatal unless treated . In these patients clinical response to treatment with drugs that are effective in vitro against these organisms has usually been dramatic . Of these agents those that penetrate cells and are found in high concentrations intracellularly, such as erythromycin, clarithromycin, azithromycin, rifampin, doxycycline and gentamicin, appear to be most effective . These agents not only appear to provide the most dramatic treatment response in patients with BA, BP and PRFB and other manifestations of B . henselae (and B . quintana as well) in immunocompromised persons, they appear to be the most promising agents for treatment of persons with both typical and atypical CSD . Further studies will be necessary to more clearly elucidated the mechanisms responsible for the diverse clinical presentations of infection with these organisms in human hosts relative to their immune status . In addition clarification of the epidemiology of B . elizabethae infections in humans may be helpful in understanding the nature of infection with Bartonella organisms.

Nat Biotechnol, 1997 Feb, 15(2), 137 - 41
Transgenic plants: an emerging approach to pest control; Estruch JJ et al.; Insect pests are a major cause of damage to the world's commercially important agricultural crops . Current strategies aimed at reducing crop losses rely primarily on chemical pesticides . Alternatively transgenic crops with intrinsic pest resistance offer a promising alternative and continue to be developed . The first generation of insect-resistant transgenic plants are based on insecticidal proteins from Bacillus thuringiensis (Bt) . A second generation of insect-resistant plants under development include both Bt and non-Bt proteins with novel modes of action and different spectra of activity against insect pests.

Br J Pharmacol, 1997 Feb, 120(3), 502 - 8
The role of B1 and B2 kinin receptors in oedema formation after long-term treatment with Mycobacterium bovis bacillus Calmette-Guérin (BCG); Campos MM et al.; 1 . The present study was designed to investigate the influence of long-term systemic treatment with Mycobacterium bovis bacillus Calmette-Guerin (BCG, 1 dose per animal, containing 6 x 10(4) colony-forming-units (CFu), 5 to 75 days beforehand) on oedema formation induced by intradermal injection of B1 and B2 selective agonists . The interaction between the B1 agonist des-Arg9-bradykinin and bradykinin was also investigated . 2 . Intradermal injection (i.d.) of the B2 selective agonist tyrosine8-bradykinin (1-10 nmol) in naive (saline pretreated) animals, or in animals that had received BCG (30 days beforehand), caused dose-related and very similar oedema formation (ED50; 1.1 and 1.0 nmol/paw, respectively) . I.d . injection of the selective B1 agonists des-Arg9-bradykinin (100 nmol) or des-Arg10-kallidin in naive animals caused very little paw oedema (0.04 +/- 0.06 and 0.07 +/- 0.02 ml, respectively, n = 5) . However, i.d . injection of des-Arg9-bradykinin (10-300 nmol) or des-Arg10-kallidin (3-100 nmol) in animals pretreated with BCG, 30 days previously, resulted in dose-related and marked oedema formation, with mean ED50 values of 20.1 and 5.5 nmol/paw, respectively . 3 . Oedema caused by i.d . injection of des-Arg9-bradykinin (100 nmol/paw) in rats pretreated with BCG was evident 5 days after treatment, reaching the maximum 30 days later, remaining stable for up to 45 days, and reduced markedly at 75 days . 4 . The i.d . co-injection of the selective B1 antagonists des-Arg9{Leu8}-bradykinin (200 nmol), des-Arg10{Leu9}-bradykinin (30 nmol) and des-Arg9-NPC 17731 (30 nmol) significantly (18 +/- 3, 34 +/- 2 and 56 +/- 4%, respectively) prevented the paw oedema caused by i.d . injection of des-Arg9-bradykinin (100 nmol) in rats treated with BCG . These effects were selective, because the i.d . injection of the B1 selective antagonist des-Arg10{Leu9}-kallidin (30 nmol), at the same dose that consistently antagonized des-Arg9-bradykinin (100 nmol)-mediated paw oedema, had no significant effect against tyrosine8-bradykinin (3 nmol)-induced oedema in animals that had been treated previously with BCG . On the other hand, the i.d . co-injection of the selective B2 antagonist, Hoe 140 (10 nmol) at a dose which markedly inhibited tyrosine8-bradykinin (3 nmol)-induced oedema by 55 +/- 4%, did not significantly affect des-Arg9-bradykinin-induced paw oedema in animals pretreated with BCG . 5 . Treatment of animals with dexamethasone (0.5 mg kg-1, s.c.) every 24 h, from day 0 to day 30, inhibited significantly (67 +/- 4%) the oedema caused by des-Arg9-bradykinin (100 nmol), but did not affect the paw oedema caused by tyrosine8-bradykinin (3 nmol) in animals pretreated with BCG . 6 . Indomethacin (2 mg kg-1, i.p.), administered 1 h before experiments, significantly inhibited des-Arg9-bradykinin (100 nmol)-induced oedema formation, and, to a lesser extent, the paw oedema caused by tyrosine8-bradykinin (3 nmol) (44 +/- 4 and 20 +/- 4%, respectively) . 7 . These findings show that the long-term systemic treatment of rats with BCG promoted a time-dependent and consistent paw oedema formation to B1 agonists, des-Arg9-bradykinin and des-Arg10-kallidin, leaving responses to the B2 agonist tyrosine8-bradykinin unaffected . The upregulation of B1 receptors after BCG treatment was inhibited by dexamethasone, suggesting the possible involvement of de novo protein synthesis . Finally, our results also show that in BCG-treated animals, the B1 agonist des-Arg9-bradykinin interacts in a synergistic manner with bradykinin . Therefore, both B1 and B2 kinin receptors appear to play a relevant role in modulating chronic inflammatory processes.

Appl Environ Microbiol, 1997 Feb, 63(2), 779 - 84
A recombinase-mediated system for elimination of antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains; Sanchis V et al.; A TnpI-mediated site-specific recombination system to construct genetically modified Bacillus thuringiensis strains was developed . Recombinant B . thuringiensis strains from which antibiotic resistance genes can be selectively eliminated were obtained in vivo with a new vector based on the specific resolution site of transposon Tn4430 . For example, a cryIC gene, whose product is active against Spodoptera littoralis, was introduced into B . thuringiensis Kto harboring a cryIA(c) gene active against Ostrinia nubilalis . The resulting strain had a broader activity spectrum than that of the parental strain . It contained only B . thuringiensis DNA and was free of antibiotic resistance genes . This should facilitate regulatory approval for its development as a commercial biopesticide.

Appl Environ Microbiol, 1997 Feb, 63(2), 763 - 6
Two amino acid amidohydrolase genes encoding L-stereospecific carbamoylase and aminoacylase are organized in a common operon in Bacillus stearothermophilus; Batisse N et al.; The L-carbamoylase gene (amaB) upstream of the previously detected L-aminoacylase gene (amaA) in the Bacillus stearothermophilus NCIB8224 strain was identified in this study . The amaB and amaA genes are cotranscribed as a single mRNA from the same transcriptional start . The two-ama-gene operon is conserved in B . stearothermophilus strains . A cross-activity of L-carbamoylase towards respective substrates for L-aminoacylase supports the hypothesis of a common ancestor for both amino acid amidohydrolase genes.

Appl Environ Microbiol, 1997 Feb, 63(2), 532 - 6
The Bacillus thuringiensis vegetative insecticidal protein Vip3A lyses midgut epithelium cells of susceptible insects; Yu CG et al.; The Vip3A protein is a member of a newly discovered class of vegetative insecticidal proteins with activity against a broad spectrum of lepidopteran insects . Histopathological observations indicate that Vip3A ingestion by susceptible insects such as the black cutworm (Agrotis ipsilon) and fall armyworm (Spodoptera frugiperda) causes gut paralysis at concentrations as low as 4 ng/cm2 of diet and complete lysis of gut epithelium cells resulting in larval death at concentrations above 40 ng/cm2 . The European corn borer (Ostrinia nubilalis), a nonsusceptible insect, does not develop any pathology upon ingesting Vip3A . While proteolytic processing of the Vip3A protein by midgut fluids obtained from susceptible and nonsusceptible insects is comparable, in vivo immunolocalization studies show that Vip3a binding is restricted to gut cells of susceptible insects . Therefore, the insect host range for Vip3A seems to be determined by its ability to bind gut cells . These results indicate that midgut epithelium cells of susceptible insects are the primary target for the Vip3A insecticidal protein and that their subsequent lysis is the primary mechanism of lethality . Disruption of gut cells appears to be the strategy adopted by the most effective insecticidal proteins.

Appl Environ Microbiol, 1997 Feb, 63(2), 468 - 73
Identification of a gene for Cyt1A-like hemolysin from Bacillus thuringiensis subsp . medellin and expression in a crystal-negative B . thuringiensis strain; Thiery I et al.; A gene designated cyt1Ab1, encoding a 27,490-Da protein, was isolated from Bacillus thuringiensis subsp . medellin (H30 serotype) by using an oligonucleotide probe corresponding to the cyt1Aa1 gene . The sequence of the Cyt1Ab1 protein, as deduced from the sequence of the cyt1Ab1 gene, was 86% identical to that of the Cyt1Aa1 protein and 32% identical to that of the Cyt2Aa1 protein from B . thuringiensis subsp . kyushuensis . The cyt1Ab1 gene was flanked upstream by a p21 gene, in the same orientation, encoding a 21,370-Da protein that showed 84% similarity to the putative chaperone P20 protein from B . thuringiensis subsp . israelensis and downstream, on the opposite strand, by a sequence showing 85% identity to the IS240A insertion sequence . The cyt1Ab1 gene was expressed at a high level in a nontoxic strain of B . thuringiensis subsp . israelensis in which large inclusions of the Cyt1Ab1 protein were produced . Purified Cyt1Ab1 crystals were as hemolytic as those of the Cyt1Aa1 protein and were twice as hemolytic as those from the wild-type strain . Mosquitocidal activity toward Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae was assayed . The toxicity of the Cyt1Ab1 protein was slightly lower than that of the Cyt1Aa1 protein for all three mosquito species, and Cyt1Ab1 was 150, 300, and 800 times less active toward Culex, Anopheles, and Aedes larvae, respectively, than were the native crystals from B . thuringiensis subsp . medellin.

J Bacteriol, 1997 Feb, 179(4), 1023 - 8
X-ray photoelectron spectroscopy analysis of whole cells and isolated cell walls of gram-positive bacteria: comparison with biochemical analysis; Dufrene YF et al.; The surface chemical composition of whole cells and isolated cell walls of four coryneform bacteria and of a Bacillus brevis strain has been determined by X-ray photoelectron spectroscopy (XPS) . The XPS data were converted into concentrations of model compounds: peptides, polysaccharides, and hydrocarbonlike compounds . The composition of the surface of B . brevis differed markedly from that of coryneforms: the peptide concentration was about twice higher in the former case, which is attributed to the presence of an S-layer at the cell surface; in contrast, the surface of coryneforms was rich in hydrocarbonlike compounds (about 40%), which was concomitant with a high water contact angle . The peptide surface concentration of the isolated cell walls of the five strains deduced from XPS data fitted well with the total peptide content determined by biochemical analysis, which supports the validity of XPS to determine the overall macromolecular composition of the bacterial cell surface . Compared to biochemical analysis of isolated cell walls, XPS analysis of whole cells provides information which concerns directly the cell surface (2- to 5-nm-thick layer) and is less subject to alteration via losses of cell wall constituents or contamination by intracellular compounds.

Infect Immun, 1997 Feb, 65(2), 676 - 84
Induction of cytotoxic T-cell responses against culture filtrate antigens in Mycobacterium bovis bacillus Calmette-Guérin-infected mice; Denis O et al.; CD8+ T cells are essential for protection against mycobacteria, as is clearly demonstrated by the fatal outcome of experimental infection of beta-2 microglobulin knockout mice . However, the mechanisms and antigens (Ags) leading to CD8+ T-cell activation and regulation have been poorly characterized . Here we show that, upon immunization of major histocompatibility complex (MHC)-congenic mice with Mycobacterium bovis bacillus Calmette-Guerin (BCG), a cytotoxic response against BCG culture filtrate (CF) Ags (CFAgs) is induced in H-2b and H-2bxd haplotypes but not in H-2d haplotype . This response is mediated by CD8+ T cells and absolutely requires the activation of CD4+ T cells and their secretion of interleukin 2 . The lack of cytotoxic response in H-2d mice cannot be explained by impaired cytokine production or by a defect in Ag presentation by H-2d macrophages . Using the MHC class I mutant B6.C-H-2bm13 mouse strain, we demonstrate that cytotoxic T lymphocytes (CTLs) recognize CFAgs exclusively in association with D(b) molecules . These Ags are cross-reactive in mycobacteria, since BCG-induced CTLs also recognize macrophages pulsed with CF from Mycobacterium tuberculosis H37Rv and H37Ra and from two virulent strains of M . bovis . Moreover, immunization with Mycobacterium kansasii induces CTLs able to lyse macrophages pulsed with BCG CF . Finally, we have found that these Ags can be characterized as hydrophilic proteins, since they do not bind to phenyl-Sepharose CL-4B . Our results indicate that MHC-linked genes exert a profound influence on the generation of CD8+ CTLs following BCG vaccination.

J Bacteriol, 1997 Feb, 179(3), 863 - 70
Construction and characterization of a mutant of alkaliphilic Bacillus firmus OF4 with a disrupted cta operon and purification of a novel cytochrome bd; Gilmour R et al.; The caa3-type terminal oxidase of Bacillus firmus OF4 has been proposed to play an important role in the growth and bioenergetics of this alkaliphile (A . A . Guffanti and T . A . Krulwich, J . Biol . Chem . 267:9580-9588, 1992) . A mutant strain was generated in which the cta operon encoding the oxidase was disrupted by insertion of a spectinomycin resistance cassette . The mutant was unable to oxidize ascorbate in the presence of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) . Absorption spectra of membranes confirmed the loss of the enzyme and indicated the presence of a cytochrome bd-type terminal oxidase . The mutant could grow on glucose but was unable to grow on malate or other nonfermentative carbon sources, despite the presence of the cytochrome bd . The cytochrome bd was purified from the mutant . The enzyme consisted of two subunits and, with menadiol as substrate, consumed oxygen with a specific activity of 12 micromol of O2 x min(-1) x mg(-1) . In contrast to both cytochromes bd of Escherichia coli, the enzyme did not utilize TMPD as an electron source . A number of additional features, including subunit size and spectral properties, distinguish this cytochrome bd from its counterparts in E . coli and Azotobacter vinelandii.

J Clin Microbiol, 1997 Feb, 35(2), 504 - 7
Fulminating bacteremia and pneumonia due to Bacillus cereus; Miller JM et al.; We present two cases of rapidly progressing, fatal pneumonia caused by Bacillus cereus . These cases are interesting in that B . cereus, even from blood or sputum specimens, may often be considered a contaminant and receive inadequate attention . Also of interest was the fact that the two patients resided in the same area of the state, were welders by trade, and became ill within a few days of each other, yet there was no epidemiologic link between them.

J Clin Microbiol, 1997 Feb, 35(2), 489 - 91
Bacteremia caused by Arcobacter cryaerophilus 1B; Hsueh PR et al.; Arcobacter cryaerophilus group 1B, a gram-negative, curved or helical bacillus primarily known as a bovine and porcine pathogen, was isolated from the blood of a uremic patient with hematogenous pneumonia . The patient was treated successfully with ceftizoxime and tobramycin.

Curr Microbiol, 1997 Feb, 34(2), 118 - 21
Cloning and expression of a Bacillus thuringiensis (L1-2) gene encoding a crystal protein active against Glossina morsitans morsitans and Chilo partellus; Omolo EO et al.; A local isolate of Bacillus thuringiensis,designated L1-2, that is toxic to Chilo partellus was found to be toxic to the adult tsetse fly, Glossina morsitans morsitans . The delta-endotoxin crystals derived from the isolate gave a major protein band with a molecular weight of Mr 130,000-140,000 on denaturing polyacrylamide gel electrophoresis . The sequence of the cloned gene was found to be similar to that of the B . thuringiensis subsp . kurstaki HD-73 cryIA(c) gene, having one amino acid difference at position 148 and four additional DNA differences.

J Urol, 1997 Feb, 157(2), 487 - 91
A 12 versus 6-week course of bacillus Calmette-Guerin prophylaxis for the treatment of high risk superficial bladder cancer; Gruenwald IE et al.; PURPOSE: We examined the efficacy of a 12-week prophylactic course of bacillus Calmette-Guerin (BCG) on superficial bladder cancer . MATERIALS AND METHODS: From August 1992 until July 1994, 70 evaluable patients 41 to 80 years old (mean age 68.5) with high risk transitional cell carcinoma of the bladder were prospectively randomized to a 12-week prophylactic course of BCG (group 2) versus a traditional 6-week course (group 1) . Mean followup was 28 months . RESULTS: A 70% tumor-free rate (21 patients) and mean interval of 12.9 months to recurrence were achieved in group 2 compared to 55% (22 patients) and 12.3 months, respectively, in group 1 . Group 2 patients had an overall longer disease-free survival, although no statistical significance was achieved . A subgroup of patients with stage Ta cancer in whom at least 1 tumor was resected 12 month before treatment showed the most benefit from long-term prophylactic treatment in terms of disease-free survival . Side effects were only slightly more prominent in group 2, rendering the longer course fairly acceptable . CONCLUSIONS: Our findings suggest a difference for better overall results with the 12-week course of BCG . However, a larger number of patients are needed to demonstrate a statistically significant difference between the 2 groups.

Proc Natl Acad Sci U S A, 1997 Jan 21, 94(2), 443 - 7
A point mutation leads to altered product specificity in beta-lactamase catalysis; Lewis ER et al.; beta-Lactamases are the primary cause of beta-lactam antibiotic resistance in many pathogenic organisms . The beta-lactamase catalytic mechanism has been shown to involve a covalent acyl-enzyme . Examination of the structure of the class A beta-lactamase from Bacillus licheniformis suggested that replacement of Asn-170 by leucine would disrupt the deacylation reaction by displacing the hydrolytic water molecule . When N170L beta-lactamase was reacted with penicillins, a novel product was formed . We postulate that with leucine at position 170 the acyl-enzyme undergoes deacylation by an intramolecular rearrangement (rather than hydrolysis) to form a thiazolidine-oxazolinone as the initial product . The oxazolinone subsequently undergoes rapid breakdown leading to the formation of N-phenylacetylglycine and N-formylpenicillamine . This appears to be the first reported case where a point mutation leads to a change in enzyme mechanism resulting in a substantially altered product, effectively changing the product specificity of beta-lactamase into that of D-Ala-D-Ala-carboxypeptidase interacting with benzylpenicillin.

J Biol Chem, 1997 Jan 17, 272(3), 1601 - 7
Altering substrate specificity of Bacillus sp . SAM1606 alpha-glucosidase by comparative site-specific mutagenesis; Inohara-Ochiai M et al.; The Bacillus sp . SAM1606 alpha-glucosidase with a broad substrate specificity is the only known alpha-glucosidase that can hydrolyze alpha,alpha'-trehalose efficiently . The enzyme exhibits a very high sequence similarity to the oligo-1,6-glucosidases (O16G) of Bacillus thermoglucosidasius and Bacillus cereus which cannot act on trehalose . These three enzymes share 80% identical residues within the conserved regions (CR), which have been suggested to be located near or at the active site of the alpha-amylase family enzymes . To identify by site-specific mutagenesis the critical residues that determine the broad substrate specificity of the SAM1606 enzyme we compared the CR sequences of these three glucosidases and selected five targets to be mutagenized in SAM1606 alpha-glucosidase, Met76, Arg81, Ala116, Gly273, and Thr342 . These residues have been specifically replaced by in vitro mutagenesis with Asn, Ser, Val, Pro, and Asn, respectively, as in the Bacillus O16G . The 12 mutant enzymes with single and multiple substitutions were expressed and characterized kinetically . The results showed that the 5-fold mutation virtually abolished the affinity of the enzyme for alpha, alpha'-trehalose, whereas the specificity constant for the hydrolysis of isomaltose, a good substrate for both the SAM1606 enzyme and O16G, remained essentially unchanged upon the mutation . This loss in affinity for trehalose was critically governed by a Gly273 --> Pro substitution, whose effect was specifically enhanced by the Thr342 --> Asn substitution in the 5-fold and quadruple mutants . These results provide evidence for the differential roles of the amino acid residues in the CR in determining the substrate specificity of the alpha-glucosidase.

Anal Biochem, 1997 Jan 15, 244(2), 291 - 300
Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ionspray ionization tandem mass spectrometry; Mano N et al.; Sphingolipid metabolites ceramide, sphingomyelin, sphingosine, psycosine, sphingosylphosphorylcholine, and dimethylsphingosine were separated and simulataneously quantitated by liquid chromatography/ionspray ionization tandem mass spectrometry (LC/MS/ MS) . The use of glassware throughout minimized losses due to adsorption and the pretreatment of this method consisted of simple liquid-liquid extraction procedure with a mixture of chloroform and methanol . After separation on a short C18 silica column eluted in a gradient mode, the metabolites were detected by MS/ MS . This assay allows simultaneously quantification of these metabolites over a range of at least 0.1 to 100 ng/ 10(6) cells . The LC/MS/MS analyses took 10 to 15 min per sample and we could examine up to 50 samples per day . We also detected endogenous sphingosine 1-phosphate in HL-60 cells . The utility of the method was demonstrated by examining changes in metabolites levels in HL-60 cells after treatment with sphingomyelinase . It was found that sphingomyelinase from Bacillus cereus may have selectivity for acyl chain length.

Arch Biochem Biophys, 1997 Jan 15, 337(2), 308 - 16
Intracellular and extracellular forms of alkaline pullulanase from an alkaliphilic Bacillus sp . S-1; Lee MJ et al.; Bacillus sp.S-1 alkaline pullulanase (AP) exists in two forms: a precursor form (PUL-Ia, M(r) 180,000) and a processed form (PUL-Ib, M(r) 140,000) . PUL-Ia was accumulated intracellularly in large amounts, and PUL-Ib was detected in both the membrane fraction and the fraction trapped between the cytoplasmic membrane and the cell wall . Two forms of AP were purified to homogeneity and their properties were compared with previously purified PUL-E (140 kDa) . PUL-Ib showed similar properties, such as the M(r) value, the pI value (5.7), specific activity, substrate specificity, the NH2-terminal amino acid sequence (Phe-Leu-Asn-Met-Ser), and biophysical characters . However, in the case of PUL-Ia, even though the patterns of optimum pH and temperature, substrate specificity, and enzyme inhibition and activation were similar to those for PUL-Ib and PUL-E, the M(r) value and the pI value (5.97) were different . Furthermore, the NH2-terminal amino acid sequence of PUL-Ia was completely blocked, and the stabilities over pH and temperature ranges were decreased . The catalytic activities of PUL-Ia were distinguishable in the Km and Vmax values for various substrates and in the specific activity (71.4 U/mg) for pullulan hydrolysis . PUL-Ib and PUL-E showed 10-fold higher specific activities (744.6 for PUL-E and 736 for PUL-Ib) than PUL-Ia . However, PUL-Ia was immunologically identical to PUL-E and PUL-Ib . Therefore, it was concluded that PUL-Ib and PUL-E are the same form of the enzyme, suggesting that PUL-Ia is initially synthesized and proteolytically processed to the mature form of PUL-E . On the other hand, the translocation of AP required processing of the AP protein and the processing facilitated enzymatic activation and stabilization through a complete conformational change, resulting in an increase in affinity for substrates of PUL-E.

Structure, 1997 Jan 15, 5(1), 95 - 108
Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 A resolution; Kiefer JR et al.; BACKGROUND: The study of DNA polymerases in the Pol l family is central to the understanding of DNA replication and repair . DNA polymerases are used in many molecular biology techniques, including PCR, which require a thermostable polymerase . In order to learn about Pol I function and the basis of thermostability, we undertook structural studies of a new thermostable DNA polymerase . RESULTS: A DNA polymerase large, Klenow-like, fragment from a recently identified thermostable strain of Bacillus stearothermophilus (BF) was cloned, sequenced, overexpressed and characterized . Its crystal structure was determined to 2.1 A resolution by the method of multiple isomorphous replacement . CONCLUSIONS: This structure represents the highest resolution view of a Pol I enzyme obtained to date . Comparison of the three Pol I structures reveals no compelling evidence for many of the specific interactions that have been proposed to induce thermostability, but suggests that thermostability arises from innumerable small changes distributed throughout the protein structure . The polymerase domain is highly conserved in all three proteins . The N-terminal domains are highly divergent in sequence, but retain a common fold . When present, the 3'-5' proofreading exonuclease activity is associated with this domain . Its absence is associated with changes in catalytic residues that coordinate the divalent ions required for activity and in loops connecting homologous secondary structural elements . In BF, these changes result in a blockage of the DNA-binding cleft.

Biochemistry, 1997 Jan 14, 36(2), 347 - 55
Activation of phosphatidylinositol-specific phospholipase C toward inositol 1,2-(cyclic)-phosphate; Zhou C et al.; Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in discrete steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate . Water-soluble cIP was used as the substrate to study the cyclic phosphodiesterase activity and interfacial behavior of PI-PLC . Different detergent micelles and phospholipid vesicles were used to examine if "interfacial activation" of the enzyme could occur toward a soluble substrate . Almost all detergents examined activated the enzyme at least 2-fold, with PC species yielding the largest increases in PI-PLC specific activity . Kinetic parameters were measured in the absence and presence of several representative detergents (e.g., Triton X-100 and diheptanoylphosphatidylcholine (diC7PC)) . Gel filtration experiments showed that, under these conditions, the cIP did not partition to any measurable extent with these detergent micelles . The concentration at which half the maximum activation was observed occurred near the detergent CMC . Both Km and Vmax were altered by the presence of a surface: Km decreased to different degrees depending on the detergent, while Vmax increased substantially . The Km for cIP was 90 mM without detergent and decreased to 29 mM with diC7PC micelles added; Vmax increased almost 7-fold in the presence of diC7PC micelles . The enzyme efficiency (Vmax/Km) in the presence of diC7PC increased more than 21-fold, but it was still 20-fold lower than initial phosphotransferase activity for monomeric dihexanoylphosphatidylinositol . The poor efficiency of the cyclic phosphodiesterase activity is largely due to substrate binding affinity . The dependence of rate on substrate concentration exhibits cooperative behavior, especially without detergent . This cooperativity is discussed in terms of protein aggregation and ligand binding sites on the enzyme.

Biochemistry, 1997 Jan 14, 36(2), 333 - 40
Is the structure of the N-domain of phosphoglycerate kinase affected by isolation from the intact molecule?
Hosszu LL, Craven CJ, Spencer J, Parker MJ, Clarke AR, Kelly M, Waltho JP.
The structural integrity of the isolated N-domain (residues 1-174) of Bacillus stearothermophilus 3-phosphoglycerate kinase (PGK) has been investigated using heteronuclear NMR spectroscopy . Analysis of 13C chemical shifts, amide protection, and comparison of observed and expected sequential NOE intensities calculated from the crystal structure of the domain in the intact protein indicate that the secondary structure of the isolated domain is unchanged from that found in the intact molecule . Markedly shifted 1H resonances, amide protection, and long-range NOEs indicate that the tertiary structure is similarly unaffected . These results are confirmed by an excellent agreement (standard deviation 0.28 ppm) between observed H alpha chemical shifts and those calculated from the high-resolution (1.6 A) crystal structure of intact PGK {Davies et al . (1994) Acta Crystallogr . D50, 202-209} . The only region perturbed by loss of interactions with the C-domain is a small portion of the substrate-binding site (residues 148-152) whose amide protons are poorly protected from solvent . These results provide a structural basis for the analysis of the folding of the domains of PGK as isolated units and within the intact molecule {Parker et al . (1996) Biochemistry (in press)} and contrast with the notion that the native tertiary fold of the N-domain of PGK requires the whole polypeptide chain, including the entire C-domain {Mas et al . (1995) Biochemistry 34, 7931-7940} . Assignments of backbone 13C, 15N, HN, and H alpha resonances are provided.

Int J Cancer, 1997 Jan 6, 70(1), 128 - 34
Bacillus Calmette-Guérin mycobacteria stimulate human blood dendritic cells; Thurnher M et al.; Bacillus Calmette-Guerin (BCG) mycobacteria have been used as adjuvant in the active immunotherapy of various human cancers . In addition, dendritic cells, which are the most potent antigen-presenting cells, have been shown to be capable of initiating anti-tumor immune responses . Here we investigated the effects of BCG on dendritic cells cultured from human blood . Addition of BCG resulted in rapid homotypic adhesion of dendritic cells . Moreover, BCG concentrations ranging from 10(4) to 10(6) bacteria/ml enhanced expression of the dendritic-cell-maturation antigen CD83 and of the T-cell co-stimulator CD86 (B7-2) in a dose-dependent manner . Concomitant with the increase of CD83 and CD86 expression, the cells lost the ability to capture soluble antigens, as determined by the exclusion of fluoresceinated Dextran molecules . Strikingly, the same dosages of BCG-bacteria stimulated TNF-alpha-gene transcription and TNF-alpha-protein release from dendritic cells in a dose-dependent fashion . BCG infection of dendritic cells in the presence of a neutralizing antibody directed against TNF-alpha inhibited CD83 expression by more than 50% indicating that the BCG-induced maturation of dendritic cells was at least partially mediated by dendritic-cell-derived TNF-alpha . The finding that BCG activates the most potent antigen-presenting cells reveals a plausible immunological mechanism of the occasionally observed anti-tumor activity of BCG.

J Biol Chem, 1997 Jan 3, 272(1), 233 - 9
Tripartite hemolysin BL from Bacillus cereus . Hemolytic analysis of component interactions and a model for its characteristic paradoxical zone phenomenon; Beecher DJ et al.; Hemolysin BL (HBL) is a unique membrane-lytic toxin from Bacillus cereus composed of three distinct proteins, designated B, L1, and L2 . HBL produces a paradoxical zone phenomenon in gel diffusion assays in sheep blood agar . Lysis does not begin immediately adjacent to the source of diffusion; rather, it begins several millimeters away . Cells near the source and at intersections of lysis zones remain intact longer . Here, we developed a spectrophotometric hemolysis assay system that measures the activities of the individual HBL components and used it to analyze the mechanisms of hemolysis and the paradoxical zone phenomenon . The B component was rate-limiting, and erythrocytes were slowly primed by B at an optimal concentration of about 1.3 nM to rapid lytic action by the combination of the L components (L(1+2)) . All of the individual components bound to cells independently, and membrane-associated HBL components were neutralized by specific antibodies, suggesting that lysis was caused by formation of a membrane attack complex on the cell surface . Osmotic protection experiments indicate a colloid osmotic lysis mechanism . Concentrations of the B component above 1.3 nM caused inhibition of L1-mediated lysis, and L1 inhibited the priming reaction of B over a similar concentration range . From analyses of spectrophotometric and diffusion assays we constructed a basic model for the interactions between HBL components and for the paradoxical zone phenomenon in blood agar . In the latter, areas of slow lysis near diffusion sources are caused primarily by the accumulation of inhibitory levels of L1 reached before cells are primed by B.

Vestn Ross Akad Med Nauk, 1997, (6), 20 - 4
{Elucidation of functionally active domains in molecules of protective antigen bacillus anthracis's toxin}; Noskov AN et al.; Limited proteolysis has established that the protective antigen (PA) molecule consists of four functional-active domains . So, the shielding domain borrows an area in the linear structure of the PA molecule with NH2 of the end up to Lys 166 and plays a conducting role in the proteolytic activation of PA . The associative domain, engaging in the area Arg 167-Met266, plays a key role in the interaction with LF or EF at self-assembly toxic complexes LT or ET . The stabilizing domain borrows in the linear structure of the PA molecule are with Gly351 up to Met434 . On the one hand, this area promotes formation with LF conformationally steady toxic complex's, and, on the other, takes a direct participation in the formation of a hydrophobic channel by which the molecule LF or EF enters the target cell . The receptor domain, representing a COOH-terminal area, starting from Leu663 amino acid, begins to interact with specific receptors on the macrophages and thus delivers the toxic complex to the target cell . It has been found that in the molecule of lethal factor there are 3 functionally active domains located in the linear structure of the molecule as follows: the associative domain borrows an area from Lys39 up to Met242, stabilizing and effector domains occupy areas from Leu517 to Lys614 and from that point to Lys COOH-terminal amino acid.

Pneumoftiziologia, 1997 Jan-Mar, 46(1), 9 - 13
{The tuberculosis endemic and its control in the city of Bucharest in 1996}; Stoicescu P et al.; Tuberculosis is one of the main problems of public health and the population has to face it in one of the biggest crowded town in Romania, Bucharest . It is also the capital of the country . An increasing tendency has appeared in the number of cases and deaths due to tuberculosis . The overall incidence of tuberculosis has increased by 5% in 1996, attaining finally the level of 128.6/100,000 . The level is exceeded only by a few towns in Romania . The increase is due almost exclusively to the new cases of disease . The program of chemotherapy is facing a more reduced rate of cure and, implicitly, a high rate of failure by treatment . The periodical prevalence of the bacilliferous patients has attained 129.0/100,000 inhabitants . The deaths by tuberculosis have been increasing for a few years; the mortality was of 13.6/100,000 inhabitants in 1995 more than double as that in 1995 (6.5/100,000).

Respiration, 1997, 64(4), 304 - 6
Disseminated pulmonary granulomas after intravesical bacillus Calmette-Guérin immunotherapy; De Diego A et al.; Intravesical instillation of bacillus Calmette-Guerin (BCG) vaccine has been shown to be an effective treatment of superficial bladder cancer . However, it is not free of side-effects and complications . We present the case of a 62-year-old man who developed disseminated pulmonary granulomas after local BCG immunotherapy for recurrent papillary bladder cancer.

Respiration, 1997, 64(4), 296 - 9
Atypical locations of pulmonary tuberculosis and the influence of the roentgenographic patterns and sample type in its diagnosis; Navio P et al.; In 97 cases of pulmonary tuberculosis (PTB), we analyzed the incidence of atypical roentgenographic locations, roentgenographic patterns, the correlation between the diagnostic yield and the roentgenographic pattern and the usefulness of simple or induced sputum (82 cases), bronchoaspirate (BAS; 29 cases), postfiberoptic bronchoscopy sputum (PFBS; 16 cases) and how the different tests supplemented each other . Atypical locations were defined as those not corresponding to classic primary and postprimary PTB . This atypical-location PTB index was 8.2%, and roentgenographic patterns found most frequently were: destructive 52.5%, destructive-alveolar 20.6% and alveolar 12.3% . Lowenstein-Jensen (LJ) culture of the sputum of alveolar-pattern cases improved acid-fast bacillus (AFB) diagnosis by 46% (p < 0.005), in contrast to other radiologic patterns . Simple or induced sputum proved to be a very good diagnostic specimen in 98% of the cases (AFB staining 73.1% and LJ culture 89%) . BAS increased the sputum yield by 21% and PFBS contributed only 1 additional case to the results obtained with BAS . Therefore, BAS is a very good supplemental test in cases of false-negative findings.

Antibiot Khimioter, 1997, 42(4), 16 - 20
{Cytotoxic properties of the complex of the antineoplastic antibiotic bleomycetin and Bacillus intermedius ribonuclease}; Kalacheva NV et al.; It was shown possible to change the cytotoxic properties of the antitumor antibiotic bleomycetin by its binding to Bacillus intermedius RNAse . The complexing lowered the antibiotic effect on DNA in the cells of the human amnion . At the same time the experiments with human red blood cells indicated that RNAse of B . intermedius in complex with bleomycetin-Fe(II) increased the antibiotic capacity for the cell membrane break down.

Int J Urol, 1997 Jan, 4(1), 68 - 73
Immunohistochemical study of tumor-infiltrating lymphocytes before and after intravesical bacillus Calmette-Guérin treatment for superficial bladder cancer; Honda S et al.; BACKGROUND: This study investigated changes in the phenotypic characteristics of tumor-infiltrating lymphocytes during intravesical bacillus Calmette-Guerin (BCG) treatment using an immunohistochemical technique . METHODS: A total of 16 patients with superficial bladder cancer underwent intravesical BCG treatment for therapeutic purposes . Tissue specimens were obtained from these patients before and after BCG treatment by cold cup biopsies . RESULTS: The numbers of CD3+ cells, CD4+ cells, CD8+ cells, and CD19+ cells significantly increased after treatment compared with numbers before treatment (P < 0.01) . Although gamma/delta T cells were not observed before treatment, they appeared after treatment in 6 patients . In all these patients, the tumors disappeared or their size was reduced by more than 50%, and none of the tumors recurred . The induction of CD25+ cells after treatment was seen in 11 of the 16 patients . CONCLUSION: gamma/delta T cells may play an important role in the immune response of the host to the tumor in intravesical BCG treatment (although this correlation was statistically insignificant.

Rev Argent Microbiol, 1997 Jan-Mar, 29(1), 1 - 6
Solid state production of a Mucor bacilliformis acid protease; Fernandez Lahore HM et al.; Acid protease production by a local strain of Mucor bacilliformis was performed by solid state cultivation on different agricultural by-products as substrate . The effects of different parameters on enzyme biosynthesis were studied: Wheat bran wetted at 120% with a 200 mM HCl solution and inoculated with 5 x 10(5) spores/g produced a milk clotting activity of 7500 U/g bran after 72 h cultivation at 24 degrees C.

Mol Membr Biol, 1997 Jan-Mar, 14(1), 13 - 8
Channel activity caused by a Bacillus thuringiensis delta-endotoxin preparation depends on the method of activation; Smedley DP et al.; The spontaneous insertion of Bacillus thuringiensis Cry delta-endotoxins into planar lipid bilayers to form discrete channels in the absence of receptors is the subject of conflicting reports in the literature . Because these proteins are synthesized as protoxins requiring proteolytic activation for conversion to the active form, differences in the in-vitro protocol used for this activation could be responsible for the contradictory results . To investigate this, CrylA(c) toxin was activated by different procedures, and its ability to release glucose entrapped within liposomes and to form channels in planar lipid bilayers assessed . The toxin preparations exhibited widely differing activities on the lipid membranes; SDS-PAGE and immunoblot analysis suggested that variations in the protein profile of the activated samples could be responsible . These findings raise important practical considerations for further in-vitro studies into the mechanism of action of these toxins.

World J Urol, 1997, 15(2), 96 - 102
Biologic response modifiers in the management of superficial bladder cancer; Serels S et al.; For the treatment of existing transitional-cell carcinoma or for prophylaxis of recurrent disease, intravesical therapy should be chosen according to stage . Papillary disease (stages Ta, Tl) may be treated effectively either with an alkylating agent or with bacillus Calmette-Guerin (BCG) . BCG is the agent of choice for the treatment of Hat carcinoma in situ (Tis), with the recommended treatment course comprising 12 weekly and 12 monthly instillations . Intravesical interferon and many of the other biologic response modifiers mentioned herein may be effective for patients with Ta disease who have failed BCG therapy.

Biochemistry (Mosc), 1997 Jan, 62(1), 49 - 53
Enzymatic properties of thiol-dependent serine proteinase of Bacillus intermedius 3-19; Itskovich EL et al.; Effects of a thiol-dependent serine proteinase of Bacillus intermedius on peptide substrates and insulin B-chain were studied . The enzyme preferably splits peptide bonds formed by carboxyl groups of hydrophobic amino acids . Ca2+ increases the thermal stability of the proteinase significantly . The kinetic characteristics of hydrolysis of Z-Ala-Ala-Leu-pNA by this enzyme was determined as K(m) = 1.25 mM and kcat = 0.15 sec-1 . The enzyme has high stability to DMFA and isopropanol, and is able to catalyze peptide bond synthesis.

Parasitol Res, 1997, 83(3), 209 - 13
Biological control studies of soft and hard ticks in Egypt . I . The effect of Bacillus thuringiensis varieties on soft and hard ticks (ixodidae); Hassanain MA et al.; The potential activity of three varieties of Bacillus thuringiensis (kurstaki, israeliensis, and thuringiensis) against the soft tick Argas persicus and the hard tick Hyalomma dromedarii was investigated . Soft ticks succumbed within a period ranging from 36 h to 5 days and hard ticks died at between 48 h and 10 days posttreatment, depending on the dose . Concentrations lethal to 50% of tick populations (LC50 values) indicated that Dipel 2x (B . thuringiensis var . kurstaki) was the most potent, followed by Vectobac (B . thuringiensis var . israeliensis), then HD 703 (B . thuringiensis var . thuringiensis) . A . persicus was more affected than H . dromedarii by B: thuringiensis varieties . Eggs were mostly affected at 16 and 25 days after deposition for A . persicus and H . dromedarii, respectively.

J Med Entomol, 1997 Jan, 34(1), 5 - 10
Biological fitness of Culex quinquefasciatus (Diptera:Culicidae) susceptible and resistant to Bacillus sphaericus; Rodcharoen J et al.; Biological fitness of laboratory and field-collected strains of southern house mosquito, Culex quinquefasciatus Say, susceptible and resistant (37- and 31-fold upon selection) to the microbial agent, Bacillus sphaericus, were compared in the absence of B . sphaericus . The resistant strains showed significantly lower fecundity and fertility, but they had significantly higher survival rates than the susceptible strains . The preadult stages from females of resistant strains developed at slightly faster rates than those of the susceptible strains, which could result in a shorter generation time . However, lower fecundity was likely to lead to overall lower population growth rates than in the susceptible strains . Data provided evidence that the resistant strains exhibited fitness disadvantages in the absence of B . sphaericus . We suggest that once resistance to B . sphaericus is detected in the field, its use should be discontinued until the mosquito population becomes susceptible again because of the decline in number of resistant individuals . A strategy of resistance management by rotation of insecticides is discussed.

Urol Res, 1997, 25(1), 19 - 24
Local expression of cytokines in rat bladder carcinoma tissue after intravesical treatment with Nocardia rubra cell wall skeleton and bacille-Calmette-Guerin; Rohde D et al.; This study aimed to investigate local immunotherapy with Nocardia rubra cell wall skeleton (N-CWS) and bacille-Calmette-Guerin (BCG) in chemically induced rat bladder carcinoma . After being fed with 0.025% N-butyl-N-(4-hydroxybutyl) nitrosamine for 26 weeks, female Wistar rats were treated once a week by intravesical instillation of 100 microg N-CWS or 5 x 10(6) Colony-Forming Units (CFU) BCG . Tissue specimens were obtained 4 h after the ninth instillation and analyzed by reverse transcription polymerase chain reaction (RT-PCR) for mRNA expression of rat cytokines . The analysis showed high expression of interleukin (IL)-1 alpha, tumor necrosis factor (TNF)-alpha, IL-2, and interferon (IFN)-tau in BCG (7/7, 7/7, 7/7, 5/7) and N-CWS (9/9, 9/9, 8/9, 8/9) treated tumors in comparison to low expression in controls (3/9, 0/9, 3/9, 3/9) . Interestingly, intravesical instillation of N-CWS tended to be less effective at preventing local invasion of the tumors . It is speculated that this difference may result from a more strongly induced expression of T-helper cell-derived lymphokines (IL-2, IFN-tau) by BCG.

Glycoconj J, 1997 Jan, 14(1), 75 - 80
Enzymatic syntheses of GlcNAc beta 1-2Man and Gal beta 1-4GlcNAc beta 1-2Man as components of complex type sugar chains; Fujimoto H et al.; GlcNAc beta 1-2Man and GlcNAc beta 1-6Man were synthesized using the reverse hydrolysis activity of beta-N-acetylglucosaminidase from both jack beans and Bacillus circulans . In turn, Gal beta 1-4GlcNAc beta 1-2Man and Gal beta 1-4GlcNAc beta 1-6Man were synthesized regioselectively using the transglycosylation activity of beta-galactosidase from Diplococcus pneumoniae and B . circulans, respectively . These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies.

Plasmid, 1997, 37(1), 15 - 21
Characterization of pBP614, a putative rolling-circle plasmid from Bacillus popilliae; Longley M et al.; A plasmid, pBP614 (5.647 kb), has been isolated from the New Zealand Bacillus popilliae strain 17 and characterized by physical mapping, cloning, and sequencing . An open reading frame was found which could encode a protein with homology to the replication (Rep) proteins of rolling-circle plasmids . The predicted B . popilliae Rep protein shows closest homology to Rep proteins from Bacillus plasmids of the pC194 family . Sequences homologous to plus- and minus-strand replication origins were found . The minus-strand origin shows similarities to the pal-T type family . Taken together, these observations suggest that pBP614 is a rolling-circle plasmid, the first such plasmid characterized from B . popilliae.

Cancer Immunol Immunother, 1997 Jan, 43(6), 324 - 30
Tumor cell reactivity mediated by IgM antibodies in sera from melanoma patients vaccinated with GM2 ganglioside covalently linked to KLH is increased by IgG antibodies; Livingston P et al.; Natural IgM antibodies against the melanoma cell-surface ganglioside GM2, and IgM antibodies induced by vaccination with GM2 adherent to bacillus Calmette-Guerin, have been correlated with increased disease-free and overall survival in melanoma patients in previous phase I and II clinical trials . A vaccine containing GM2 covalently attached to keyhole limpet hemocyanin (KLH) plus the immunological adjuvant QS-21 now induces higher-titer, longer-lasting IgM antibodies against GM2 and has recently entered phase III clinical trials . For the first time this new vaccine also induces IgG antibodies against GM2 in the majority of immunized patients . With regard to immunity against bacteria, IgM antibodies have been described to be 1000-fold more effective than IgG antibodies at opsonification, complement-mediated cytotoxicity and protection from bacterial challenge . Though IgG antibodies have the theoretical advantage of being able to mediate antibody-directed cell-mediated cytotoxicity (ADCC), they may inhibit complement mediated IgM effector mechanisms against melanoma cells . Our goal was to confirm the functional characteristics of the anti-GM2 IgM and IgG antibodies induced by vaccination and to determine the impact that IgG antibodies might have on IgM antibody reactivity with GM2-positive tumor cells . Post-immunization sera from seven immunized patients were separated by size-exclusion chromatography into IgM and IgG fractions and a variety of serological assays were performed with the individual fractions and their combinations . Assays identifying specific IgM or IgG reactivity demonstrated partial inhibition by the opposite fraction . However, when the endpoint was complement-mediated lysis or overall antibody binding, which may more faithfully predict in vivo complement-mediated opsonification and lysis, the combinations of IgM and IgG fractions consistently demonstrated higher reactivity than either fraction alone . In addition, ADCC was induced in all seven patients . The results were the same whether the sera were obtained after 2 months or 2 years of immunizations . These findings suggest that IgG antibodies induced by the GM2-KLH plus QS-21 vaccine will not inhibit and should further augment the clinical impact of induced IgM antibodies.

Anticancer Res, 1997 Jan-Feb, 17(1A), 445 - 50
Activity of a mycobacterial antineoplastic glycan against human breast cancer; Donmez C et al.; BACKGROUND: Attenuated Mycobacterium bovis, Bacillus Calmette Guerin, BCG vaccine, is a general immune stimulant and is now an approved clinical treatment for superficial bladder cancer . Isolation and characterization of a series of complex polysaccharides (glycans) from BCG and other mycobacteria has shown that these materials are remarkably heat stable and have considerable in vivo activity against a number of animal cancer models . This present communication describes the testing of a glycan, PS1, obtained from the Tice substrain of BCG against the hormonal dependent human breast cancer cell line MCF-7 and the hormonally independent BT-20 line, using 5-fluorouracil (5-FU) as a positive control . MATERIALS AND METHODS: The PS1 was obtained by methods previously described . Cells were obtained from the American Type Culture Collection (Rockville, MD) and athymic nu/nu mice from Frederick (MD) . The cells were implanted into the flanks of 20g female nude mice (n = 10) . After two weeks, volumes of phosphate buffered saline (control), 5-FU (positive control) or PS1 solutions were injected and the tumor growth rates followed for up to six weeks . RESULTS: The 5-FU was effective in slowing tumor growth of both tumors . The MCF-7 cell line was markedly affected by the PS1, especially in the presence of estradiol . The BT-20 cell line was only marginally affected by PS1, with or without estradiol . CONCLUSIONS: Since PS1 is known to have macrophage stimulating activity and nude mice are deficient in both T-cells and natural killer cells, the mechanism of activity is postulated to involve MHC-1 antigen secretion by the hormonal-dependent tumor cells, enhanced in the presence of hormone . These cells are then actively identified and destroyed by local macrophages.

Medicine (Baltimore), 1997 Jan, 76(1), 30 - 41
Chryseobacterium meningosepticum: an emerging pathogen among immunocompromised adults . Report of 6 cases and literature review; Bloch KC et al.; Chryseobacterium meningosepticum is a ubiquitous Gram-negative bacillus historically associated with meningitis in premature neonates . We report 15 positive cultures and 6 cases of infection among immunocompromised adults at our institution over a 10-year period and review the English-language literature on C . meningosepticum . Excluding the present series, there are 308 reports of positive cultures in the literature, of which 59% were determined to represent true infections . Sixty-five percent of those infected were younger than 3 months of age . Meningitis was the most common infectious syndrome among neonates, seen in 84% of cases and associated with a 57% mortality rate . Less commonly reported infections among infants included sepsis (13%) and pneumonia (3%) . Pneumonia was the most frequent infection among the postneonatal group, accounting for 40% of cases, followed by sepsis (24%), meningitis (18%), endocarditis (3%), cellulitis (3%), abdominal infections (3%), eye infections (3%), and single case reports of sinusitis, bronchitis, and epididymitis . The 6 cases in our series were all adults, with a mean age of 58.7 years . Sites of C . meningosepticum infection were limited to the lungs, bloodstream, and biliary tree . Infection in our series was associated with prolonged hospitalization, prior exposure to multiple antibiotics, and host immunocompromise, particularly neutropenia . C . meningosepticum is resistant to multiple antibiotics, and disk dilution is notoriously unreliable for antibiotic sensitivity testing . Sensitivity testing on the 15 isolates from our institution revealed the most efficacious antibiotics to be minocycline (100% sensitive), rifampin (93%), trimethoprim-sulfamethoxazole (67%), and ciprofloxacin (53%) . In contrast to reports in the literature, the isolates in our series displayed widespread resistance to vancomycin (100% resistant or intermediately sensitive), erythromycin (100%), and clindamycin (86%) . These findings have important implications for the clinician when choosing empiric antibiotic regimens for patients with risk factors for C . meningosepticum infection.

APMIS, 1997 Jan, 105(1), 48 - 54
A preliminary study on the pathogenicity of Bacillus licheniformis bacteria in immunodepressed mice; Agerholm JS et al.; The pathogenicity of 13 strains of Bacillus licheniformis was studied in immunodepressed mice . The strains had been isolated from cases of bovine abortions (n = 5), bovine feedstuffs (n = 3), soil (n = 1), and grain products (n = 2) . The origin of two strains was unknown . Groups of 10 mice were inoculated intravenously with B . licheniformis bacteria at doses from < 10(6) to 10(10) colony-forming units . Following 7 days of infection, the animals were euthanized and examined bacteriologically, histologically, and immunohistochemically using a PAP technique based on primary polyclonal rabbit anti-B . licheniformis antibodies . B . licheniformis bacteria were reisolated from the liver, spleen or kidneys of mice in all groups . Inflammatory lesions were present in mice of all immunodepressed groups, but only brain and pulmonic lesions were definitely attributed to B . licheniformis infection, as strong immunostaining was found within these lesions . It is concluded that all strains of B . licheniformis examined were pathogenic for immunodepressed mice, and that spontaneous infections may be established by bacterial strains to which susceptible individuals are accidentally exposed.

Mol Microbiol, 1997 Jan, 23(2), 313 - 22
Mycobacterium bovis BCG genes involved in the biosynthesis of cyclopropyl keto- and hydroxy-mycolic acids; Dubnau E et al.; The resurgence of tuberculosis and the emergence of multidrug-resistant mycobacteria necessitate the development of new antituberculosis drugs . The biosynthesis of mycolic acids, essential elements of the mycobacterial envelope, is a good target for chemotherapy . Species of the Mycobacterium tuberculosis complex synthesize oxygenated mycolic acids with keto and methoxy functions . In contrast, the fast-growing Mycobacterium smegmatis synthesizes oxygenated mycolic acids with an epoxy function . We describe the isolation and sequencing of a cluster of four genes from Mycobacterium bovis bacillus Calmette-Guerin (BCG), coding for methyl transferases, and which, when transferred into M . smegmatis, allow the synthesis of ketomycolic acid, in addition to an as yet undescribed mycolic acid, hydroxymycolic acid . These oxygenated mycolic acids, unlike the regular mycolic acids of M . smegmatis, and similar to the mycolic acids of M . bovis, are highly cyclopropanated . Furthermore, there is a perfect match between the structures of the keto- and the hydroxy-mycolic acids . We propose a biosynthetic model in which there is a direct relationship between these two types of mycolic acid.

Br J Dermatol, 1997 Jan, 136(1), 60 - 5
Bacillary angiomatosis: presentation of six patients, some with unusual features; Schwartz RA et al.; Bacillary angiomatosis (BA) is an unusual systemic vascular proliferation seen predominantly in patients with the acquired immunodeficiency syndrome . These vascular lesions are probably due to infection with a Bartonella species, most often B . henselae and, in some patients, B . quintana . BA is treatable and often curable, but without therapy, may be life-threatening . Clinically, the lesions, when superficial, are said to often resemble pyogenic granulomas, appearing polypoid histologically with an epidermal collarette . We now report six patients, three of whom showed lesions of BA morphologically and histologically distinct from the other patients reported to date . Two patients lesions appeared clinically as violaceous plaques and tumours resembling Kaposi's sarcoma; one of them had lesions histologically reminiscent of a papular angiokeratoma; and the other had lesions histologically suggestive of a combination of Kaposi's sarcoma and BA . Another patient presented with soft subcutaneous nodules which histologically showed extensive acute inflammation characteristic of an acute abscess, but which also displayed proliferating dilated small blood vessels with bulbous endothelial cells adjacent to numerous bacteria and also containing them . The Grocott-methenamine silver stain and the Warthin-Starry stain showed the organisms to better advantage in lesions of all six patients, although bacteria were also evident with the haematoxylin and eosin, periodic acid-Schiff and alcian blue stains.

New Microbiol, 1997 Jan, 20(1), 69 - 76
Distribution and characterization of Bacillus thuringiensis in the environment of the olive in Greece; Aptosoglou SG et al.; The distribution of Bacillus thuringiensis in Greece, was studied and the presence of this organism was found in 11.2% of the samples collected . The characterization of the different isolates was based on their whole-cell protein profiles . Some of the isolates form quite unusual parasporal inclusions.

J Invertebr Pathol, 1997 Jan, 69(1), 24 - 30
A 16S rRNA gene oligonucleotide probe for identification of Bacillus thuringiensis isolates from sheep fleece; Akhurst RJ et al.; Larvae of the Australian sheep blowfly Lucilia cuprina are susceptible to some strains of Bacillus thuringiensis, including some isolates from sheep fleeces from South Australia and Western Australia . Larvicidal strains of B . thuringiensis include var . thuringiensis, tolworthi, darmstadiensis, morrisoni, and toumanoffi . As part of an ecological study, a large number of B . thuringiensis fleece isolates from untreated sheep and sheep previously treated with larvicidal B . thuringiensis were screened for larvicidal activity . A ribotyping method to distinguish larvicidal B . thuringiensis jetted (sprayed) onto sheep from the natural B . thuringiensis flora of the fleece was developed . A cloned fragment of the 16S rRNA gene used as a probe for B . thuringiensis DNA digested with restriction endonucleases enables separation of B . thuringiensis serovars and separation within the serovars thuringiensis, tolworthi, and kurstaki . The 16S gene probe method is useful for distinguishing between reisolates of larvicidal B . thuringiensis jetted onto sheep and the background B . thuringiensis population present prior to jetting.

Biosci Biotechnol Biochem, 1997 Jan, 61(1), 202 - 3
High efficiency transformation of Bacillus brevis by electroporation; Okamoto A et al.; Various conditions in the Bacillus brevis transformation by electroporation were investigated to increase the efficiency . Competent cell volume and presence of polyethylene glycol 6000 and single stranded DNA on pulsing were critical for the efficiency . As a result, we established the improved method by which 10(7)-10(8) transformants/microgram plasmid DNA could be obtained.

Eur J Immunol, 1997 Jan, 27(1), 183 - 8
Bacille Calmette Guérin and interleukin-12 down-modulate interleukin-4-producing CD4+ NK1+ T lymphocytes; Emoto M et al.; Early production of interleukin-12 (IL-12) by macrophages and of IL-4 from CD4+ NK1+ T cells influence development of the acquired immune response against infectious agents, namely differentiation of interferon-gamma-secreting T helper 1 (Th1) cells against intracellular pathogens and of IL-4-producing Th2 cells against helminths . Evidence has been presented for transient convertibility of Th1 and Th2 cells in the presence of the polarizing cytokines IL-4 or IL-12, respectively . Moreover, it is likely that IL-4 dominates over IL-12, suggesting that Th2 cell development is preferred in the presence of both cytokines . Mycobacterium bovis Bacille Calmette Guerin (BCG) and IL-12 are potent inducers of Th1 responses . Here we show that BCG and IL-12 down-modulate IL-4-producing CD4+ NK1+ TCR alpha/beta(intermediate) liver lymphocytes . Our data provide further insights into the mechanisms by which BCG and IL-12 may promote unrestricted development of Th1 responses in vivo: BCG and IL-12 not only provide the positive stimuli for Th1 cell differentiation, but also interfere with antagonizing signals.

Pharmacol Ther, 1997, 73(1), 1 - 50
Tuberculosis in Africa: clinical presentation and management; Harries AD; In the last decade, sub-Saharan Africa has experienced an explosive increase in tuberculosis (TB) cases, largely as a result of the co-epidemic of human immunodeficiency virus (HIV) infection . This article reviews the essential background epidemiology of TB in sub-Saharan Africa . The clinical features and diagnostic problems of pulmonary/extrapulmonary TB in adults and children are discussed, particularly in relation to HIV infection . Different treatment regimens, their cost, adverse reactions, the ways in which HIV infection influences treatment response and the extent of drug resistance are reviewed . The recommended approaches to TB control in Africa, including methods used to prevent TB through Bacillus Calmette-Guerin and chemoprophylaxis are examined . The success achieved by good National TB Control Programmes in some African countries allows cautious optimism that this epidemic can be controlled.

Scand J Immunol, 1997 Jan, 45(1), 21 - 7
Inhibition of neutrophil apoptosis by antioxidants in culture medium; Oishi K et al.; Neutrophil apoptosis is an important mechanism that has implications for understanding the life span and toxic potentials of neutrophils at inflamed sights . In this study the authors examined the possibility that reactive oxygen species (ROS) released by neutrophils can regulate neutrophil survival . Cu,Zn-superoxide dismutase (Cu,Zn-SOD), Mn-SOD, and catalase in culture media were significantly effective in delaying the spontaneous apoptosis, suggesting that ROS play an important role in the resolution of inflammation by inducing neutrophil apoptosis . In this experiment, boiled Cu,Zn-SOD had no effect on inhibiting the apoptosis, but boiled Mn-SOD from Bacillus stearothermophilus was more effective in inhibiting the apoptosis than untreated Mn-SOD at the same dose . However, the boiled Mn-SOD showed only 80% of O2- inhibitable activity compared with the untreated Mn-SOD . This effect may be attributed to the partial liberation of manganese because MnCl2 inhibited the apoptosis effectively . Furthermore, Cu,Zn-SOD was effective in delaying apoptosis only when added to the culture within the first 3 h of incubation, suggesting that the isolation of neutrophils from peripheral blood enhances apoptosis of neutrophils.

Arch Microbiol, 1997 Jan, 167(1), 24 - 31
Haem O and a putative cytochrome bo in a mutant of Bacillus cereus impaired in the synthesis of haem A; Del Arenal IP et al.; In a spontaneous mutant (PYM1) of Bacillus cereus impaired in the synthesis of haem A, no haem-A-containing cytochromes were detected spectroscopically . The haem A deficiency was compensated by high levels of haem O and a CO-reactive cytochrome o in membranes; no other oxidases were detected . In contrast, the wild-type strain had considerable amounts of haem A and negligible levels of haem O . The mutant PYM1 exhibited normal colony morphology, growth, and sporulation in nonfermentable media, whereas on fermentable media, the mutant overproduced acid, which led to poor growth and inhibition of sporulation . External control of the pH of the medium in fermentable media allowed close-to-normal growth and massive sporulation of the mutant . The presence of membrane-bound cytochrome caa3-OII and aa3-II subunits in strain PYM1 was confirmed by Western blots and haem C staining (COII subunit) . Western blotting also revealed that in contrast to the wild-type - strain PYM1 contained the membrane-bound subunits caa3-COI and aa3-I, but in low amounts . The effect of several respiratory inhibitors on the respiratory system of strain PYM1 suggested that the terminal oxidase is highly resistant to KCN and CO and that a c-type cytochrome might be involved in the electron transfer sequence to the putative cytochrome bo.

FEMS Microbiol Lett, 1997 Jan 1, 146(1), 47 - 51
Discrimination between Bacillus cereus and Bacillus thuringiensis using specific DNA probes based on variable regions of 16S rRNA; te Giffel MC et al.; Identification of Bacillus cereus and differentiation between B . cereus and closely related species are currently based on biochemical tests . The main problem is to discriminate between B . cereus and B . thuringiensis . Sequencing part of the 16S rRNA showed that several B . cereus isolates present in food and involved in food poisoning, confirmed according to the classical biochemical methods, were in fact B . thuringiensis . As this organism is the most commonly used microbial insecticide worldwide, the results of this study emphasize the need for accurate identification methods and for careful screening of strains for use as insecticides . Therefore, specific DNA probes based on the variable region VI of 16S rRNA of B . cereus and B . thuringiensis were designed . The probes were used in hybridization experiments with the variable region amplified using the polymerase chain reaction . In this way, a rapid and sensitive method was developed to distinguish B . cereus and B . thuringiensis.

J Bacteriol, 1997 Jan, 179(1), 170 - 9
Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation; Okamoto S et al.; Morphological differentiation in microorganisms is usually accompanied by a decrease in intracellular GTP pool size, as has been demonstrated in bacillaceae, streptomycetaceae, and yeasts . The obg gene, which codes for a GTP-binding protein belonging to the GTPase superfamily of proteins, was cloned from Streptomyces griseus IFO13189 . The gene is located just downstream of the genes for ribosomal proteins L21 and L27, encoded a protein of 478 amino acids (51 kDa), and possessed three consensus motifs which confer GTP-binding ability; Obg protein expressed in Escherichia coli bound GTP, as demonstrated using a UV cross-linking method . Introduction of multiple copies of obg into wild-type S . griseus suppressed aerial mycelium development in cells on solid media . However, no effect on streptomycin production was detected, indicating that Obg is involved in the regulation of the onset of morphological but not physiological differentiation . Multiple copies of obg also suppressed submerged spore formation in liquid culture . Southern hybridization studies indicated that genes homologous to obg exist widely in streptomycetes, and an obg homolog was successfully cloned from S . coelicolor A3(2) . We propose that by monitoring the intracellular GTP pool size, the Obg protein is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation.

Antimicrob Agents Chemother, 1997 Jan, 41(1), 135 - 40
Inhibition of metallo-beta-lactamases by a series of mercaptoacetic acid thiol ester derivatives; Payne DJ et al.; A series of mercaptoacetic acid thiol esters have been identified as metallo-beta-lactamase inhibitors . Electrospray mass spectrometry (ESMS) has shown that irreversible inhibition of the Bacillus cereus II metallo-beta-lactamase by SB214751, SB214752, and SB213079 was concomitant with a 90-Da increase in mass of the enzyme . Tryptic digestion of the B . cereus II inhibited with SB214751 illustrated that the peptide fragment, containing the only cysteine of the enzyme, had undergone a mass increment of 90 Da . It was further demonstrated that B . cereus II hydrolyzed this type of compound across the thiol ester bond to yield mercaptoacetic acid . Mercaptoacetic acid is the only molecular fragment common to SB214751, SB214752, and SB213079, and free mercaptoacetic acid does not bind covalently to B . cereus II . Therefore, it is concluded that these compounds inhibit B . cereus II by the mechanism-based delivery of mercaptoacetic acid, forming a disulfide linkage with the active sites cysteine (predicted mass shift = +90 Da) under the aerobic conditions of the assay . The different thiol esters examined had a broad range of potencies against the metallo-beta-lactamases tested . For example SB214751, SB214752, and SB213079 all had 50% inhibitory concentrations of < 10 and > 1,000 microM for the Stenotrophomonas maltophilia L-1 and Bacteroides fragilis CfiA enzymes, respectively . SB216968 was particularly active against the Aeromonas hydrophila CphA metallo-beta-lactamase and was found to be an uncompetitive inhibitor of this enzyme (Ki = 3.9 microM), whereas it exhibited irreversible inhibition of the L-1 enzyme . These observations with this series of compounds have revealed subtle differences between the active sites of different metallo-beta-lactamases . Finally, a novel application for isothermal titration calorimetry for assessing the zinc chelating activity of candidate inhibitors is also presented.

J Immunol, 1997 Jan 1, 158(1), 330 - 7
IL-6 produced by macrophages infected with Mycobacterium species suppresses T cell responses; VanHeyningen TK et al.; The ability of Mycobacterium bovis Calmette-Guerin bacillus-infected bone marrow-derived macrophages to process and present exogenously added Ags to T cells and stimulate their growth and production of IL-2 was examined . The infected macrophages were inhibited in their ability to activate T cells, and this inhibition could be transferred to uninfected macrophages with filtered supernatants from mycobacteria-infected macrophages . The inhibition was not due to decreases in macrophage viability, Ag uptake, or cell surface expression of MHC class II or other accessory molecules necessary for Ag presentation . Other intracellular pathogens such as Listeria monocytogenes and Leishmania mexicana did not induce the soluble inhibitory factor, while Mycobacterium avium strain 101 did, suggesting the factor is specific to infection with mycobacteria . The inhibitory effect was reversed completely by preincubation with neutralizing Abs against IL-6, and rIL-6 partially restored the effect . Approximately 10,000-fold more IL-6 was produced by mycobacteria-infected macrophages compared with uninfected controls . Such sustained levels of IL-6 may account for the immune unresponsiveness apparent in both human and murine mycobacterial disease.

J Immunol, 1997 Jan 1, 158(1), 315 - 21
T cell-derived IL-10 antagonizes macrophage function in mycobacterial infection; Murray PJ et al.; Pathogenic mycobacteria survive within macrophages despite T cell responses that activate host defenses against most pathogens . Among cytokines produced by T cells, IL-10 is known to negatively regulate Th1 cells as well as macrophages . IL-10 has been shown to inhibit the anti-mycobacterial activity of macrophages in vitro and could account for the ability of mycobacteria to survive intracellularly . To test the inhibitory functions of IL-10 in vivo, transgenic mice that secrete IL-10 from the T cell compartment were constructed and infected with Calmette-Guerin bacillus (Mycobacterium bovis) . These mice were unable to clear the infection and developed large bacterial burdens . Nonetheless, their T cells produced abundant amounts of IFN-gamma and IL-2 in response to Ag challenge . These results indicate that the presence of excess IL-10 had little, if any, effect on T cell function or development during the immune response to Calmette-Guerin bacillus . Rather, the data suggest that IL-10 helps maintain mycobacterial infections by acting primarily at the level of the macrophage, overriding anti-mycobacterial signals delivered by IFN-gamma.

J Immunol, 1997 Jan 1, 158(1), 308 - 14
Albumin nitrosylated by activated macrophages possesses antiparasitic effects neutralized by anti-NO-acetylated-cysteine antibodies; Mnaimneh S et al.; Activated macrophages exert an L-arginine-dependent cytostatic effect on the extracellular parasite, Trypanosoma musculi . This effect is not observed in the absence of albumin in the culture medium but is restored by the addition of albumin, indicating the presence of an albumin-nitric oxide (NO) adduct acting as an effector molecule . Since L-cysteine represents a privileged target for NO, an immunochemical approach was performed using an acetylated-cysteine-BSA conjugate . This conjugate was nitrosylated using sodium nitrite as a NO donor . Binding of NO to the conjugated haptens was assayed using spectrophotometry . It was completely abolished by mercuric chloride, confirming the presence of an S-NO bond . Polyclonal Abs were obtained after immunizing rabbits with S-nitroso-acetylated-cysteine (NO-ac-Cys) conjugates . Using the enzyme-linked immunosorbent assay method, Ab avidity and specificity were determined by competition experiments between NO-ac-Cys-conjugated compounds and other nitrosylated or non-nitrosylated compounds . The resulting cross-reactivity ratios showed that conjugated NO-ac-Cys-BSA was the best recognized compound . These Ab were used for an in vitro study of the kinetics of NO-derived compounds from activated murine macrophages . Anti-NO-ac-Cys Ab inhibited the antimicrobial effect of activated macrophages on the extracellular parasite, T . musculi . Moreover, the L-arginine-dependent antiparasitic activity of supernatants from Calmette-Guerin bacillus-activated macrophages required the presence of albumin and was also inhibited by anti-NO-ac-Cys Ab, showing the effector role of S-nitroso-albumin.

J Biol Chem, 1996 Dec 27, 271(52), 33390 - 3
Localization of a conformational energy-coupling determinant near the C terminus of the beta subunit of the F1F0-ATPase; Schemidt RA et al.; Escherichia coli mutants in the beta subunit of the F1F0-ATPase can be complemented with the beta subunit from the obligate aerobe Bacillus megaterium . It has been shown that cells carrying such hybrid ATPases have an unusual energy-coupling phenotype . Although they are able to grow on minimal succinate medium, and therefore carry a functional ATP synthase, they are defective in the ability to grow anaerobically, indicating some defect in ATP-driven proton pumping (Scarpetta, M., Hawthorne, C . A., and Brusilow, W . S . A . (1991) J . Biol . Chem . 266, 18567-18572) . In this study, chimeric beta subunits were constructed consisting of the E . coli or the B . megaterium beta subunit carrying the C-terminal 18% of the other's beta subunit . The phenotypes of an E . coli beta mutant complemented with these chimeric subunits showed that the energy-coupling defect was located in this C-terminal region . The E . coli beta subunit carrying the B . megaterium C-terminal region displayed the energy-coupling defect, while the B . megaterium beta subunit carrying the E . coli C-terminal region did not . In ATP-dependent fluorescence quenching assays, membranes isolated from cells displaying the energy-coupling defect also pumped protons less well than membranes isolated from cells that were able to grow anaerobically . These results demonstrate that the C terminus of the beta subunit is involved in the conformational coupling pathway, which, through the polypeptide backbone of the beta subunit, physically links ATP synthesis or hydrolysis to the energy of proton translocation.

Biochemistry, 1996 Dec 24, 35(51), 16863 - 70
Competitive interaction of component enzymes with the peripheral subunit-binding domain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus: kinetic analysis using surface plasmon resonance detection; Lessard IA et al.; The interactions of the peripheral enzymes (E1, a pyruvate decarboxylase, and E3, dihydrolipoyl dehydrogenase) with the core component (E2, dihydrolipoyl acetyltransferase) of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus have been analyzed using a biosensor based on surface plasmon resonance detection . A recombinant di-domain (lipoyl domain plus peripheral subunit-binding domain) from E2 was attached to the biosensor chip by means of the pendant lipoyl group . The dissociation constant (Kd) for the complex between the peripheral subunit-binding domain and E3 (5.8 x 10(-10) M) was found to be almost twice that for the complex with E1 (3.24 x 10(-10) M) . This was due to differences in the rate constants for dissociation (kdiss); these were 1.06 x 10(-3) and 1.87 x 10(-3) s-1 for the complexes with E1 and E3, respectively, whereas the rate constants for association (kass) were identical (3.26 x 10(6) M-1 s-1) . Separate studies using non-denaturing polyacrylamide gel electrophoresis confirmed the difference in affinity and demonstrated that E1 can rapidly displace E3 from an E3-di-domain complex and vice versa . The peripheral subunit-binding domain showed no detectable interaction with the E1 alpha subunit of E1 (alpha 2 beta 2) but exhibited a strong affinity for E1 beta (Kd = 8.5 x 10(-9) M), confirming that the E1 beta subunit is responsible for binding E1 to E2 . These measurements introduce new features of potential importance into the assembly and mechanism of the multienzyme complex.

Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15012 - 7
A synthetic cryIC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobacco; Strizhov N et al.; Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis delta-endotoxins used thus far as insecticides in transgenic plants . Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method . This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required . Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable pfu DNA ligase using repeated cycles of melting, annealing, and ligation . The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA . Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua) . To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.

Chem Phys Lipids, 1996 Dec 20, 84(2), 87 - 92
Kinetics of phosphatidylinositol-specific phospholipase C with vesicles of a thiophosphate analogue of phosphatidylinositol; Hendrickson HS et al.; 1,2-Dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI) was synthesized as a substrate for the continuous spectrophotometric assay of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus . Release of thio-diglyceride is followed by a coupled reaction with 4,4'-dithiopyridine to produce a chromophore, 4-thiopyridine, measured by its absorption at 324 nm . Sonicated vesicles of D-thio-DMPI gave sigmoidal Michaelis-Menten kinetics with PI-PLC as a function of bulk concentration of substrate (Hill plot: Vmax = 132 mumol min-1 mg-1, apparent Km = 0.115 mM, h = 1.8) . Addition of dimyristoyl phosphatidylcholine (DMPC) or dimyristoyl phosphatidylmethanol to vesicles of D-thio-DMPI resulted in an initial increase in rate followed by a decrease at higher concentrations of non-substrate lipid . Binding of PI-PLC to vesicles of DMPC with 10 mol% of N-dansyl phosphatidylethanolamine was demonstrated by fluorescence resonance energy transfer from tryptophan in the enzyme to the dansyl lipid probe.

J Mol Biol, 1996 Dec 20, 264(5), 1058 - 71
Ribosomal protein L9: a structure determination by the combined use of X-ray crystallography and NMR spectroscopy; Hoffman DW et al.; The structure of protein L9 from the Bacillus stearothernophilus ribosome has been determined at 2.5 A resolution by refinement against single crystal X-ray diffraction data with additional constraints provided by NMR data . This highly elongated protein consists of two domains separated by a nine-turn connecting helix . Conserved aromatic and positively charged amino acid residues on the surface of each domain are likely to be directly involved in binding 23 S ribosomal RNA . The shape of the protein, with its two widely spaced RNA-binding sites, suggests that it may serve as a "molecular strut", most likely playing a role in ribosome assembly and/or maintaining the catalytically active conformation of the ribosomal RNA . The combined use of X-ray and NMR data in the refinement procedure was essential in defining the N-terminal domain of the protein, which was relatively poorly determined by the X-ray data alone . In addition to resolving the ambiguities in defining the hydrophobic core and side-chain conformations with the N-terminal domain, this combined NMR-X-ray analysis provides the first detailed and accurate view of the N-terminal RNA-binding site . NMR data also showed that the N-terminal domain is stable in solution, indicated by amide protons that are protected from solvent exchange . The lack of definition of the N-terminal domain in the X-ray structure is therefore likely due to packing disorder within the crystal rather than structural instability . This combined NMR-X-ray analysis provides a useful model as to how X-ray and NMR data can be practically and logically combined in the determination of the structure of a single protein molecule.

J Biol Chem, 1996 Dec 20, 271(51), 32777 - 84
The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251; Penninga D et al.; The E-domain of cyclodextrin glycosyltransferase (CGTase) (EC 2.4.1.19) from Bacillus circulans strain 251 is a putative raw starch binding domain . Analysis of the maltose-dependent CGTase crystal structure revealed that each enzyme molecule contained three maltose molecules, situated at contact points between protein molecules . Two of these maltoses were bound to specific sites in the E-domain, the third maltose was bound at the C-domain . To delineate the roles in raw starch binding and cyclization reaction kinetics of the two maltose binding sites in the E-domain, we replaced Trp-616 and Trp-662 of maltose binding site 1 and Tyr-633 of maltose binding site 2 by alanines using site-directed mutagenesis . Purified mutant CGTases were characterized with respect to raw starch binding and cyclization reaction kinetics on both soluble and raw starch . The results show that maltose binding site 1 is most important for raw starch binding, whereas maltose binding site 2 is involved in guiding linear starch chains into the active site . beta-Cyclodextrin causes product inhibition by interfering with catalysis in the active site and the function of maltose binding site 2 in the E-domain . CGTase mutants in the E-domain maltose binding site 1 could no longer be crystallized as maltose-dependent monomers . Instead, the W616A mutant CGTase protein was successfully crystallized as a carbohydrate-independent dimer; its structure has been refined to 2.2 A resolution . The three-dimensional structure shows that, within the error limits, neither the absence of carbohydrates nor the W616A mutation caused significant further conformational changes . The modified starch binding and cyclization kinetic properties observed with the mutant CGTase proteins thus can be directly related to the amino acid replacements.

EMBO J, 1996 Dec 16, 15(24), 6798 - 809
Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution; Das AK et al.; Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes . The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices . Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction . Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase . The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.

FEBS Lett, 1996 Dec 16, 399(3), 203 - 6
Complete synthesis of 3'-sialyl-N-acetyllactosamine by regioselective transglycosylation; Vetere A et al.; Transglycolytic synthesis of 3'-sialyl-N-acetyllactosamine by sequential use of beta-galactosidase from Bacillus circulans and trans-sialidase from Trypanosoma cruzi was described . These reactions depicted the first complete synthesis of a biologically important oligosaccharide with high regioselectivity avoiding use of glycosyltransferases and NDP sugars.

FEMS Microbiol Lett, 1996 Dec 15, 145(3), 333 - 9
Isolation of Cry1Ab protein mutants of Bacillus thuringiensis by a highly efficient PCR site-directed mutagenesis system; Meza R et al.; A site-directed mutagenesis method was designed and used to create Cry1Ab mutant proteins in two of the five highly conserved blocks present in the Cry protein family . Region 1 comprises the central alpha-helix 5 of domain I and has been implicated in the pore formation activity of the toxin . Substitution of arginine by serine at position 173 (R173S) affects neither structural integrity nor toxicity . Region 2 comprises the major part of the domain I/domain II interface, characterized by the presence of numerous hydrogen bonds and electrostatic interactions . Mutations in the salt bridge formed by aspartic acid 242 and arginine 265 (D242N, D242C, R265C, and D242C/R265C) resulted in structurally unstable mutant proteins as is shown by their increased protease sensitivity and lack of biological activity.

Carbohydr Res, 1996 Dec 13, 295, 91 - 101
Structure of the cyclic glucan produced from amylopectin by Bacillus stearothermophilus branching enzyme; Takata H et al.; The thermostable branching enzyme (BE, EC 2.4.1.18) from Bacillus stearothermophilus TRBE14 produces large cyclic glucans from waxy rice amylopectin similar to those obtained from amylose as described elsewhere {H . Takata, T . Takaha, S . Okada . M . Takagi, and T . Imanaka, J . Bacteriol., 178 (1996) 1600-1606} . The structure of the product (P-1) from the late-stage reaction was analyzed in detail . The weight-average degree of polymerization (dpw) of P-1 was 900 . Its chain-length distribution was not significantly changed compared with that of amylopectin, although the amount of long chains (dp > 38) was slightly decreased . The cyclic component of P-1, which was isolated by the extensive action of glucoamylase, had dpw of 49 . Three point five alpha-1,6 linkages were directly involved in the formation of the ring structure with several non-cyclic side chains linked to the ring . Based on these results, the action and new roles of BE are discussed.

Gene, 1996 Dec 12, 183(1-2), 97 - 101
Phenobarbital-dependent protein binding to Barbie box-like sequences in the coding region of cytochrome P450BM-3 gene from Bacillus megaterium; Gaidamakova EK et al.; Phenobarbital-dependent protein binding was shown to occur to DNA fragments from the coding region of the cytochrome P450BM-3 gene from Bacillus megaterium . Incubation of the DNA fragments from the coding region of the gene with total cell extract from Bacillus megaterium revealed two DNA regions with protein-binding capacity: +237/+318 and +319/+425 considering 'O' as the start of cytochrome P450BM-3 translation . DNaseI footprint analysis of the fragment +319/+425 with the total cell extract showed that some protein(s) protected DNA stretches from the position +373 up to the position +389 on the transcribed strand and from the position +378 up to the position +398 on the non-transcribed strand . DNaseI footprint analysis of the fragment +237/+318 revealed the protection in the region +262/+277 on the non-transcribed strand . Three regions protected by cell extract protein(s) from DNaseI hydrolysis (+262/+277, +373/+389 and +378/+398) appeared to be strongly homologous to the Barbie box sequence . Barbie-box-like sequences were found in the majority of regulatory regions of phenobarbital-inducible genes whose regulatory sequences had been reported (Fulco et al., 1994) . Our results suggest that a functional role of Barbie box sequence takes place not only in regulatory but also in the coding region of the gene . In line with that hypothesis we analyzed all cytochrome P450 genes in respect to the presence of Barbie box-like sequences in their coding parts . At least one cytochrome P450 gene (CYP6A1, phenobarbital-inducible gene from Musca domestica) was shown to contain Barbie box sequence in the coding part of the gene.

Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14338 - 43
Protein engineering of Bacillus thuringiensis delta-endotoxin: mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae; Rajamohan F et al.; Substitutions or deletions of domain II loop residues of Bacillus thuringiensis delta-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar) . Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) approximately 4-fold . Deletion of N372 (D3), however, substantially reduced toxicity (> 21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding . Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin . The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations . Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type . However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency . Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s) . The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets . These results offer an excellent model system for engineering delta-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.

Biochemistry, 1996 Dec 10, 35(49), 15941 - 8
A low-barrier hydrogen bond in subtilisin: 1H and 15N NMR studies with peptidyl trifluoromethyl ketones; Halkides CJ et al.; The N delta 1 proton of His 64 forms a hydrogen bond with Asp 32, as part of the catalytic triad in serine proteases of the subtilisin family . His 64 in subtilisin has been studied by 1H and 15N NMR spectroscopy in the presence and absence of peptidyl trifluoromethyl ketones (TFMKs) that are transition state analog inhibitors . For subtilisin Carlsberg, the downfield resonance of the imidazolium N delta 1 proton is approximately 18.3 ppm and the D/H fractionation factor is 0.55 +/- 0.04 at pH 5.5 (11 degrees C), and 0.63 +/- 0.04 (5 degrees C) and 0.68 +/- 0.04 at pH 6 (11 degrees C) . In the complex between subtilisin Carlsberg and Z-L-leucyl-L-leucyl-L-phenylalanyltrifluoromethyl ketone (Z-LLF-CF3) at pH values between 6.5 and 10.6, His 64 remains positively charged, and the D/H fractionation factor of its N delta 1 proton is 0.85 +/- 0.05 . In the complex between a subtilisin variant from Bacillus lentus and Z-LLF-CF3, the proton resonance at 18.8 ppm is correlated with a 15N resonance at 197.6 ppm downfield from liquid NH3 with a 1JNH of 81 Hz . The chemical shifts of subtilisin complexes with peptidyl TFMKs are among the most downfield shifts reported for any protein . At pH 9.5, His 64 is neutral and the D/H fractionation factor increases to 1.2 with a chemical shift of 15.0 . His 64 is positively charged in the free enzyme at low pH, the inhibitor hemiketal complex at neutral pH, and the transition state for amide bond hydrolysis . These data thus provide indirect evidence for the presence of a low-barrier hydrogen bond in the catalytic mechanism of subtilisin proteases.

Biochemistry, 1996 Dec 10, 35(49), 15740 - 52
Domain behavior during the folding of a thermostable phosphoglycerate kinase; Parker MJ et al.; Bacillus stearothermophilus phosphoglycerate kinase (bsPGK) is a monomeric enzyme of 394 residues comprising two globular domains (N and C), covalently linked by an interdomain alpha-helix (residues 170-185) . The molecule folds to the native state in three stages . In the first, each domain rapidly and independently collapses to form an intermediate in which the N-domain is stabilized by 5.1 kcal mol-1 and the C-domain by 3.3 kcal mol-1 over their respective unfolded conformations . The N-domain then converts to a folded state at a rate of 1.2 s-1 (delta GI-F = 3.8 kcal mol-1), followed by the C-domain at 0.032 s-1 (delta GI-F = 12.1 kcal mol-1) . It is this last step that limits the rate of acquisition of enzyme activity . In the dynamics of unfolding in water, the N-domain converts to the intermediate state at a rate of 8 x 10(-4) s-1, some 10(7) times faster than the C-domain . Consequently, the most populated intermediate in the folding reaction has a native-like N-domain, while that in the unfolding direction has a native-like C-domain . In a conventional sense, therefore, the folding/unfolding kinetics of bsPGK can be described as random order . Consistent with these observations, cutting the molecule in the interdomain helix produces two, independently stable units comprising residues 1-175 and 180-394 . A detailed comparison of their folding behavior with that of the whole molecule reveals that true interdomain contacts are relatively weak, contributing approximately 1.4 kcal mol-1 to the stability of the active enzyme . The only interactions which contribute to the stability of rapidly formed intermediates or to transition states along the productive folding pathways are those within domain cores . Contacts formed either between domains or with the interdomain helix are made only in the folded ground state, but do not constitute a separate step in the folding mechanism . Intriguingly, the most pronounced effect of interdomain contacts on the kinetics of folding is inhibitory; the presence of the C-domain appearing to reduce the effective rate of acquisition of native structure within the N-domain.

J Biol Chem, 1996 Dec 6, 271(49), 31317 - 21
Membrane topology of the C-terminal half of the neuronal, glial, and bacterial glutamate transporter family; Slotboom DJ et al.; Secondary glutamate transporters in neuronal and glial cells in the mammalian central nervous system remove the excitatory neurotransmitter glutamate from the synaptic cleft and prevent the extracellular glutamate concentration to rise above neurotoxic levels . Secondary structure prediction algorithms predict 6 transmembrane helices in the first half of the transporters but fail in the C-terminal half where no clear helix-loop-helix motif is resolved in the hydropathy profile of the primary sequences . A number of previous studies have emphasized the importance of the C-terminal half of the molecules for the function . Here we determine the membrane topology of the C-terminal half of the glutamate transporters by applying the phoA gene fusion technique to the homologous bacterial glutamate transporter of Bacillus stearothermophilus . High sequence conservation and very similar hydropathy profiles in the C-terminal half warrant a similar folding as in the glutamate transporters of the mammalian central nervous system . The C-terminal half contains four putative transmembrane helices . The strong hydrophobic moment and substitution moment of the most C-terminal helix X that point to opposite faces of the helix suggest that the helix faces the lipid environment with its least conserved, hydrophobic face and the interior of the protein with its well conserved, hydrophilic face . Residues that were shown before to be critical for function cluster in helix X and VII.

J Biol Chem, 1996 Dec 6, 271(49), 31145 - 53
The 2'-5' RNA ligase of Escherichia coli . Purification, cloning, and genomic disruption; Arn EA et al.; An RNA ligase previously detected in extracts of Escherichia coli is capable of joining Saccharomyces cerevisiae tRNA splicing intermediates in the absence of ATP to form a 2'-5' phosphodiester linkage (Greer, C., Javor, B., and Abelson, J . (1983) Cell 33, 899-906) . This enzyme specifically ligates tRNA half-molecules containing nucleoside base modifications and shows a preference among different tRNA species . In order to investigate the function of this enzyme in RNA metabolism, the ligase was purified to homogeneity from E . coli lysate utilizing chromatographic techniques and separation of proteins by SDS-polyacrylamide gel electrophoresis . A single polypeptide of approximately 20 kilodaltons exhibited RNA ligase activity . The amino terminus of this protein was sequenced, and the open reading frame (ORF) encoding it was identified by a data base search . This ORF, which encodes a novel protein with a predicted molecular mass of 19.9 kDa, was amplified from E . coli genomic DNA and cloned . ORFs coding for highly similar proteins were detected in Methanococcus jannaschii and Bacillus stearothermophilus . The chromosomal gene encoding RNA ligase in E . coli was disrupted, abolishing ligase activity in cell lysates . Cells lacking ligase activity grew normally under laboratory conditions . However, moderate overexpression of the ligase protein led to slower growth rates and a temperature-sensitive phenotype in both wild-type and RNA ligase knockout strains . The RNA ligase reaction was studied in vitro using purified enzyme and was found to be reversible, indicating that this enzyme may perform cleavage or ligation in vivo.

Biochem Biophys Res Commun, 1996 Dec 4, 229(1), 139 - 46
Involvement of two amino acid residues in the loop region of Bacillus thuringiensis Cry1Ab toxin in toxicity and binding to Lymantria dispar; Lee MK et al.; Two amino acids, Gly and Ser, at positions 282 and 283 in the loop region of domain II of Cry1Ab2 toxin are substituted with Ala and Leu in the Cry1Ab9-033 toxin . Cry1Ab2 exhibited about a 10-fold increase in toxicity and a 9-fold increase in binding affinity to Lymantria dispar compared to Cry1Ab9-033 . However, these toxins showed similar toxicity and binding affinity to Manduca sexta and Spodoptera exigua . Heterologous competition assays and brush border membrane vesicle (BBMV) ligand blotting experiments demonstrated that Cry1Ab2 and Cry1Ab9-033 toxins recognized the same 210-kDa L dispar BBMV protein . No measurable differences in dissociation binding assays were observed between these two toxins . Digestion of these toxins with L dispar gut enzymes and BBMV proteases indicated no differences in stability . Ala and Leu residues in Cry1Ab9-033 were substituted with Gly and Ser by site-directed mutagenesis to produce mutant Cry1Ab alpha 8 . This toxin exhibited full recovery of toxicity and binding affinity for L dispar . These data suggested that the residues Gly and Ser in the loop region might be directly involved in receptor binding and toxicity in L dispar.

J S Afr Vet Assoc, 1996 Dec, 67(4), 182 - 7
Bartonella (Rochalimaea) henselae in southern Africa--evidence for infections in domestic cats and implications for veterinarians; Kelly PJ et al.; Substantial evidence has recently accumulated showing domestic cats to be the principal reservoirs of Bartonella henselae, the aetiological agent of human diseases including cat-scratch disease, bacillary angiomatosis, bacillary peliosis and a febrile bacteraemia syndrome . To determine the prevalence of antibodies reactive with Bartonella henselae in cats from southern Africa, indirect fluorescent antibody assays were carried out on feline sera from South Africa and Zimbabwe . Overall, 23% (39/171) of cats had antibody titres > or = 1/64, with cats from Zimbabwe (24%; 28/119) having higher seroprevalences than those from South Africa (21%; 11/52) although this difference was not statistically significant . The implications of these findings for veterinarians in southern Africa are discussed.

Indian J Exp Biol, 1996 Dec, 34(12), 1279 - 82
Thermostable alpha-amylase production using Bacillus licheniformis NRRL B14368; Bose K et al.; Maximum amount of extracellular alpha-amylase of B . licheniformis NRRL B14368 was obtained at the stationary phase . Highest yield of alpha-amylase was achieved with high level of crude protein and low carbohydrate level . There was a catabolite repression in the organism . Protease was produced concurrently with alpha-amylase . It was also observed that soyabean acts as an inhibitor of the protease . Optimum pH and temperature of alpha-amylase were 5-7 and 76 degrees C respectively . It was also observed that alpha-amylase production was a non-growth associated product . Maltose was an excellent inducer for alpha-amylase production . Ca2+ (0.01 M) increased the thermostability of the enzyme . Alpha-amylase purification studies were carried out by using isopropanol, acetone, ammonium sulphate solution, ion exchange chromatography . Acetone was found most suitable for the separation of alpha-amylase . Protein recovery and relative enzyme activity (as compared to that of the maximum activity of the crude enzyme) were 30.77% and 3.03 respectively.

Indian J Exp Biol, 1996 Dec, 34(12), 1241 - 4
Effect of insecticidal crystal proteins of Bacillus thuringiensis on human erythrocytes in vitro; Rani SS et al.; Effect of intact and alkali solubilized insecticidal crystal protein (ICP) preparations from a mutant strain of B . thuringiensis var . israelensis (VCRC MB24) and the wild type strain (VCRC B17) in vitro on human erythrocytes with respect to lipid peroxidation, osmofragility and membrane bound enzymes was determined . The alkali solubilized ICPs of both B . thuringiensis strains caused increased lipid peroxidation, decreased resistance to hypotonic lysis and reduction in the activity of membrane bound enzymes . On the contrary, the intact ICPs did not produce any such adverse effect on RBCs under the same experimental conditions . It is suggested that the ICPs are safe when they are intact when compared with solubilized ones.

Onderstepoort J Vet Res, 1996 Dec, 63(4), 289 - 304
Control of pest blackflies (Diptera:Simuliidae) along the Orange River, South Africa: 1990-1995; Palmer RW et al.; The efficacy of Bacillus thuringiensis var . israelensis (B.t.i.) and temephos in controlling the pest blackfly Simulium chutteri Lewis along the middle Orange River between 1990 and 1995, was assessed . Larvicides were applied by helicopter to rapids and riffles between Hopetown and Onseepkans, a river distance of 807 km . Larvicidal efficacy was based on the change in larval abundance at selected sites before and after each treatment . The success of the control programme was assessed independently by local farmers, who ranked adult blackfly annoyance on a 4-point scale . Before treatment, blackfly annoyance showed consistent peaks in spring, and sometimes in autumn, and levels were unacceptably high for between 17 and 36 weeks of the year . After treatment started, blackfly annoyance levels were reduced significantly . The number of annual treatments necessary to reduce blackfly annoyance to acceptable levels was highly variable (3-13), and depended on river conditions, as well as the efficacy and timing of each treatment . During low-flow conditions (< 50 m3/s), applications became increasingly difficult in braided sections of the river, and dosage calculations were inaccurate because of local abstraction and return flows . Both larvicides worked well in winter (water temperature 11-13 degrees C) . Control of the spring outbreak can be planned well in advance, with the first treatment starting in mid July . A flexible protocol is required to control outbreaks at other times of the year . We recommended the use of B.t.i . for most applications, with increased dosages during algal blooms (> 1500 cells/ml) . The use of temphos in the Orange River should be considered only during algal blooms or when flows exceed 300 m3/s . We conclude that helicopter application of larvicides is an effective method or controlling blackflies, along the middle Orange River.

Bioorg Khim, 1996 Dec, 22(12), 900 - 6
{Thermodynamic analysis of domain organization of Bacillus thuringiensis toxins}; Loseva OI et al.; The structure of delta-endotoxins CryIA(c) and CryIIIA from Bacillus thuringiensis was studied by differential scanning microcalorimerty . The analysis of molecular melting showed that the N- and C-terminal halves of the CryIA(c) protoxin from B . thuringiensis subspecies kurstaki HD-73 are thermodynamically independent subunits, with the C-terminal fragment being denatured at a much lower temperature than the N-terminal fragment . The tertiary structure of the N-terminal fragment undergoes no changes during the protoxin-toxin transition . The melting of the native structure of CryIA(c) at pH 9.7-11.0 suggests that it consists of two domains . In CryIIIA from B . thuringiensis subspecies tenebrionis, the transition from the native to denatured state under alkaline conditions (pH 9.7-11.0) proceeds by the "two-state" principle; i.e., the protein melts as one cooperative domain . The melting of the CryIIIA toxin at pH 2.2-3.5 is described by two transitions overlapping by temperatures, indicating the presence of two domains.

J Am Mosq Control Assoc, 1996 Dec, 12(4), 721 - 4
Acute toxicity of selected pesticides to the estuarine shrimp Leander tenuicornis (Decapoda:Palaemonidae); Brown MD et al.; The shrimp Leander tenuicornis is abundant in southeastern Queensland intertidal marsh pools and was chosen as an indicator species for toxicological studies with pesticides . Acute toxicity to this crustacean of temephos and 3 pesticide compounds under evaluation for registration in Australia (Bacillus thuringiensis var . israelensis, s-methoprene, and pyriproxyfen) was tested in 96-h laboratory trials . Temephos was the most toxic compound, with a median lethal concentration (LC50) of 0.01 ppm (0.33 times the estimated field concentration {EFC} for a 15-cm-deep pool) . s-Methoprene was the least toxic compound, with an LC50 of 14.32 ppm (1,790 times the EFC) . Bacillus thuringiensis var . israelensis and pyriproxyfen produced LC50 values of 60.9 x 10(6) ITU (176 times the EFC) and 0.098 ppm (12.25 times the EFC), respectively.

J Am Mosq Control Assoc, 1996 Dec, 12(4), 651 - 5
Ultralow volume application of Bacillus thuringiensis ssp . israelensis for the control of mosquitoes; Lee HL et al.; Evaluation of the effectiveness of Bacillus thuringiensis ssp . israelensis (B.t.i.) against mosquito larvae dispersed by ultralow volume (ULV) spraying was conducted in simulated field trials . Effectiveness was measured using 3 different indicators: larval mortality, colony-forming unit enumeration, and droplet analysis . B.t.i . was dispersed with a ULV generator using 2 different flow rates: 0.3 and 0.5 liter/min on 2 different days . Based on the results of this study, it can be concluded that an output of 0.3 liter/min is effective for controlling Aedes aegypti . although a dosage of 0.5 liter/min can be used when high residual activity is desired . For Culex quinquefasciatus control, both dosages were effective but with low residual activity . For Anopheles maculatus control, only a discharge rate of 0.5 liter/min was effective with low residual activity . B.t.i . application at both dosages penetrated tires well, indicating that B.t.i . ULV application is an effective method for controlling container-inhabiting mosquitoes . Good coverage of target area and penetration were attributed to satisfactory droplet profiles.

J Am Mosq Control Assoc, 1996 Dec, 12(4), 627 - 31
Raising activity of Bacillus thuringiensis var . israelensis against Anopheles stephensi larvae by encapsulation in Tetrahymena pyriformis (Hymenostomatida:Tetrahymenidae); Manasherob R et al.; Toxicity of Bacillus thuringiensis var israelensis (B.t.i.) against surface-feeding mosquito larvae of Anopheles stephensi was enhanced by encapsulation in the protozoan Tetrahymena pyriformis . In the laboratory, larvae died about 8 times faster when exposed to protozoan cells filled with B.t.i . than when exposed to the same concentrations of B.t.i . alone . Best larvicidal activities were achieved with ratios of 1:200-1:500 T . pyriformis cells to B.t.i . spores . The concentration of B.t.i . needed to kill 50% of exposed populations was 4-fold lower with T . pyriformis than with B.t.i . alone in 100 ml-test cups . Toxicity enhancement is very likely a consequence of concentrating B.t.i . insecticidal crystal proteins in T . pyriformis cells and floating them to the water surface in the larval feeding zone . Reduction in the exposure time of B.t.i . to unfavorable field conditions, as a result of the decrease in larval mortality time, might improve the persistence of this biological control agent in nature.

Tuber Lung Dis, 1996 Dec, 77(6), 486 - 90
Why sequence the genome of Mycobacterium tuberculosis?
Cole ST.
Radical measures are required to prevent the grim predictions of the World Health Organisation for the deterioration of the global tuberculosis epidemic in the next century from becoming reality . Study of the nerve centre of the tubercle bacillus, its genome, by means of systematic deoxyribonucleic acid sequence analysis will provide a wealth of information about Mycobacterium tuberculosis that will undoubtedly fuel the next generation of research . In the coming year, this highly cost-effective approach will deliver an unprecedented amount of knowledge to catalyse the development of new, more efficient diagnostic tools and therapeutic interventions to detect, control and, ultimately, eliminate tuberculosis.

Vaccine, 1996 Dec, 14(17-18), 1707 - 11
Expression and secretion of the S2 subunit of pertussis toxin in Bacillus brevis; Kozuka S et al.; We have been trying to develop a mass production system for each of the subunits (S2, S3, S4, S5) of the pertussis toxin B oligomer (PTB) by using a Bacillus brevis-pNU212 system . In consequence a moderately efficient expression-secretion system for S2 was constructed by fusing the mature S2 gene from Bordetella pertussis Tohama with the signal-peptide coding region of pNU212 and by introducing the plasmid pNU212-S2 into B . brevis HPD31 by electroporation . The clone producing S2 secreted about 70 mg of recombinant S2 (rS2) per liter of PY-erythromycin medium after 5 days incubation at 37 degrees C . The rS2 purified by an ammonium sulfate fractionation at 30-50% saturation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was identical to the native S2 in respect to the molecular weight determined by SDS-PAGE and the amino terminal amino acid sequence . The nucleotide sequence of the S2 gene in B . pertussis Tohama inserted into pNU212 was identical with that of the S2 gene in other virulent B . pertussis strains except that an adenine at position 52 of the latter was replaced by a guanine in the former, causing an amino acid substitution (glycine in the former for serine in the latter) at position 18.

Protein Eng, 1996 Dec, 9(12), 1197 - 202
Relationship between thermal stability, degradation rate and expression yield of barnase variants in the periplasm of Escherichia coli; Kwon WS et al.; An advantage of exporting a recombinant protein to the periplasm of Escherichia coli is decreased proteolysis in the periplasm compared with that in the cytoplasm . However, protein degradation in the periplasm also occurs . It has been widely accepted that the thermodynamic stability of a protein is an important factor for protein degradation in the cytoplasm of E.coli . To investigate the effect of the thermodynamic stability of an exported protein on the extent of proteolysis in the periplasm, barnase (an extracellular ribonuclease from Bacillus amyloliquefaciens) fused to alkaline phosphatase leader peptide was used as a model protein . A set of singly or doubly mutated barnase variants were constructed for export to the E.coli periplasm . It was found that the half-life of the barnase variants in vivo increased with their thermodynamic stability in vitro . A dominant factor for the final yield of exported barnase was not exportability but the turnover rate of the barnase variant . The yield of a stabilized mutant was up to 50% higher than that of the wild type . This suggests that exporting a protein to the periplasm and using protein engineering to enhance the stability can be combined as a strategy to optimize the production of recombinant proteins.

Protein Eng, 1996 Dec, 9(12), 1181 - 9
Analysis of structural determinants of the stability of thermolysin-like proteases by molecular modelling and site-directed mutagenesis; Veltman OR et al.; The thermolysin-like protease (TLP) produced by Bacillus stearothermophilus CU21 (TLP-ste) differs at 43 positions from the more thermally stable thermolysin (containing 316 residues in total) . Of these differences, 26 were analysed by studying the effect of replacing residues in TLP-ste by the corresponding residues in thermolysin . Several stabilizing mutations were identified but, remarkably, considerable destabilizing mutational effects were also found . A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertional difference between the two enzymes) appeared to make an important contribution to the stability of the enzyme . Mutations with large effects on stability were all localized in the beta-pleated N-terminal domain of TLP-ste, confirming observations that this domain has a lower intrinsic stability than the largely alpha-helical C-terminal domain . Rigidifying mutations such as Gly58-->Ala and Ala69-->Pro were among the most stabilizing ones . Apart from this observation, the analyses did not reveal general rules for stabilizing proteins . Instead, the results highlight the importance of context in evaluating the stability effects of mutations.

Microbiology, 1996 Dec, 142 ( Pt 12), 3425 - 36
Genotypic and phenotypic analysis of zwittermicin A-producing strains of Bacillus cereus; Raffel SJ et al.; Many strains of Bacillus cereus produce zwittermicin A, a novel antibiotic that contributes to the ability of B . cereus to suppress certain plant diseases . The purpose of this study was to identify molecular indicators of zwittermicin A production in B, cereus strains, contribute to an understanding of the ecology and evolution of this group of bacteria, and identify potential agents for control of plant disease . The fatty acid composition of 20 strains known to be zwittermicin A producers and 20 strains known to be non-producers was determined . Cluster analysis of the fatty acid methyl ester (FAME) profiles revealed that zwittermicin A producers grouped together in two clusters, apart from most non-producers . Discriminant analysis of the FAME profiles generated models that correctly predicted the zwittermicin A-production phenotype in 17 of 20 zwittermicin A producers and 17 of 20 non-producers . Sixteen random oligonucleotide primers were tested in PCR, and one primer was identified that generated a fragment of 0.48 kb or 0.49 kb from total DNA from 26 of 28 strains known to produce zwittermicin A, whereas PCR with this primer did not generate bands of that size from 16 of 20 non-producing strains . PCR with primers designed to amplify zmaR, a gene from B . cereus that confers resistance to zwittermicin A, generated DNA fragments of 1.1 kb and 1.0 kb in all 29 zwittermicin A-producing strains tested, amplified a fragment of 0.3 kb in some of the zwittermicin A-producing strains, and amplified no fragments in 20 of 23 non-producing strains in a stock collection of B . cereus strains . The zmaR primers were tested for their ability to identify new zwittermicin A-producing isolates of B . cereus from two soils . All 12 of the isolates that produced the banding pattern characteristic of this primer pair produced zwittermicin A, and none of the 12 isolates that did not have the banding pattern produced detectable zwittermicin A . Seven of the 12 isolates initially identified as zwittermicin A producers with the zmaR primers significantly suppressed damping-off of alfalfa, whereas only one of the non-producers suppressed this disease . The results show that FAME and PCR analyses distinguish B . cereus strains that produce zwittermicin A from other B . cereus strains, that PCR with the primers designed to amplify zmaR is the most reliable method of those tested for identification of zwittermicin A producers, and that this method can be used to identify new strains with disease-suppressive activity.

J Vet Med Sci, 1996 Dec, 58(12), 1219 - 21
Demonstration of rat CAR bacillus using a labelled streptavidin biotin (LSAB) method; Oros J et al.; Immunohistochemical detection of rat CAR bacillus antigen in paraffin-embedded experimentally infected rat lungs, using an immunoperoxidase technique based on the labelled streptavidin biotin (LSAB) method and 3-amino-9-ethylcarbazole (AEC) as substrate is described in this paper . The pattern of immunostaining was confined to the ciliated bronchial epithelium and the specificity of this technique was confirmed . The use of AEC as substrate was evaluated more efficient than diaminobenzidine (DAB) . The usefulness of this immunoperoxidase technique for the detection of CAR bacillus in rats and its advantages compared to the indirect immunofluorescence (IF) are discussed.

Am J Dermatopathol, 1996 Dec, 18(6), 597 - 600
Bacillary angiomatosis in an immunocompetent child; Smith KJ et al.; Bacillary angiomatosis (BA) is a pathological process characterized by prominent vascular proliferation secondary to organisms of the genus Rochalimaea . BA has been most commonly associated with HIV-1+ patients, but has also been reported rarely in other immune-suppressed patients and in a small group of patients with no demonstrated immune suppression . Even in immune-suppressed children, BA is extremely rare . We report a 5-year-old girl with no apparent immune suppression and no risk factors for HIV-1+ disease, who presented with a skin lesion that histopathologically was diagnostic of BA.

Md Med J, 1996 Dec, 45(12), 1019 - 22
Pulmonary tuberculosis in a high school student and a broad contact investigation: lessons relearned; Rodriguez EM et al.; A case of acid-fast bacillus smear-positive cavitary tuberculosis (TB) was diagnosed in a high school senior (Student A) who lived in a community with a low prevalence for TB . A broad TB investigation was conducted in July 1994 among persons who attended the high school graduation with Student A . Follow-up investigations three months later focused on close contacts at highest risk . A positive tuberculin skin test (TST) was defined as induration of > or = 5 mm after placement of purified protein derivative . We determined the TST results and the estimated costs incurred by the local health department for the broad screening that was conducted . TST results were available for 122/161 (75%) close contacts, and for 1804 persons with nonclose contact with Student A . Her family members were known to have had prior positive TSTs . Positive TSTs were found among 3/122 (2.5%) close contacts, versus 34/1804 (1.9%) persons with nonclose contact . Only one close contact had conversion of TST from negative to positive, and no other active TB case was identified . We estimate the broad TST screening cost the local health department $36,507 . Broad TST screening was costly and diverted staff from their customary public health service priorities . Local health departments and clinicians should follow the recommendations of the American Thoracic Society and the Centers for Disease Control and Prevention regarding TB contact investigations.

Br J Dermatol, 1996 Dec, 135(6), 982 - 7
Bacillary angiomatosis in an HIV seronegative patient on systemic steroid therapy; Schwartz RA et al.; Bacillary angiomatosis is an unusual systemic vascular proliferation seen predominantly in patients with the acquired immunodeficiency syndrome . These vascular lesions are due to infection with a Bartonella species, most commonly B . henselae, but sometimes B . quintana . It is treatable and often curable, but without therapy may be life-threatening . Clinically, the disorder often resembles several different vascular disorders, particularly pyogenic granuloma and Kaposi's sarcoma . We now report a clinically typical patient with bacillary angiomatosis who was HIV seronegative, but who had idiopathic thrombocytopenic purpura, was status-post splenectomy and to whom long-term systemic prednisone had been administered.

Am Ind Hyg Assoc J, 1996 Dec, 57(12), 1154 - 62
Exposure assessment for a field investigation of the acute respiratory effects of metalworking fluids . I . Summary of findings; Woskie SR et al.; The exposure assessment summarized here is part of an epidemiologic study of the acute respiratory health effects of metalworking fluid (MF) exposures . Exposures were measured as the inhalable concentrations of the MF aerosol, a variety of metals and elements, and endotoxin as well as the level of culturable bacteria in the aerosol size fraction less than 8 microns . Bulk samples of soluble MFs were tested for pH, mineral and tramp oil fraction, endotoxin, culturable bacteria, and lipopolysaccharide levels . The MF exposed workers had higher geometric mean inhalable aerosol exposures (0.181 mg/m3) than the MF unexposed workers (0.046 mg/m3) . The MF exposed workers had higher geometric mean (GM) airborne culturable microbial counts (102 colony-forming units (CFU)/m3 for bacteria < 8 microns) than the unexposed workers (GM = 14 CFU/m3) . Among the unexposed, Bacillus was the predominant airborne species, while among the exposed workers, Pseudomonas predominated . Exposed workers also had higher geometric mean airborne endotoxin levels (GM = 7.1 endotoxin units (EU)/m3) than the unexposed workers (GM = 1.9 EU/m3) . Elemental concentrations of iron, chlorine, and sulfur were substantially higher among the exposed workers compared to the unexposed workers . For soluble metalworking fluids, the levels of bulk constituents were examined by three categories of time since the machine sump was refilled with fresh MF (< 4 days, 4-21 days, > 21 days) . Univariate analyses of percent oil, pH, culturable bacteria, tramp oil percent, endotoxin, or fatty acid levels all showed no statistically significant changes in level over time.

Protein Sci, 1996 Dec, 5(12), 2459 - 67
A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I; Matsuo Y et al.; The three-dimensional structure of bacterial sphingomyelinase (SMase) was predicted using a protein fold recognition method; the search of a library of known structures showed that the SMase sequence is highly compatible with the mammalian DNase I structure, which suggested that SMase adopts a structure similar to that of DNase I . The amino acid sequence alignment based on the prediction revealed that, despite the lack of overall sequence similarity (less than 10% identity), those residues of DNase I that are involved in the hydrolysis of the phosphodiester bond, including two histidine residues (His 134 and His 252) of the active center, are conserved in SMase . In addition, a conserved pentapeptide sequence motif was found, which includes two catalytically critical residues, Asp 251 and His 252 . A sequence database search showed that the motif is highly specific to mammalian DNase I and bacterial SMase . The functional roles of SMase residues identified by the sequence comparison were consistent with the results from mutant studies . Two Bacillus cereus SMase mutants (H134A and H252A) were constructed by site-directed mutagenesis . They completely abolished their catalytic activity . A model for the SMase-sphingomyelin complex structure was built to investigate how the SMase specifically recognizes its substrate . The model suggested that a set of residues conserved among bacterial SMases, including Trp 28 and Phe 55, might be important in the substrate recognition . The predicted structural similarity and the conservation of the functionally important residues strongly suggest a distant evolutionary relationship between bacterial SMase and mammalian DNase I . These two phosphodiesterases must have acquired the specificity for different substrates in the course of evolution.

J Leukoc Biol, 1996 Dec, 60(6), 692 - 703
Rabbit vascular endothelial adhesion molecules: ELAM-1 is most elevated in acute inflammation, whereas VCAM-1 and ICAM-1 predominate in chronic inflammation; Abe Y et al.; Activation of the microvasculature is a major component of the inflammatory response . During inflammation the vascular endothelium not only becomes more permeable to plasma proteins but also develops adhesion molecules that initiate the local immigration of leukocytes . We describe herein the in vivo changes in the three major vascular adhesion molecules during the development and healing of two types of rabbit dermal inflammatory lesions: (1) acute lesions produced in rabbits by the topical application of 1% sulfur mustard (SM, the military irritant/toxicant); and (2) chronic (immune-mediated) lesions produced in rabbits by intradermal injections of Mycobacterium bovis (BCG), the vaccine strain of tubercle bacillus . In each case, frozen tissue sections were made from lesions of various ages and stained immunohistochemically for von Willebrand (vW) factor to measure the total functional microvasculature . The sections were also stained immunohistochemically for the vascular endothelial adhesion molecules ICAM-1, ELAM-1 (E-selectin), and VCAM-1, and for the leukocyte ligands for ICAM-1: LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) . Infiltrating monocytes and lymphocytes expressed the LFA-1 ligand and infiltrating PMN expressed the MAC-1 ligand . The area of stained microvasculature per square millimeter of tissue section was determined with the use of a computerized image analyzer . Edema and cell infiltration spread apart the microvessels, changing the number of microvessels per square millimeter of tissue section . Three methods of assessing such changes are presented . In SM lesions, endothelial ICAM levels were decreased from normal by about 50% at 1 and 2 days (when the lesions reached their peak size) and returned to normal at 3 and 6 days (during the healing process) . ELAM rose in peak SM lesions and remained high during healing . VCAM levels, however, were only elevated in the 6-day (almost healed) lesions . In BCG lesions the levels of endothelial ICAM and VCAM (and to a lesser extent ELAM) were increased at 9 days and remained so as the size of the lesions peaked at 23 days . During the healing phase at 37 days, the elevated ICAM and VCAM levels decreased but the slightly increased ELAM levels persisted . These findings indicate that ELAM plays a major role in acute inflammation and that VCAM and ICAM play major roles in chronic inflammation . VCAM is known to be monocyte and lymphocyte selective.

Mol Phylogenet Evol, 1996 Dec, 6(3), 391 - 407
Evolution of the diatoms (Bacillariophyta) . IV . A reconstruction of their age from small subunit rRNA coding regions and the fossil record; Kooistra WH et al.; Small subunit ribosomal RNA (ssu rRNA) coding regions from 30 diatoms, 3 oomycetes, and 6 pelagophytes were used to construct linearized trees, maximum-likelihood trees, and neighbor-joining trees inferred from both unweighted and weighted distances . Stochastic accumulation of sequence substitutions among the diatoms was assessed with relative rate tests . Pennate diatoms evolved relatively slowly but within the limits set by a stochastic model; centric diatoms exceeded those limits . A rate distribution test was devised to identify those taxa showing an aberrant distribution of base substitutions within the ssu rRNA coding region . First appearance dates of diatom taxa from the fossil record were regressed against their corresponding branch lengths to infer the average and earliest possible age for the origin of the diatoms, the pennate diatoms, and the centric diatom order Thalassiosirales . Our most lenient age estimate (based on the median-evolving diatom taxon in the maximum-likelihood tree or on the average branch length in a linearized tree) suggests that their average age is approximately 164-166 Ma, which is close to their earliest fossil record . Both calculations suggest that it is unlikely that diatoms existed prior to 238-266 Ma . Rate variation among the diatoms' ssu rRNA coding regions and uncertainties associated with the origin of extant taxa in the fossil record contribute significantly to the variation in age estimates obtained . Different evolutionary models and the exclusion of fast or slow evolving taxa did not significantly affect age estimates; however, the inclusion of aberrantly fast evolving taxa did . Our molecular clock calibrations indicate that the rRNA coding regions in the diatoms are evolving at approximately 1% per 18 to 26 Ma, which is the fastest substitution rate reported in any pro- or eukaryotic group of organisms to date.

Urology, 1996 Dec, 48(6), 957 - 61; discussion 961-2
Bacillus Calmette-Guérin plus intravesical interferon alpha-2b in patients with superficial bladder cancer; Stricker P et al.; OBJECTIVES: Bacillus Calmette-Guerin (BCG) and interferon alpha-2b (IFN alpha 2b) have been used individually for the treatment of bladder cancer . We used a low dose of BCG combined with IFN alpha 2b to determine the safety and to assess the efficacy of this combination therapy . METHODS: A study of 12 patients with superficial bladder cancer evaluated the safety and efficacy of a combination of low-dose BCG and IFN alpha 2b, given weekly for 6 weeks . Three patients were assigned to each of four groups in which 60 mg of BCG was combined with 10, 30, 60, or 100 x 10(6) IU of IFN alpha 2b . RESULTS: The combination BCG/IFN alpha 2b therapy was well tolerated, with adverse effects being mild to moderate and resolved at the end of treatment . At 12 months post-treatment there has been no tumor progression . Two patients with previous multifocal transitional cell carcinoma have had solitary recurrences . One patient has had recurrent carcinoma in situ . CONCLUSIONS: This preliminary study found combination BCG/IFN alpha 2b induction therapy to be safe and well tolerated . These early results show a high response rate, but efficacy can only be determined with Phase II and III studies.

J Virol, 1996 Dec, 70(12), 8411 - 21
Efficient transcription from the rice tungro bacilliform virus promoter requires elements downstream of the transcription start site; Chen G et al.; Elements downstream of the transcription start site enhance the activity of the rice tungro bacilliform virus (RTBV) promoter in protoplasts derived from cultured rice cells . This enhancer region was located to the first 90 nucleotides of the RTBV leader sequence . Within this region, at least two components which act together to enhance expression from the RTBV promoter could be identified . One is a position- and orientation-independent DNA element within a CT-rich region, and the other is a position-dependent element . Either element was found to be capable of acting independently on a heterologous promoter . The enhancer activity of the DNA element correlates with specific binding of nuclear proteins . Nuclear proteins also recognize an RNA transcript covering the first 90 nucleotides of the RTBV leader.

Semin Oncol, 1996 Dec, 23(6), 773 - 81
Active specific immunization in the treatment of patients with melanoma; Mastrangelo MJ et al.; The bonafide albeit infrequent examples of tumor regression observed with whole tumor cell vaccines give evidence for the hypothesis that active specific immunization can induce a therapeutically effective immune response in melanoma patients . A dinitrophenyl-conjugated autologous whole tumor cell plus bacille Calmette-Guerin (BCG) vaccine administered in conjunction with low dose cyclophosphamide has produced clinically significant prolongation of disease free survival when used as a postsurgical adjuvant in patients with stage III melanoma . However, tumor cell-based vaccines are cumbersome and consequently of limited applicability . Improvements in our understanding of the antimelanoma immune response and technological advances have allowed investigators to explore better defined immunogens and antigenic targets; these include anti-idiotypic antibodies, gangliosides, and tumor associated/specific proteins and derived peptides . The rationale for, promise of, and progress to date with these materials are reviewed.

Arch Surg, 1996 Dec, 131(12), 1344 - 6
Mycobacterium fortuitum infection of the sternum . Review of the literature and case illustration; Samuels LE et al.; Sternal wound infection with atypical mycobacteria following open heart surgery is a rare occurrence . Previous reports have described infection by Mycobacterium fortuitum, an acid-fast bacillus and member of a larger family of rapidly growing mycobacteria . The source and mode of transmission have not been identified . Surgical debridement and the combination of aminoglycosides and quinolones have been shown to be effective methods of treatment . More recently, clarithromycin has been shown to be the drug of choice against rapidly growing mycobacteria . We describe a 49-year-old woman who underwent infundibular stenosis repair and in whom M fortuitum sternal osteomyelitis developed . Total sternectomy, muscle flap reconstruction, and antibiotic treatment successfully eradicated the infection.

J Bacteriol, 1996 Dec, 178(24), 7254 - 9
Cytometric detection of mycobacterial surface antigens: exposure of mannosyl epitopes and of the arabinan segment of arabinomannans; Ozanne V et al.; The physical arrangement of cell envelope components leads to the exposure of selected structural motifs which in turn may influence host-parasite interactions . To gain insight into the exposed epitopes, the present study describes a flow cytometric method designed to probe defined molecules on dispersed mycobacteria . The hydrophobic fluorophore N-hexadecanoyl aminofluorescein inserted in the mycobacterial cell envelope permitted focusing of fluorescence-activated cell sorter analysis on cells that were further labeled with defined monoclonal antibodies and fluorochrome-coupled streptavidin . The use of antibodies directed against the lipooligosaccharide of Mycobacterium tuberculosis demonstrated the specific detection of the antigen on the cell surface of a Canetti-like strain of M . tuberculosis, and not on those of mycobacterial strains that were devoid of the glycolipid . Thus, the method was applied to investigate the relative amounts of surface-exposed mannosylated compounds and D-arabinan-containing substances of different strains of the tubercle bacillus and a strain of the rapidly growing nonpathogenic species Mycobacterium smegmatis . Both M . tuberculosis and M . smegmatis are endowed with mannosyl and arabinan epitopes on their surfaces, although there are many differences in terms of exposed mannosyl epitopes between the various strains of the tubercle bacillus examined . These differences are correlated with the amounts of terminal mannosyl residues that cap the surface-exposed arabinomannans (A . Ortalo-Magne, A . B . Andersen, and M . Daffe, Microbiology 142:927-935, 1996) but not with the degrees of virulence of the strains . This novel approach could provide new insights into the distribution of defined surface-exposed antigens and thereby into the architecture of the cell envelopes.

Appl Environ Microbiol, 1996 Dec, 62(12), 4367 - 73
Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system; Baum JA et al.; The cry genes of Bacillus thuringiensis encode a diverse group of crystal-forming proteins that exhibit insecticidal activity, particularly against the larvae of lepidopteran, coleopteran, and dipteran insects . The efficacy of B . thuringiensis-based biopesticides may be improved through the genetic manipulation of these genes . A gene transfer system has been developed for the introduction and maintenance of cloned insecticidal cry genes on small plasmids in B . thuringiensis . This vector system combines a B . thuringiensis plasmid replicon and an indigenous site-specific recombination system that allows for the selective removal of ancillary or foreign DNA from the recombinant bacterium after introduction of the Cry-encoding plasmid . The site-specific recombination system is useful for engineering strains with unique combinations of cry genes, resulting in new active ingredients with improved insecticidal properties.

J Bioenerg Biomembr, 1996 Dec, 28(6), 495 - 501
A flash-photolysis study of the reactions of a caa3-type cytochrome oxidase with dioxygen and carbon monoxide; Hirota S et al.; The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase from Bacillus PS3 in the presence of O2 has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase . The PS3 enzyme has a covalently bound cytochrome c subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme . In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 microseconds, 30 microseconds, 100 microseconds, and I ms at 445 nm . The initial, 10-microsecond absorbance change at 445 nm is similar in the two enzymes . The subsequent phases involving heme a and CuA are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochrome c, as indicated by absorbance changes at 550 nm . A reaction scheme consistent with the experimental observations is presented . In addition, internal electron-transfer reactions in the absence of O2 were studied following flash-induced CO dissociation from the mixed-valence enzyme . Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from heme a3 to a in PS3 oxidase compared to the bovine enzyme.

Cytometry, 1996 Dec 1, 25(4), 381 - 7
Double fluorescent flow cytometric assessment of bacterial internalization and binding by epithelial cells; de Boer EC et al.; This study describes a new flow cytometric method for assessment of phagocytosis of specific bacteria (bacillus Calmette-Guerin (BCG) and Escherichia coli) by bladder epithelial cells . The internalization assay consisted of labeling bacteria chemically with fluorescein isothiocyanate (FITC) . Subsequent to incubation of fluoresceinated bacteria with internalizing cells, adherent nonphagocytosed bacteria were marked by two-step labeling using specific antibodies and phycoerythrin (PE)-conjugated antibodies . Double fluorescent FACS analysis differentiated between bacterial phagocytosis and adherence . The validity of the method was shown by inhibition of BCG phagocytosis at 4 degrees C by cytochalasin B, by removal of excess free bacteria, and by anti-BCG antibodies . BCG-phagocytizing and -nonphagocytizing cell lines were discriminated by applying this technique to a series of bladder carcinoma cell lines . There seemed to be a relationship between phagocytic capacity and grade of differentiation in these cell lines, which may have implications for topical BCG immunotherapy in superficial bladder cancer . In conclusion, a new, reliable, rapid, and relatively simple double fluorescent method is described for quantification of specific bacterial internalization by large numbers of (bladder) epithelial cells . This method should be generally applicable to the study of in vitro interaction between bacteria and different types of host cells.

J Biol Chem, 1996 Nov 29, 271(48), 30748 - 54
A role of the amino acid residue located on the fifth position before the first aspartate-rich motif of farnesyl diphosphate synthase on determination of the final product; Ohnuma S et al.; Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP . Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T . (1996) J . Biol . Chem . 271, 10087-10095) . Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them . All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate . The final products and the product distributions are different from each other . Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product . The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl diphosphates more than geranylgeranyl diphosphate . Substitution of tryptophan does not affect the product specificity of FPP synthase . The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid . However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed . These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the omega-terminal of an elongating allylic product . This interaction must prevent further condensation of isopentenyl diphosphate.

Nature, 1996 Nov 28, 384(6607), 379 - 83
Crystal structure of a DExx box DNA helicase; Subramanya HS et al.; There are a wide variety of helicases that unwind helical DNA and RNA substrates . The twelve helicases that have been identified in Escherichia coli play a role in almost all cellular processes involving nucleic acids . We have solved the crystal structure of a monomeric form of a DNA helicase from Bacillus stearothermophilus, alone and in a complex with ADP, at 2.5 and 2.9 A resolution, respectively . The enzyme comprises two domains with a deep cleft running between them . The ATP-binding site, which is situated at the bottom of this cleft, is formed by motifs that are conserved across the superfamily of related helicases . Unexpected structural homology with the DNA recombination protein, RecA, suggests how ATP binding and hydrolysis may drive conformational changes of the enzyme during catalysis, and implies that there is a common mechanism for all helicases.

Mol Gen Genet, 1996 Nov 27, 253(1-2), 11 - 9
Mapping of the entomocidal fragment of Spodoptera-specific Bacillus thuringiensis toxin CryIC; Strizhov N et al.; Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects . A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects . However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide . To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro . In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627 . Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615 . However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S . littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases . Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI proteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins.

J Mol Biol, 1996 Nov 22, 264(1), 97 - 110
Guided evolution of enzymes with new substrate specificities; el Hawrani AS et al.; A gene library was constructed coding for all possible variants of two amino acids (101, 102) in a solvent-exposed surface return loop (alpha E-beta D) of Bacillus stearothermophilus L-lactate dehydrogenase (bsLDH) . All but one of 38 enzyme variants examined were thermally stable and had native-like hydrodynamic properties . In this sample, there was no bias detected in either the DNA or amino acid sequences encoded . We argue that the alpha E-beta D surface loop sequence is unimportant for protein folding or stability and can be fully varied to select enzymes with new substrate specificities . The selection of NAD-dependent dehydrogenases with specificity for: malate, phenyllactate, hydroxyisocaproate and 4-phenyl-2-hydroxy-butanoate from two bsLDH libraries is described . This required a highly discriminatory screen for 2-hydroxy acid dehydrogenase activity to select enzymes which, in the absence of the natural allosteric activator fructose-1,6-bisphosphate (FBP), maintained high temperature stability and catalytic activity without substrate inhibition . In general the amino acid residues at positions 101 and 102 which determined substrate specificity were as expected from hydrophobic and ionic complementarity to the substrate . For example, a bsLDH variant with Asn101Va1102 is as efficient with phenylpyruvate as is the wild-type enzyme (Asn101Gln102) with pyruvate . Using molecular modelling, the valine at position 102 can be fitted into the active site without significant structural distortion caused by the aromatic side-chain of the substrate . Similarly, nine out of ten malate dehydrogenases (MDHs) selected had an arginine residue at position 102 to complement the negatively charged carboxyl group in oxaloacetate . One, Arg101Arg102 (Kcat/Kmoxaloacetate = 1.6 x 10(6) M-1 S-1) is 25% more active than the previous best synthetic MDH . There were surprises: present understanding would not have predicted the oxaloacetate transforming activity of Ser101Leu102 or the phenylpyruvate activity of Pro101Lys102 . The former is about one-third as efficient as the best malate dehydrogenase selected, whilst the latter had about one-seventh of the best phenylpyruvate dehydrogenase activity.

Gene, 1996 Nov 21, 180(1-2), 157 - 63
A novel gene family defined by human dihydropyrimidinase and three related proteins with differential tissue distribution; Hamajima N et al.; We have isolated cDNA clones encoding dihydropyrimidinase (DHPase) from human liver and its three homologues from human fetal brain . The deduced amino acid (aa) sequence of human DHPase showed 90% identity with that of rat DHPase, and the three homologues showed 57-59% aa identity with human DHPase, and 74-77% aa identity with each other . We tentatively termed these homologues human DHPase related protein (DRP)-1, DRP-2 and DRP-3 . Human DRP-2 showed 98% aa identity with chicken CRMP-62 (collapsin response mediator protein of relative molecular mass of 62 kDa) which is involved in neuronal growth cone collapse . Human DRP-3 showed 94-100% aa identity with two partial peptide sequences of rat TOAD-64 (turned on after division, 64 kDa) which is specifically expressed in postmitotic neurons . Human DHPase and DRPs showed a lower degree of aa sequence identity with Bacillus stearothermophilus hydantoinase (39-42%) and Caenorhabditis elegans unc-33 (32-34%) . Thus we describe a novel gene family which displays differential tissue distribution: i.e., human DHPase, in liver and kidney; human DRP-1, in brain; human DRP-2, ubiquitously expressed except for liver; human DRP-3, mainly in heart and skeletal muscle.

J Biotechnol, 1996 Nov 15, 51(3), 259 - 64
Overexpression and single-step purification of a thermostable xylanase from Bacillus stearothermophilus T-6; Lapidot A et al.; Xylanase T-6 is a thermostable alkaline-tolerant enzyme that is produced by Bacillus stearothermophilus T-6 . Xylanase T-6 was found to bleach pulp effectively at pH 9 and 65 degrees C and was used successfully on an industrial-scale mill trial . To facilitate the future characterization of the protein via X-ray analysis and protein engineering, it was necessary to overexpress the enzyme in Escherichia coli . The xylanase gene was cloned into T-7 polymerase expression vectors and its expression was optimized . The enzyme was found to constitute over 70% of the cell protein and it was efficiently purified from the host proteins by a single heating step . Over 2 g soluble and active enzyme per 1 culture were achieved.

Anal Biochem, 1996 Nov 15, 242(2), 221 - 7
Active-site titration of serine proteases using a fluoride ion selective electrode and sulfonyl fluoride inhibitors; Hsia CY et al.; We report a general procedure for the determination of active enzyme concentrations for serine proteases . The method relies on the measurement of fluoride ion released from sulfonyl fluorides upon reaction with the active-site serine using an ion selective electrode . The results have been independently confirmed by amino acid analyses of subtilisins and by spectrofluorometric and spectrophotometric titrations . The minimal enzyme concentration detectable is 1-10 microM protease . The method is insensitive to color and turbidity of the sample and is therefore useful for measuring protease concentration in broth solutions . The active enzyme concentration of subtilisin BPN' from Bacillus amyloliquefaciens determined by titration with phenylmethylsulfonyl fluoride is 25% higher than the concentration determined using the spectrophotometric burst titrant N-trans-cinnamoylimidazole . Analysis of the pre-steady-state burst amplitude and kinetics suggests that the extinction coefficient for the cinnamoyl acyl-enzyme is larger than previously measured and a significant fraction of the enzyme is present as an unproductive ES2 complex . The molar extinction coefficient at 280 nm for subtilisin BPN' is 26.5 mM-1 cm-1 and for subtilisin from Bacillus lentus is 22.5 mM-1 cm-1.

Biochim Biophys Acta, 1996 Nov 14, 1298(1), 58 - 68
Vanadate is a potent competitive inhibitor of phospholipase C from Bacillus cereus; Tan CA et al.; Monomeric vanadate is a potent competitive inhibitor of phospholipase C from Bacillus cereus, much better than other oxyanions (e.g., phosphate or iodate) . The apparent efficiency of inhibition depends on the substrate aggregate structure . The measured inhibition constant with respect to monomeric phosphatidylcholine substrate is 0.21 mM under conditions where the K(m) is 0.12 mM; for micellar substrate the apparent Ki appears much lower and in fact tracks the apparent K(m) which decreases 10-fold . Vanadate inhibition is removed by addition of exogenous diacylglycerol, which by itself is an inhibitor . In contrast to its effect with monomeric or micellar substrate, vanadate does not strongly inhibit the PLC-catalyzed hydrolysis of small unilamellar vesicles of phosphatidylcholine . These results are interpreted in terms of the surface binding of the enzyme . Because of its ability to mimic the transition state of phosphate ester hydrolysis vanadate is also used to investigate the constraints on the occurrence of strained cyclic intermediates in phospholipid hydrolysis by PLC.

Forensic Sci Int, 1996 Nov 11, 83(1), 1 - 13
Is the exploding powder gas of the propellant from blank cartridges sterile?
Rothschild MA, Liesenfeld O.
Shots from blank weapons loaded with blank cartridges, when fired from close range or as a contact shot, almost always cause the skin to burst open and lead to injuries to structures below the surface . Subsequently, wound infections are often observed . In addition to the introduction of skin germs, the possibility exists that contaminated propellants may enter into consideration as a source of infection . Using step-by-step experimental procedures we were able to demonstrate that: 1 . Blank cartridge propellants were almost always contaminated with Bacillus cereus (nitrocellulose powder more so than black powder); 2 . When the shot is fired numerous bacteria survive and are forced out with the gunsmoke from the weapon and thus find their way into the wound . In principle, blank cartridge propellant thus exhibits as much potential for wound infection as the skin germs . Clearly, the species B . cereus is prominent in this context . For open injuries even with 'harmless' blank weapons, an antibiotic prophylaxis should always be administered.

Tidsskr Nor Laegeforen, 1996 Nov 10, 116(27), 3231 - 2
{Arthritis after BCG treatment of bladder cancer . A rare complication}; Rodevand E et al.; Since 1976 intravesical instillation of bacillus Calmette-Guerin (BCG) has been used after surgical treatment of bladder cancer . Local side effects like cystitis are common, but a small share of the patients develop influenza-like symptoms, arthritis and other complications . We describe two patients with arthritis.

Gene, 1996 Nov 7, 179(1), 119 - 26
Expression of chitinase-encoding genes from Aeromonas hydrophila and Pseudomonas maltophilia in Bacillus thuringiensis subsp . israelensis; Wiwat C et al.; Fifty isolates of chitinase (Cts)-producing bacteria were collected from soil samples and tested for their ability to degrade chitin using colloidal chitin agar as the primary plating medium . The results indicated that three isolates could degrade chitin at high pH . Further studies also demonstrated that crude Cts preparations from Bacillus circulans (Bc) No . 4.1 could enhance the toxicity of Bacillus thuringiensis subsp . kurstaki (Bt-k) toward diamondback moth larvae . Thus, it might be useful to increase the toxicity of B . thuringiensis (Bt) toward target insects by introducing a Cts-encoding gene (cts) into Bt . To investigate the expression of cts in Bt, cloned cts from Aeromonas hydrophila (pHYA1) and Pseudomonas maltophilia (pHYB1, pHYB2 and pHYB3) were cloned into the shuttle vector pHY300PLK and transformed into Escherichia coli DH5 alpha using 4-methylumbelliferyl beta-D-N,N'-diacetylchitobioside (4-MUF GlcNAc) as the detecting substrate . The four plasmids were then introduced into B . thuringiensis subsp . israelensis (Bt-i) strain c4Q272 by electroporation . Various transformants harboring cloned cts were selected, and expression and stability of the plasmids in Bt were studied.

Gene, 1996 Nov 7, 179(1), 111 - 7
Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins by site-directed mutagenesis--a minireview; Dean DH et al.; The current model of the mechanism of action of several Bacillus thuringiensis insecticidal crystal proteins (Cry) is reviewed and tested by site-directed mutagenesis experiments . Amino acid (aa) residues were substituted in each of the three domains of Cry toxins and the effects on toxin stability, binding to receptors, irreversible insertion into the membrane, and ion channel activity were examined . Mutant proteins with aa altered on the putative membrane-proximal surface of domain I are affected in insertion into the membrane and toxicity, but not in binding to the receptor . Alterations in the putative receptor-binding loops of domain II show an effect on the initial (reversible) binding to the receptor when certain aa are altered, while affecting irreversible binding when other aa are altered . Mutant proteins with aa altered in a conserved track of aa of domain III have altered ion channel properties, as measured by the voltage clamping of insect midguts and the K+ permeability of brush border membrane vesicles . In summary, domain I is involved in insertion into the membrane and affects ion channel function, domain II is involved in receptor binding and insertion into the membrane, and domain III is involved ion channel function, receptor binding, and insertion into the membrane.

Mem Inst Oswaldo Cruz, 1996 Nov-Dec, 91(6), 771 - 6
Characterization and biological activity of a Brazilian isolate of Bacillus sphaericus (Neide) highly toxic to mosquito larvae; Vilarinhos Pde T et al.; Primary powders of Bacillus sphaericus strain S2 isolated from soil samples in Brazil, and strain 2362 were produced in a 14 liter fermentor . Growth patterns and sporulation observed in three trials with strains S2 and 2362 in the fermentor were similar . Second-instar larvae of Culex quinquefasciatus, Anopheles albimanus, Anopheles quadrimaculatus, and Aedes aegypti exposed for 48 hr to strain S2 responded with LC50 values of 0.25, 5.95, 12.28 and 140.0 ppb of lyophilized primary powder, respectively . Under the same conditions, strain 2362 resulted in LC50 values of 0.39, 7.16, 16.93 and 307.0 ppb of lyophilized primary powder, respectively, in those mosquito larvae . Statistical analysis of the bioassay data did not show significant differences among LC50 values observed in B . sphaericus strains S2 and 2362, at the 0.05 level . Toxins of strains S2 and 2362 were extracted at pH 12 with NaOH . Electrophoresis of the extracts in polyacrylamide gel under denaturing conditions revealed the 51 and 42 kDa toxins in both S2 and 2362 B . sphaericus strains . The presence of the 42 kDa peptide in the extracts was confirmed by Western blot and Elisa, with anti-42 kDa IgG previously prepared from strain 2362.

Boll Soc Ital Biol Sper, 1996 Nov-Dec, 72(11-12), 303 - 8
The carotenoid pigments of a marine Bacillus firmus strain; Pane L et al.; As carotenoids have important biological functions, it is important to discover new natural sources of these pigments . The bacterial strains isolated from a sea water rock pool were cultivated on marine agar containing yeast extract and identified by conventional methods . The bacterial pigments were extracted with methanol and analyzed by reversed-phase HPLC with diode array detection . The major pigment of a Bacillus firmus strain was identified as astaxanthin; the results obtained suggest potential use of this bacterium in aquaculture and in pharmaceutical field.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Nov-Dec, (6), 16 - 20
{The detection of Peptostreptococcus asaccharolyticus and Bacillus cereus in clinical materials from a child who died of sepsis with a fulminant clinical course}; Beleva S et al.; A case of an acute disease with a rapid clinical course and a fatal outcome in the presence of irreversible toxicoinfectious shock, appearing in two children after the consumption of sheep kidneys, is described . The post mortem examination of the children revealed the presence of hemorrhagic, erosive and necrotic areas on the mucous membrane of the stomach, the duodenum and the upper section of the small intestine . From the material obtained by probing the stomach of one of the children 6 hours before death P.asaccharolyticus and B.cereus were isolated . Hemorrhage, erosions and necrosis were also found in experimental mice, injected with the centrifugates of the gastric secretions of the patient and the cultures of the isolated bacteria, which was indicative of the presence of highly active exotoxin . On the basis of the above facts, compared with similar data in the literature, this case was considered to be etiologically related anaerobic Peptostreptococcus in symbiosis with B.cereus.

Mikrobiologiia, 1996 Nov-Dec, 65(6), 855 - 64
{Determination of the taxonomic position of bacteria from Lake Baikal using sequence analysis of 16S rRNA fragments}; Belikov SI et al.; The taxonomic position of seven strains of aquatic bacteria from Lake Baikal was determined based on the analysis of the nucleotide sequences of 16 S rRNA gene fragments . Three strains belonged to the gamma-sub-class of Proteobacteria, one strain, to the beta-subclass, and the other three stains were assigned to the genus Bacillus (gram-positive bacteria with a low G+C content).

Mikrobiologiia, 1996 Nov-Dec, 65(6), 782 - 9
{Formation of a resting form of Bacillus cereus and Micrococcus luteus}; Muliukin AL et al.; Under certain cultivation conditions, the bacteria Bacillus cereus and Micrococcus luteus form cystlike refractive cells (up to 60% of the total number) that retain viability over a long time, are metabolically inactive and thermotolerant and possess specific ultrastructure . These properties allow them to be attributed to a new type of resting forms of microorganisms.

Plasmid, 1996 Nov, 36(3), 191 - 9
Two related rolling circle replication plasmids from salt-tolerant bacteria; Hasnain S et al.; In a range of salt-tolerant bacteria isolated from soil, rhizosphere, and phyloplane, cross-hybridization tests revealed a group of seven plasmids which were chosen for further study . Two plasmids (pSH1418 and pSH1451) were picked as representative examples of the two related subgroups . Restriction mapping and Southern blotting identified a region common to the two plasmids which later was identified as encoding the replication functions . We were unable to join these plasmids to high-copy-number vectors but this was possible with low-copy-number IncP vectors . In Escherichia coli, cloned plasmids pSH1418 and pSH1451 conferred salt tolerance but the phenotype was unstable, with the loss of salt tolerance apparently being correlated with structural instability of the plasmid DNA . Plasmid pSH1451 was sequenced and shown to be closely related to RCR plasmids of Gram-positive bacteria . The host of this plasmid was classified as Bacillus pumilus by rDNA typing and lipid profiling by gas chromatography . A number of open reading frames (orfs) which could code for salt tolerance or other functions were identified in the plasmid sequence . Sequence similarity to previously sequenced genes suggested that the products of orf4 and orf5 may work together to transport a molecule such as aspartate ion that may promote osmotolerance.

J Photochem Photobiol B, 1996 Nov, 36(2), 233 - 6
Electromotive diffusion (EMD) and photodynamic therapy with delta-aminolaevulinic acid (delta-ALA) for superficial bladder cancer; Stenzl A et al.; Recent reports have shown that topical application of delta-aminolaevulinic acid (delta-ALA) can be used for the photodynamic diagnosis and therapy of superficial bladder tumours . Electromotive diffusion (EMD) increases the cellular uptake of polar substances . In six patients with biopsy-proven recurrent carcinoma in situ of the bladder after Bacillus Calmette-Guerin (BCG) treatment, 100 cm3 of a 0.5% delta-ALA solution at pH approximately 6 was instilled into the bladder . With a special Foley catheter containing a stainless steel electrode, EMD, with a 15 mA, pulsed current, positive polarity at the catheter, was created for 20 min in the sedated patient . Thereafter a laser fibre with a spheric diffuser was inserted into the bladder over a cystoscope . The bladder surface was irradiated at a wavelength of 632 nm with 350 mW s-1 (total dose, 30-50 J cm-2) . Follow-up consisted of a cystoscopy and biopsy after 6 weeks and cystoscopy and cytology every 3 months thereafter . The whole procedure was well tolerated . Currently, five patients are tumour free after a follow-up of 10-16 months . One patient has recurred after 10 months with a Ta G3 superficial tumour at the bladder neck which was resected . In a transwell cell culture model, we investigated the effect of delta-ALA treatment with and without application of EMD . In this in vitro system, application of an electric field showed only a small increase in delta-ALA uptake compared with uptake by passive diffusion.

Vet Immunol Immunopathol, 1996 Nov, 54(1-4), 127 - 31
PR-39, a proline-rich peptide antibiotic from pig, and FALL-39, a tentative human counterpart; Agerberth B et al.; The peptide antibiotic PR-39 was originally isolated from the upper part of pig intestine . It has antibacterial activity against Gram negative bacteria at concentrations comparable with tetracycline . Studies of the mechanism of action showed that PR-39 inhibits both DNA and protein synthesis . Recently, PR-39 was found in wound fluid and was shown to have inductive activity on matrix components as part of the wound repair process . We have now sequenced the complete gene and possible mediators of its expression will be discussed . Our attempts to characterize the human counterpart of PR-39 by probing for the well conserved prepro-part led to a different peptide antibiotic . A clone containing the coding information for this new peptide was isolated from a human bone marrow cDNA library . The putative human peptide antibiotic was designated FALL-39 after the first four residues and the total number of residues . All human peptide antibiotics previously isolated (or predicted) belong to the defensin family with three disulfide bridges, while FALL-39 lacks cysteine . The clone for the prepro-FALL-39 encodes a cathelin-like precursor protein with 170 amino acid residues . We have postulated a dibasic processing site for the mature FALL-39 and chemically synthesized the peptide . In the presence of the basal medium E, synthetic FALL-39 was highly active against Escherichia coli D21 and Bacillus megaterium Bm11 . Residues 13-34 in FALL-39 can be predicted to form a perfect amphipatic helix and CD spectra showed that medium E induced 30% helix formation in FALL-39 . By Northern blot analyses the transcript was located in bone marrow and testis . The structure of the gene and the chromosomal location is under investigation.

Vet Immunol Immunopathol, 1996 Nov, 54(1-4), 123 - 6
NK-lysin, structure and function of a novel effector molecule of porcine T and NK cells; Andersson M et al.; NK-lysin (NKL), a 78-residue antimicrobial peptide, was isolated from pig small intestine . Standard methods identified the peptide as basic, with six half-cystine residues in three intrachain disulphide bonds . The sequence showed 33% identity with a part of a putative gene product (NKG5) from activated T and NK cells, NK-lysin showed antibacterial activity against Escherichia coli and Bacillus megaterium and marked lytic activity against YAC-1, a NK sensitive tumour cell line, while sheep red blood cells were unaffected . The cDNA clone corresponding to NK-lysin has been characterized . We have also analyzed the cell and tissue specific expression and the induction of the gene . A lymphocyte fraction enriched in T and NK cells, stimulated by human interleukin-2 (IL-2), showed a 30-fold increase of the NKL transcript . NK-lysin specific mRNA is also detectable in spleen, bone marrow and colon . Immunostaining showed NKL to be present in different types of lymphocytes . Our results strongly suggest that NK-lysin is involved in the inducible cytotoxicity of T and NK cells.

Lett Appl Microbiol, 1996 Nov, 23(5), 287 - 9
Identification of flagellar (H) antigenic subfactors in Bacillus thuringiensis H serotypes 10, 18 and 24 isolated in Japan; Ohba M; A total of 95 Bacillus thuringiensis strains isolated from Japan and belonging to H serotypes 10, 18 and 24, were examined for their H antigenic subfactors . Of 84 H serotype 10 isolates, 83 were identified as the H serotype 10a: 10b (serovar darmstadiensis) and only one isolate was assigned to the H serotype 10a: 10c (serovar londrina) . Among five isolates belonging to the H serotype 18, three were allocated to the H serotype 18a: 18b (serovar kumamotoensis), while two isolates did not react to antisera against the two known H antigenic subfactors, 18b and 18c . All of the six H serotype 24 isolates were assigned to the H serotype 24a: 24b (serovar neoleonensis).

Pediatr Dermatol, 1996 Nov-Dec, 13(6), 451 - 4
Disseminated cutaneous eruption after BCG vaccination; Rositto A et al.; We present nine infants (3 to 10 months of age) with numerous small, papular, papular-lichenoid, and papulo-pustular lesions predominantly on the upper and lower limbs associated with local (axillary) lymphadenopathy which appeared after BCG vaccination . Histopathology of the lesions showed small tuberculoid granulomas mainly in the papillary dermis . The presence of BCG bacillus was demonstrated in five out of seven samples from the lymph nodes after culture and in one skin biopsy specimen . All cases, whether treated or not, evolved to complete resolution of the skin lesions . We believe that this peculiar association results from hematogenous spread of the bacillus, which regresses after an adequate immune system reaction.

Nihon Kyobu Shikkan Gakkai Zasshi, 1996 Nov, 34(11), 1244 - 8
{Tuberculous mediastinal lymphadenitis presenting as hoarseness}; Kawasaki T et al.; A 70 year-old woman was admitted to our hospital complaining of hoarseness . Bronchoscopic examination revealed that the left vocal cord was fixed in the paramedian position, and therefore left recurrent nerve paralysis was suspected . Although a chest X-ray film showed no abnormal findings, a chest computed tomogram showed that pretracheal (#3) and subaortic (#5) lymph nodes were swollen (1 to 2 cm in diameter) . An open biopsy was done . Histological examination of the resected mediastinal lymph nodes revealed an accumulation of Langhans giant cells and epithelioid cells in caseous necrosis . An acid-fast bacillus was seen after Ziehl-Neelsen staining . This was a rare case of mediastinal tuberculous lymphadenitis in which hoarseness was useful for early diagnosis . Fifty-three cases of tuberculous mediastinal lymphadenitis have been reported in Japan.

Arch Environ Contam Toxicol, 1996 Nov, 31(4), 571 - 80
Effects of 2,4-dichlorophenoxyacetic acid on Kentucky algae: simultaneous laboratory and field toxicity testings; Kobraei ME et al.; 2,4-D was applied to a cove in Kentucky Lake which was highly infested with Myriophllum spicatum (Eurasian watermilfoil) . Effects of 2,4-D on nontarget algal communities were monitored concurrently in the field and in laboratory microcosms for eight days . Results indicated that indirect effects of water temperature and increased nutrient concentrations due to lysis in milfoil plants may be more important in the field community dominated by Chlorophyta, Pyrrhophyta, and Bacillariophyta . 2,4-D applied at the label-recommended rate of 2 mg/L or less stimulated total community growth in both laboratory and field indicating a possible hormonal effect of 2,4-D on algae . Reduced community growth and metabolism at high laboratory concentrations of 100 mg/L and 1000 mg/L may indicate an inhibitory effect on photosynthesis and/or respiration in algae . 2,4-D altered the laboratory community structure and function in all concentrations tested . Heterotrophic taxa such as Nitzschia, Euglena, Chlamydomonas, Mallomonas, Anabaena, and Oscillatoria appeared to be least affected by 2,4-D at high concentrations . Scenedesmus, Pediastrum, Characiosiphon, Navicula, Melosira, and Fragilaria appeared to be more sensitive, even in the lowest concentrations.

Microbiology, 1996 Nov, 142 ( Pt 11), 3135 - 45
Physical mapping of Mycobacterium bovis BCG pasteur reveals differences from the genome map of Mycobacterium tuberculosis H37Rv and from M . bovis; Philipp WJ et al.; A Dral restriction map of the approximately 4.35 Mb circular chromosome of the vaccine strain Mycobacterium bovis BCG Pasteur was constructed by linking all 21 Dral fragments, ranging in size from 6 to 820 kb, using specific clones that spanned the Dral recognition sites as hybridization probes . The positions of 20 known genes were also established . Comparison of the resultant genome map with that of the virulent tubercle bacillus Mycobacterium tuberculosis H37Rv revealed extensive global conservation of the genomes of these two members of the M . tuberculosis complex . Possible sites of evolutionary rearrangements were localized on the chromosome of M . bovis BCG Pasteur by comparing the Asnl restriction profile with that of M . tuberculosis H37Rv . When selected cosmids from the corresponding areas of the genome of M . tuberculosis H37Rv were used as hybridization probes to examine different BCG strains, wild-type M . bovis and M . tuberculosis H37Rv, a number of deletions up to 10 kb in size, insertions and other polymorphisms were detected . In addition to the known deletions covering the genes for the protein antigens ESAT-6 and mpt64, other genetic loci exhibiting polymorphisms or rearrangements were detected in M . bovis BCG Pasteur.

Biochem Mol Biol Int, 1996 Nov, 40(5), 947 - 54
Activation of the intracellular Ca(2+)-dependent serine protease ISP1 of bacillus megaterium by purification or by high Ca2+ concentrations; Vachova L; The total proteolytic activity of ISP1 determined after its partial purification by size exclusion HPLC increased 3.0, 7.3 and 27.3 times in sporulating, growing and netropsin-treated cells, respectively, as compared with the corresponding original activity in crude cytoplasmic preparations at the same CaCl2 concentration of 3 mM . A similar rise in proteolytic activity occurred on increasing the Ca2+ concentration in the crude cytoplasm from 3 to 30 mM . This activation in the cytoplasm of netropsin-treated and sporulating cells was not reversed by subsequent lowering of CaCl2 concentration back to 3 mM by dialysis . Moreover, a similar activation appeared even after the same dialysis of cytoplasm that had not been exposed to 30 mM CaCl2 . The activation was probably due to the processing of ISP1, as established by SDS-PAGE and immunoblotting, but an involvement of additional regulation factor(s), e.g . an inhibitor or other molecules, is possible.

Oncology (Huntingt), 1996 Nov, 10(11), 1617 - 24; discussion 1624, 1627-8
Superficial bladder cancer: decreasing the risk of recurrence; Grossman HB; Bladder cancer appears to develop through two alternative pathways . Papillary bladder cancer, the most common pathway, has a less aggressive course and is frequently heralded by hematuria, whereas carcinoma in situ appears to be more aggressive and is more difficult to detect . Superficial bladder cancer has a high propensity for recurrence but a low rate of progression . Transurethral resection is frequently employed for both diagnosis and treatment . The risk of tumor recurrence is related to the number of tumors at presentation and the findings on the first follow-up cystoscopy . Even patients with a low risk of recurrence need periodic cystoscopic examinations . Patients with a higher risk of recurrence may benefit from adjuvant intravesical chemotherapy or immunotherapy . Bacillus Calmette-Guerin (BCG) appears to be the most effective drug for intravesical therapy but has the highest rate of side effects . It is the treatment of choice for carcinoma in situ . Newer treatment strategies include perioperative intravesical chemotherapy and chemoprevention.

Am J Gastroenterol, 1996 Nov, 91(11), 2289 - 92
Diagnostic yield of duodenal biopsy and aspirate in AIDS-associated diarrhea; Bown JW et al.; OBJECTIVES: To evaluate the diagnostic yield of performing duodenal biopsies and aspirates in AIDS patients with chronic diarrhea . METHODS: Retrospective review of esophagogastroduodenoscopy (EGD) records from January 1993 to March 1995 to identify those patients who underwent EGD for evaluation of AIDS associated diarrhea and had a duodenal biopsy and/or aspirate . Biopsies were examined for pathogens using routine histology and special stains, viral culture, and electron microscopy . Duodenal aspirates were evaluated for ova and parasites . All patients had previous negative stool studies . Pathology laboratory charges (hospital and professional fees) for each test and charges per positive test were determined . RESULTS: Of the 57 patients included in this study, 56 had a duodenal biopsy and 42 had a duodenal aspirate . An established pathogen was identified in only 15 (26%) patients . One patient had both Mycobacterium avium complex and microsporidia . Pathogens were identified in seven patients by hematoxylin and eosin stain, in three patients by acid-fast bacillus stain, and in six patients by electron microscopy . No pathogens were identified with Gomori's methenamine silver stain (44 patients), duodenal aspirate for ova and parasites (46 patients), immunoperoxidase stains (4 patients), or viral culture (4 patients) . Cryptosporidia were identified in six, microsporidia in five, Mycobacterium avium complex in three, and Giardia lamblia and adenovirus each in one patient . CONCLUSIONS: In this series, the diagnostic yield of EGD with duodenal biopsy and aspirate in AIDS associated diarrhea was low . Pathogens were identified in 26% of patients; predominantly Cryptosporidium organisms and microsporidia . The routine performance of aspiration of duodenal contents for parasite examination and staining of duodenal tissue with Gomori's methenamine silver stain for fungal identification are not recommended . One should consider obtaining tissue for electron microscopy whenever duodenal biopsies are performed . The utility of EGD in AIDS associated diarrhea may improve as more effective therapies become available.

J Invertebr Pathol, 1996 Nov, 68(3), 203 - 12
Interactions of Bacillus thuringiensis crystal proteins with the midgut epithelial cells of Spodoptera frugiperda (Lepidoptera: Noctuidae); Aranda E et al.; Binding of different Bacillus thuringiensis insecticidal crystal proteins (ICPs) to the midgut epithelium of Spodoptera frugiperda larvae was characterized by binding experiments with midgut tissue sections and isolated brush border membrane vesicles . Our results show that ICPs interact with the microvilli of epithelial cells of S . frugiperda in two different ways . The first is typical of highly toxic proteins (like CryIC and CryID); this interaction is saturable and specific . In contrast, some nontoxic proteins (like CryIAb) interact nonspecifically with the microvilli, since the binding of this toxin is not affected by the presence of high concentrations of homologous competitor . The CryIC toxin binds to two brush border proteins of 40 and 44 kDa and the CryIAb toxin binds to a single protein of 150 kDa . Immunological detection of ingested B . thuringiensis ICPs on gut sections of S . frugiperda larvae revealed that CryIC and CryID toxins bound along the epithelial brush border microvilli membrane . Binding of the nontoxic protein CryIAb was also observed in the epithelial brush border membrane of fed larvae, but it was extremely weak, implying that this type of interaction occurs also in vivo although it is not related to toxicity.

Protein Sci, 1996 Nov, 5(11), 2319 - 28
Characterization of a buried neutral histidine residue in Bacillus circulans xylanase: NMR assignments, pH titration, and hydrogen exchange; Plesniak LA et al.; Bacillus circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core . His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105 . These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases . To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX . Complete assignments of the 1H, 13C, and 15N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments . An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 1H epsilon 2 of His 149 . Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 x 10(-5) s-1 at pH*7.04 and 30 degrees C) is retarded by > 10(6)-fold relative to an exposed histidine . The pKa of His 156 is unperturbed at approximately 6.5, as measured from the pH dependence of the 15N- and 1H-NMR spectra of BCX . In contrast, His 149 has a pKa < 2.3, existing in the neutral N epsilon 2H tautomeric state under all conditions examined . BCX unfolds at low pH and 30 degrees C, and thus His 149 is never protonated significantly in the context of the native enzyme . The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.

Protein Sci, 1996 Nov, 5(11), 2255 - 65
Individual amino acids in the N-terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases; Welfle K et al.; Thermostability and unfolding behavior of the wild-type (1,3-1,4)-beta-glucanases from Bacillus macerans (MAC) and Bacillus amyloliquefaciens (AMY) and of two hybrid enzymes H(A12-M) delta F14 and H(A12-M) delta Y13F14A were studied by spectroscopic and microcalorimetric measurements . H(A12-M) delta F14 is constructed by the fusion of 12 N-terminal amino acids of AMY with amino acids 13-214 of MAC, and by deletion of F14 . In H(A12-M) delta Y13F14A, the N-terminal region of MAC is exchanged against the AMY sequence, Y13 is deleted, and Phe 14 is exchanged against Ala . The sequence of the N-terminal loop region from Pro 9 to amino acid 16 (or 17) is very important for the properties of the enzymes and influences the effects of Ca2+ ions on the thermostability and unfolding behavior of the enzymes . The half transition temperatures T(m) are higher in the presence of Ca2+ than in Ca2+ free buffer . Furthermore, the unfolding mechanism is influenced by Ca2+ . In Ca(2+)-free buffer, MAC, H(A12-M) delta F14 and H(A12-M) delta Y13F14A unfold in a single cooperative transition from the folded state to the unfolded state, whereas for AMY, a two-step unfolding was found . In the presence of Ca2+, the two-step unfolding of AMY is strengthened . Furthermore, for H(A12-M) delta F14, a two-step unfolding is induced by Ca2+ . These data indicate a two-domain structure of AMY and H(A12-M) delta F14, in the presence of Ca2+ . Thus, point mutations in a peripheral loop region are decisive for thermal stabilities and unfolding mechanisms of the studied glucanases in the presence of Ca2+.

Biotechniques, 1996 Nov, 21(5), 918 - 25
2-D protein crystals as an immobilization matrix for producing reaction zones in dipstick-style immunoassays; Breitwieser A et al.; In the present study, the applicability of crystalline bacterial cell-surface layers (S-layers) as novel immobilization matrices and reaction zones for dipstick-style immunoassays was investigated . For this purpose, S-layer-carrying cell-wall fragments from Bacillus sphaericus CCM 2120 were deposited on a microporous support, and the S-layer protein was cross-linked with glutaraldehyde . For developing appropriate test systems, either human IgG was directly linked to the carboxylic acid groups from the S-layer protein or it was immobilized using Protein A or, after biotinylation, using streptavidin . A clear correlation was obtained between the amount of anti-human IgG applied and the absorbance values in the immunoassays . S-layers with covalently bound recombinant major birch pollen allergen were used for quantitative and semiquantitative determination of an antibody raised against it . Using S-layers as an immobilization matrix in comparison to amorphous polymers has advantages in that the closed monolayers of functional macromolecules on their outermost surface allows for strong signals in immunoassays, almost completely eliminates background and prevents diffusion.

Eur J Immunol, 1996 Nov, 26(11), 2559 - 64
Tumor cells expressing a recall antigen are powerful cancer vaccines; Schweighoffer T; Tumor cells were engineered to express a specific recall antigen molecule to serve as a target for the host's existing memory response, and then used as immunogens as a novel form of cancer vaccine . As recall antigen, the efficiently expressible hybrid protein Heat1 was employed, that contains an antigenic fragment of the Mycobacterium bovis 65-kDa heat shock protein (hsp65), and thus can be recognized in mice immunized with live Bacillus Calmette-Guerin (BCG) or its derivatives . Mouse M-3 melanoma cells were transfected to express Heat1, irradiated, and used as anti-melanoma vaccines . Vaccination elicited anti-tumor immunity in mice, i.e . a subsequent challenge with the parental wild-type M-3 melanoma cells was rejected . Successful vaccination was dependent on the correct recognition of the recall antigen, since vaccination failed to prevent outgrowth of the challenge in mice possessing no memory response against the recall antigen . The results indicate that expression of a recall antigen can turn tumor cells into powerful vaccines . By using the Heat1 protein or other appropriate antigens, this general concept may be applied for human therapy.

J Mol Biol, 1996 Nov 1, 263(3), 463 - 74
Recognition of a surface loop of the lipoyl domain underlies substrate channelling in the pyruvate dehydrogenase multienzyme complex; Wallis NG et al.; In the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus, the interaction between the pyruvate decarboxylase (E1p) component and the lipoyl domain of the dihydrolipoyl acetyltransferase (E2) component was investigated using a combination of site-directed mutagenesis and NMR spectroscopy . Residues 11 to 15 (EGIHE) of the lipoyl domain, part of a surface loop close in space to the beta-turn containing the lipoyl-lysine residue (position 42), were deleted or replaced . The mutant domains all retained their three-dimensional structures and ability to become lipoylated, but in the absence of the loop the lipoyl-lysine residue could no longer be reductively acetylated by E1p . A mutation (N40A) in the N- terminal part of the lipoyl-lysine hairpin showed that it is involved in recognition of the domain by E1p but other mutations in the loop (E15A) and close to the lipoyl-lysine hairpin (V44S, V45S and E46A) were without effect . The heteronuclear multiple quantum coherence NMR spectra of 15N-labelled lipoyl domain in the presence and absence of B . stearothermophilus E1p were recorded . Of the 85 amino acid residues in the lipoyl domain, 13 exhibited significant differences in chemical shift . These differences, most of which were associated with residues in the surface loop between positions 8 and 15 and in, or close to, the lipoyl-lysine hairpin, indicate that E1p makes contact with the lipoyl domain in these areas . The combined results of directed mutagenesis and NMR spectroscopy point to the surface loop as a major determinant of the interaction of lipoyl domain with E1p . The specificity of this essential interaction provides the molecular basis of substrate channelling in this, the first committed, step of the enzyme reaction mechanism.

Clin Diagn Lab Immunol, 1996 Nov, 3(6), 761 - 8
A novel method for monitoring Mycobacterium bovis BCG trafficking with recombinant BCG expressing green fluorescent protein; Luo Y et al.; To better understand intracellular and extracellular trafficking of Mycobacterium bovis bacillus Calmette-Guerin (BCG) when used as an intravesical agent in the treatment of transitional cell carcinoma (TCC) of the bladder, recombinant BCG (rBCG) expressing the jellyfish green fluorescent protein (GFP) was created . When the MB49.1 murine TCC cell line was incubated with GFP-expressing rBCG, internalization of the pathogen could be directly visualized by UV microscopy and quantitated by flow cytometry . The in vitro internalization of the GFP rBCG by the bladder tumor cells was temperature dependent, occurring most readily at 37 degrees C and being severely inhibited at 4 degrees C . Optimum internalization was achieved in vitro at a 10:1 BCG-to-tumor cell ratio over 24 h during which approximately 16% of the tumor cells became infected . Cytochalasin B, a phagocytosis inhibitor, abrogated the ingestion by almost 100% at a concentration of 200 micrograms/ml, indicating that contractile microfilaments likely played an important role in this process . By using mitomycin, a DNA cross-linking reagent, to inhibit proliferation of MB49.1 cells, clearance of about 40% of the green rBCG was achieved by 3 days postinfection . No significant difference between the GFP rBCG and wild-type BCG was observed in the ability to induce the expression of cell membrane proteins of major histocompatibility classes I and II, ICAM-I and -II, B7-1 and -2, of Fas from MB49.1 cells or cytokine production from mouse spleen cells . These results indicate that GFP rBCG may serve as a useful substitute for wild-type BCG in future studies of in vivo trafficking experimental and clinical immunotherapy.

J Biol Chem, 1996 Nov 1, 271(44), 27388 - 94
Free radical generation as induced by ochratoxin A and its analogs in bacteria (Bacillus brevis); Hoehler D et al.; Lipid peroxidation is considered as one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA) . OA; its three natural analogs, OB, OC, and Oalpha; and four synthetic analogs, d-OA, the ethylamide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in bacteria with Bacillus brevis as a model system . The uptake of the different ochratoxins by B . brevis varied substantially depending on the molecular structures . Electron paramagnetic resonance spectroscopy using alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone as a spin trapping agent showed an enhanced free radical generation due to the addition of OA and most of the analogs . The EPR signals could be further enhanced by the addition of Ca2+, a calcium ionophore and an ATPase uncoupler, whereas they were eliminated by incubating the growing cells with vitamin E . The spin adduct hyperfine splitting constants indicate the presence of alpha-hydroxyethyl radicals resulting from generated hydroxyl radicals, which are trapped by alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone . The results further suggest that OA induces free radical production in this model system by enhancing the permeability of the cellular membrane to Ca2+.

Arch Ophthalmol, 1996 Nov, 114(11), 1402 - 6
Immunocytochemical features of retinoblastoma in an adult; Nork TM et al.; OBJECTIVE: To describe the clinical course and immunocytochemical characteristics of an unusual intraocular tumor . METHODS: Immunocytochemical analysis of the enucleated eye with an intraocular mass that markedly waxed and waned in size during 1 year of close observation of a 29-year-old woman . RESULTS: Most of the tumor was composed of either dying or rapidly proliferating cells . One area located near the retina consisted mostly of well-differentiated cells in uniform sheets (bacillettes) with lacelike glial processes between the tumor cells . Almost all of the differentiated tumor cells were positive for S antigen . In particular, the dominant cell type stained positively for both antibodies known to be specific for those isoforms of S antigen found only in blue cones and rods but not in red or green cones . Only a few of these cells labeled positively with an anti-rhodopsin antibody . CONCLUSIONS: This is the first case of adult retinoblastoma to be confirmed immunocytochemically . The tumor was unusual because the differentiated regions contained bacillettes composed mostly of blue cones . It is possible that this and other adult retinoblastomas may arise from previously existing retinocytomas.

FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 221 - 7
Complete nucleotide sequence of a cryptic plasmid, pBAW301, from the ruminal anaerobe Ruminococcus flavefaciens R13e2; May T et al.; The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminal bacterium Ruminococcus flavefaciens R13e2, has been determined . This plasmid is 1768 bp in size and has an overall G+C content of 43.5% . Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926 . ORF1 is preceded by Shine-Dalgarno and Escherichia coli-10 and -35 like sequences . Nine smaller open reading frames showed no significant homologies to known protein sequences . Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria . Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301 . These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.

Appl Environ Microbiol, 1996 Nov, 62(11), 4229 - 32
Epidemiological typing of Bacillus spp . isolated from food; Schraft H et al.; Biotypes, fatty acid profiles, and restriction fragment length polymorphisms of a PCR product (PCR-RFLP of the cereolysin AB gene) were compared for 62 isolates of the Bacillus cereus group . Eleven isolates originated from various foods, and 51 isolates were obtained from pasteurized milk which had been processed by two different dairies . The isolates were clustered into 6 biotypes, 10 fatty acid groups, or 7 PCR-RFLP clusters . Isolates with mesophilic or psychrotrophic characteristics were preferentially distributed into specific fatty acid or PCR-RFLP groups (P = 0.004) . Unique fatty acid clusters were predominantly found in milk samples of each dairy (P < 0.0001), suggesting that certain dairy plants may harbor plant-specific B . cereus which might constantly contribute to postpasteurization contamination.

Appl Environ Microbiol, 1996 Nov, 62(11), 4168 - 73
Altered binding of the Cry1Ac toxin to larval membranes but not to the toxin-binding protein in Plodia interpunctella selected for resistance to different Bacillus thuringiensis isolates; Mohammed SI et al.; Immunoblotting and cytochemical procedures were used to determine whether toxin binding was altered in strains of the Indianmeal moth, Plodia interpunctella, selected for resistance to various strains of Bacillus thuringiensis . Each of these B . thuringiensis subspecies produces a mixture of protoxins, primarily Cry1 types, and the greatest insect resistance is to the Cry1A protoxins . In several cases, however, there was also resistance to toxins not present in the B . thuringiensis strains used for selection . The Cry1Ab and Cry1Ac toxins bound equally well over a range of toxin concentrations and times of incubation to a single protein of ca . 80-kDa in immunoblots of larval membrane extracts from all of the colonies . This binding protein is essential for toxicity since a mutant Cry1Ac toxin known to be defective in binding and thus less toxic bound poorly to the 80-kDa protein . This binding protein differed in size from the major aminopeptidase N antigens implicated in toxin binding in other insects . Binding of fluorescently labeled Cry1Ac or Cry1Ab toxin to larval sections was found at the tips of the brush border membrane prepared from the susceptible but not from any of the resistant P . interpunctella . Accessibility of a major Cry1A-binding protein appears to be altered in resistant larvae and could account for their broad resistance to several B . thuringiensis toxins.

Appl Environ Microbiol, 1996 Nov, 62(11), 3917 - 21
Cultivation of aerobic chemoorganotrophic proteobacteria and gram-positive bacteria from a hot spring microbial mat; Nold SC et al.; The diversity of aerobic chemoorganotrophic bacteria inhabiting the Octopus Spring cyanobacterial mat community (Yellowstone National Park) was examined by using serial-dilution enrichment culture and a variety of enrichment conditions to cultivate the numerically significant microbial populations . The most abundant bacterial populations cultivated from dilutions to extinction were obtained from enrichment flasks which contained 9.0 x 10(2) primary producer (Synechococcus spp.) cells in the inoculum . Two isolates exhibited 16S rRNA nucleotide sequences typical of beta-proteobacteria . One of these isolates contained a 16S rRNA sequence identical to a sequence type previously observed in the mat by molecular retrieval techniques . Both are distantly related to a new sequence directly retrieved from the mat and contributed by a beta-proteobacterial community member . Phenotypically diverse gram-positive isolates genetically similar to Bacillus flavothermus were obtained from a variety of dilutions and enrichment types . These isolates exhibited identical 16S rRNA nucleotide sequences through a variable region of the molecule . Of the three unique sequences observed, only one had been previously retrieved from the mat, illustrating both the inability of the cultivation methods to describe the composition of a microbial community and the limitations of the ability of molecular retrieval techniques to describe populations which may be less abundant in microbial communities.

J Immunol, 1996 Nov 1, 157(9), 4094 - 9
The role of E-selectin in lymphocyte and polymorphonuclear cell recruitment into cutaneous delayed hypersensitivity reactions in sensitized pigs; Binns RM et al.; We have studied the role of E-selectin in leukocyte accumulation into Ag-specific cutaneous delayed-type hypersensitivity reactions in pigs sensitized to the topical application of 2,4-dinitro-1-fluorobenzene or to the intradermal injection of bacillus Calmette-Guerin . The delayed-type hypersensitivity reactions were shown to be specific for the sensitizing Ag and characterized by the up-regulation of E-selectin, as demonstrated by the uptake of tracer 99mTc-labeled monoclonal anti-E-selectin mAb and entry of 51Cr-labeled PBL and (111)In-labeled polymorphonuclear cells (PMN) . Intravenous injection of 5 mg/kg of a F(ab')2 preparation of a monoclonal anti-E-selectin Ab at peak times of leukocyte entry resulted in a significant inhibition of entry of both PMN and lymphocytes . The anti-E-selectin Ab inhibited PMN recruitment by 70 to 90% and lymphocyte recruitment by 50 to 60% . In comparison, an anti-CD18 treatment reduced PMN recruitment by 70 to 90% and lymphocyte recruitment by 60 to 70% in this model . These data confirm an important role for E-selectin in the recruitment of both PMN and lymphocytes to sites of immune-based dermal inflammation.

Biopolymers, 1996 Nov, 39(5), 691 - 707
Use of graph theory for secondary structure recognition and sequential assignment in heteronuclear (13C, 15N) NMR spectra: application to HU protein from Bacillus stearothermophilus; van Geerestein-Ujah EC et al.; A computer-assisted procedure, based upon a branch of mathematics known as graph theory, has been developed to recognize secondary structure elements in proteins from their corresponding nuclear Overhauser effect spectroscopy (NOESY)-type spectra and to carry out their sequential assignment . In the method, NOE connectivity templates characteristic of regular secondary structures are identified in the spectra . Resonance assignment is then achieved by connecting these NOE patterns of secondary structure together, and thereby matching connected spin systems to specific parts of the primary sequence . The range of NOE-graph templates of secondary structure motifs, incorporating alpha-helices and beta-strand motifs, has been examined for reliability and extent of secondary structure identification in a data base composed of the high resolution structures of 20 proteins . The analysis identified several robust NOE-graph templates and supports the implementation of an ordered search strategy . The method, known as SERENDIPITY, has been applied to the analysis of nuclear Overhauser effect data from a three-dimensional time-shared nuclear Overhauser effect spectroscopy (13C, 15N) heteronuclear single quantum correlation spectrum of the (alpha + beta) type protein HU from Bacillus stearothermophilus . The arrangement of the elucidated elements of secondary structure is very similar to that of the x-ray and nmr structures of HU . In addition, our analysis revealed a pattern of interstrand nuclear Overhauser effect in the beta-arm region (residues 53-76) of HU, which suggest irregularities, not reported in the x-ray structure, in both strands of the beta-arm at Ala57 and Pro72, respectively . At these residues, both strands of the beta-arm appear to flip inside out before continuing as a regular antiparallel beta-sheet.

J Urol, 1996 Nov, 156(5), 1602 - 5
Low dose Pasteur bacillus Calmette-Guerin regimen in stage T1, grade 3 bladder cancer therapy; Hurle R et al.; PURPOSE: We assessed the effectiveness of intravesical bacillus Calmette-Guerin (BCG) for high risk transitional cell carcinoma of the bladder . MATERIALS AND METHODS: A total of 51 patients with stage T1, grade 3 disease was treated with weekly instillations of 75 mg . Pasteur strain BCG for 6 weeks after transurethral resection for bladder cancer . An additional induction course was given to patients with relapse . Tumor-free patients followed a maintenance course with monthly instillations for 12 months . RESULTS: After the initial induction course 37 of 51 patients (72.5%) remained tumor-free . A second induction course was necessary in 13 patients . After 1 or 2 induction courses 44 of 51 patients (86.3%) were tumor-free . The maintenance course was administered to 44 patients, with 41 remaining tumor-free . After a median followup of 33 months (range 3 to 63) 28 patients (54.9%) were disease-free, 12 (23.5%) had recurrent tumors and 7 (13.7%) had progression . The risk of treatment failure was significantly greater for solid than papillary tumors (p = 0.0006), recurrent than primary tumors (p = 0.0052) and coexisting carcinoma in situ (p = 0.124) in multivariate analysis, and for early recurrence (p = 0.0001) in univariate analysis only . The drug was well tolerated with few side effects . CONCLUSIONS: Our data suggest that this low dose Pasteur BCG regimen is effective in the treatment of high risk superficial bladder cancer . Some tumor characteristics, such as solid appearance, coexisting carcinoma in situ, history of superficial transitional cell carcinoma and early relapse after the initial induction course, seem to be negative prognostic factors.

J Biol Chem, 1996 Oct 25, 271(43), 26477 - 81
The roles of the prosequence of thermolysin in enzyme inhibition and folding in vitro; O'Donohue MJ et al.; The zinc endopeptidase thermolysin (EC 3.4.24.27), an extracellular enzyme from Bacillus thermoproteolyticus, is synthesized as a preproprotein, with the prosequence (204 residues) being two-thirds the size of the mature enzyme (316 residues) . This prosequence, expressed in and purified from Escherichia coli, inhibited thermolysin in vitro with an IC50 value of 14 nM . It also inhibited a closely related enzyme produced by Bacillus stearothermophillus, albeit with a 16-fold higher IC50 value (220 nM) . The IC50 value for thermolysin inhibition was also increased 15-fold (210 nm) by a monoclonal antibody that recognizes an epitope close to, but not forming a part of, the active site . At a prosequence concentration of 5 microM a mammalian, thermolysin-like enzyme, neutral endopeptidase 24.11, was not inhibited . The prosequence appeared to act as a mixed, noncompetitive inhibitor of thermolysin activity, with a Ki value of 6 nM for its interaction with the enzyme alone and a Ki' value of 20 nM for its interaction with the enzyme-substrate complex . In addition, when thermolysin was denatured in 6 M guanidinium hydrochloride at acid pH and then brought to neutral pH by rapid dilution, the prosequence was found to facilitate the recovery of active enzyme in a stoichiometric manner.

J Med Chem, 1996 Oct 25, 39(22), 4366 - 76
Synthesis, structure-activity relationships, and the effect of polyethylene glycol on inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus; Ryan M et al.; Substrate analog inhibitors of Bacillus cereus phosphatidylinositol-specific phospholipase C (PI-PLC) were synthesized and screened for their suitability to map the active site region of the enzyme by protein crystallography . Analogs of the natural substrate phosphatidylinositol (PI) were designed to examine the importance of the lipid portion and the inositol phosphate head group for binding to the enzyme . The synthetic compounds contained pentyl, hexyl, or hexanoyl and octyl lipid chains at the sn-1 and sn-2 positions of the glycerol backbone and phosphonoinositol, phosphonic acid, methyl phosphonate, phosphatidic acid, or methyl phosphate at the sn-3 position . The most hydrophobic compound, dioctyl methyl phosphate 14, was also the best inhibitor with an IC50 of 12 microM . In a series of dihexyl lipids, compounds with phosphonoinositol head groups inhibited more strongly than those that do not contain inositol but are otherwise identical . Compound 29, a short-chain lipid with a phosphonoinositol head group, was found to be a competitive inhibitor and the most potent in this series with an IC50 of 18 microM (Ki = 14 microM) . Analogs with dihexyl chains were better inhibitors than those with dihexanoyl chains, presumably because the ether-linked lipids are more hydrophobic than the ester-linked lipids . No appreciable difference in inhibition was found between a phosphonoinositol lipid and the corresponding difluorophosphonoinositol lipid . Inositols and inositol derivatives that do not contain lipid moieties show IC50s about 3 orders of magnitude above those of the short-chain lipids . In this group, glucosaminyl(alpha 1-->6)-D-myo-inositol inhibited more strongly than myo-inositol, which in turn is a better inhibitor than inositol phosphate . The addition of polyethylene glycol (PEG-600) resulted in a marked decrease in inhibition by the short-chain lipids, but had little effect on the water-soluble head group analogs . This is accounted for in terms of solubilization of the amphipathic inhibitors by PEG . Since PEG is required in the crystallization, these data indicate that the best strategy for obtaining enzyme inhibitor complexes is to start by cocrystallizing PI-PLC with the head group analogs . The next step is to synthetically add the shortest possible hydrophobic moieties to the analogs and cocrystallize these with the enzyme . This strategy may be applicable to other lipolytic enzymes.

Biochim Biophys Acta, 1996 Oct 23, 1284(2), 122 - 4
Mechanism of action of hemolysin III from Bacillus cereus; Baida GE et al.; Bacillus cereus hemolysin III activity was tested in crude extracts, from Escherichia coli carrying the hly-III gene . It was concluded that hemolysin III is a pore-forming hemolysin with functional pore diameter of about 3-3.5 nm . Hemolysis occurs in at least three steps: (i) the temperature-dependent binding of the Hly-III monomers to the erythrocyte membrane; (ii) the temperature-dependent formation of the transmembrane oligomeric pore; (iii) the temperature-independent erythrocyte lysis.

Gene, 1996 Oct 17, 176(1-2), 149 - 54
A new series of mycobacterial expression vectors for the development of live recombinant vaccines; Baulard A et al.; Recombinant BCG (bacillus Calmette-Guerin) is a promising candidate as a live vaccine delivery system . Thus far, however, only autoreplicative plasmids carrying the heterologous genes to be expressed in BCG, together with antibiotic-resistance genes, have been successfully used . This could potentially lead to the spreading of antibiotic resistance among other bacteria, and might therefore be unsafe for the environment . In this study, we present a series of three Escherichia coli-Mycobacteria shuttle vectors which enable expression and secretion of antigens without the use of antibiotic-resistance markers . All these plasmids confer mercury resistance to the host bacteria as the only selectable marker and contain a unique restriction site to allow for single-step in-frame cloning of open reading frames downstream from the Mycobacterium tuberculosis 85A antigen promoter and export signal . The system was used to express the free beta-subunit of human chorionic gonadotropin (hCG beta), a potential target of an immunotherapeutic vaccine.

J Immunol Methods, 1996 Oct 16, 197(1-2), 151 - 9
Characterization of murine monoclonal antibodies specific for the 45/47 kDa antigen complex (APA) of Mycobacterium tuberculosis, M . bovis and BCG; Horn C et al.; The alanine-proline antigen (APA), representing less than 2% of the released or excreted material during Mycobacterium tuberculosis or bacillus Calmette-Guerin (BCG) growth, is a dominant antigen present during M . tuberculosis infection or BCG immunization . To obtain new tools to dissect the major epitopes of the APA molecules, seven monoclonal antibodies (mAbs) against the purified molecules were developed . Epitope maps of the mAbs were obtained on APA molecules absorbed on plastic surfaces or in solution (BIAcore technology) . The mAbs were found to be independent or to be different despite binding to adjacent or overlapping epitopes . In Western blot assays some proteins secreted in culture fluid by M . avium, M . kansasii, M . smegmatis or M . xenopi were also labelled by six of the seven antibodies . Conversely one antibody was specific for the proteins from the M . tuberculosis complex (I10-0,3) demonstrating that the APA molecules have some properties or general conformations that are specific for M . tuberculosis and M . bovis.

Eur J Biochem, 1996 Oct 15, 241(2), 538 - 45
Refolding and reassociation of glycerol dehydrogenase from Bacillus stearothermophilus in the absence and presence of GroEL; Krauss O et al.; The refolding of the tetrameric, metalloenzyme glycerol dehydrogenase (GDH) from Bacillus stearothermophilus has been investigated using stopped-flow fluorescence and circular dichroism spectroscopy . The effects of metal ions on the refolding of the native enzyme and the refolding of a monomeric mutant ({A208}GDH) have also been studied . The refolding process of the wild-type enzyme is at least biphasic; 70% of the respective signal changes occur in the first 2 ms followed by a slower process with a half-life of 3 s . The presence of the metal ion does not affect the slowest biphasic refolding rate, which is virtually the same for all three versions of the enzyme . The presence of GroEL slows down the first phase of refolding . The reassociation of subunits was examined by measuring the regain in catalytic activity and the enhancement in the fluorescence emission from NADH on binding to the oligomeric form of the enzyme . The rate and extent of reassociation is dependent on enzyme concentration and the extent of reactivation is dependent on the presence of the metal ion . The reassociation process was more efficient in the presence of NADH particularly for the metal-depleted enzyme (apo-GDH) . The presence of GroEL or GroEL plus ATP leads to a higher yield of reassociation and therefore catalytically active enzyme . The additional presence of Mg-ATP does not affect the extent of reassociation, but has a small positive effect on the rate of reassociation . These data suggest that GDH is bound weakly to GroEL and that GroES is not required for release of the protein.

Blood, 1996 Oct 15, 88(8), 3022 - 7
Identification of an unusual Fc gamma receptor IIIa (CD16) on natural killer cells in a patient with recurrent infections; de Vries E et al.; We found an unusual fc gamma receptor IIIa (CD16) phenotype on the natural killer (NK) cells of a 3-year-old boy, who suffered from recurrent viral respiratory tract infections since birth . He also had severe clinical problems after Bacille Calmette-Geerin (BCG) vaccination and following Epstein-Barr virus and Varicella Zoster virus infections . His peripheral blood lymphocytes contained a normal percentage and absolute number of CD3-CD7+ cells, which were positively stained with the CD16 monoclonal antibodies (MoAbs) 3G8 and CLBFcRgran1, but did marginally stain with the CD16 MoAb Lau11c/B73.1 . Fc gamma RillIb expression on granulocytes appeared to be normal . NK cell function, analyzed in vitro by direct cytotoxicity on K562 target cells and ADCC-activity on P815 target cells, was normal compared with an age-matched healthy control . Sequence analysis of the Fc gamma RIIIA gene, encoding CD16 on NK cells and macrophages, showed a T to A nucleotide substitution at position 230 on both alleles, predicting a leucine (L) to histidine (H) amino acid change position 48 in the first extracellular lg-like domain of Fc gamma RIIIa, which contains the Leu11c/B73.1 epitope . The combined use of CD16 and CD56 MoAbs labeled with the same fluorescent dye, as often applied in routine immunophenotyping procedures, will leave these homozygotes undiagnosed . The pattern of infections in this patient is in agreement with the postulated function of NK cells in the immunological defense against viruses and other intracellular microorganisms . Further analysis of the NK cell function in vitro and follow-up of the clinical course of Fc gamma RIIIA-48H/H homozygotes is required to ascertain whether this genotype is causally related to an NK cell immunodeficiency.

J Biol Chem, 1996 Oct 11, 271(41), 25220 - 6
Mutations at domain II, loop 3, of Bacillus thuringiensis CryIAa and CryIAb delta-endotoxins suggest loop 3 is involved in initial binding to lepidopteran midguts; Rajamohan F et al.; Alanine substitutions of loop 3 residues, 438SGFSNS443, of CryIAb toxin were constructed to study the functional role of these residues in receptor binding and toxicity to Manduca sexta and Heliothis virescens . Experiments with trypsin and insect gut juice enzyme digestions of mutant toxins showed that these mutations did not produce any gross structural changes to the toxin molecule . Bioassay data showed that mutant G439A (alanine substitution of residue Gly439) and F440A significantly reduced toxicity toward M . sexta and H . virescens . In contrast, mutants S438A, S441A, N442A, and S443A were similar or only marginally less toxic (2-3 times) to the insects compared to the wild-type toxin . Binding studies with brush border membrane vesicles prepared from M . sexta and H . virescens midgut membranes revealed that the loss of toxicity of mutants G439A and F440A was attributable to substantially reduced initial binding . Consistent with the initial binding, mutants G349A and F440A showed 3.5 times less binding to M . sexta and H . virescens brush border membrane vesicles, although the off-rate of bound toxins was not affected . The role of hydrophobic residue, Phe440, is distinctly different from our previous observation that alanine substitution of Phe371 at loop 2 of CryIAb did not affect initial binding but reduced irreversible association of the toxin to the receptor or membrane toward M . sexta (Rajamohan, F., Alcantara, E., Lee, M . K., Chen, X . J., and Dean, D . H . (1995) J . Bacteriol . 177, 2276-2282) . Likewise, deletion of relatively hydrophobic CryIAa loop 3 residues, 440AAGA443 (D3a), resulted in reduced toxicity to Bombyx mori (>62 times less) and M . sexta (28 times less) . The loss of toxicity was correlated with reduced initial binding to midgut vesicles prepared from these insects . However, alanine substitution of residues 437LSQ439 (A3a), contiguous to loop 3, altered neither toxicity nor receptor binding toward B . mori or M . sexta . These results suggest that the loop 3 residues of CryIAb and CryIAa toxins establish hydrophobic interactions with the receptor molecule, and mutations at these hydrophobic residues affect initial binding.

Acta Med Port, 1996 Oct-Dec, 9(10-12), 397 - 400
{The Calmette-Guérin bacillus (BCG) . An agent to consider in pediatric infection}; Marcelino F et al.; Complications occurring a long time after BCG vaccination in healthy children have been occasionally referred, despite being rare . Osteitis seems to be the most frequent of those complications; therefore it must be considered in the differential diagnosis of that sort of lesions . The authors report a case of a 15 month old boy, previously healthy, who suffered an enlargement of the right foot for 3 weeks, unresponsive to antibiotics . The lesion revealed to be osteitis due to bacillus Calmette-Guerin, and abated after specific treatment.

J Indian Med Assoc, 1996 Oct, 94(10), 381 - 4
Current concept in the diagnosis and treatment of childhood pulmonary tuberculosis; Singh UK et al.; PIP: In India, an estimated 80% of children are infected with tubercle bacillus by 10 years of age . Elimination of tuberculosis depends on finding all infectious patients and providing them with curative chemotherapy . Pulmonary tuberculosis--the most common form in children--is diagnosed when a child presents with fever, prolonged cough, weight loss, recurrent wheezing, or chest infection; the chest x-ray is suggestive of tuberculosis; and three or more of the following conditions exist: 1) Mantoux test result of 10 mm or more, 2) tuberculosis lymphadenitis by fine needle aspiration cytology, 3) grade III malnutrition, 4) no BCG vaccination, 5) positive family history of tuberculosis, and 6) recent history of pertussis or measles . Recommended, for children with pulmonary tuberculosis, is a regimen of isoniazid, rifampicin, and pyrazinamide daily for 2 months, followed by the first two drugs daily for an additional 4 months . The poor tuberculosis cure rates in most developing countries reflect patient non-compliance with treatment regimens .

Rev Argent Microbiol, 1996 Oct-Dec, 28(4), 170 - 4
Survival of Bacillus megaterium strains in water; Palmada FM et al.; Cultures of Bacillus megaterium strains, producers or not of poly-beta-hydroxy-butyrate (PHB+ and PHB-) were submitted to several shift-downs: nutritional (one hundred fold dilution in saline water S or artificial fresh water ADA) or nutritional and osmotic (one hundred fold dilution in water or W) . In all conditions tested, the wild type strain survived, duplicated five times and sporulated . However, the PHB- mutant strain showed a drastic loss of viability in water (< 0.1%) not observed when the shift was only nutritional (S or ADA) . Discussion was focused on the advantages of the potential use of Bacillus megaterium as host for delivering bio-insecticides in waters instead of natural hosts such as B . thuringiensis strains.

Indian J Lepr, 1996 Oct-Dec, 68(4), 349 - 61
Immunotherapy of leprosy; Katoch K; Immunotherapy aims to modify the defective cell-mediated immune response in a section of leprosy cases . This presentation reviews the various immunomodulators developed/ investigated for this purpose . Among the various mycobacterial agents, BCG, BCG + M.leprae, Mycobacterium w, ICRC bacillus and M.vaccae have been tried in leprosy patients and varying degree of beneficial effects on bacterial killing and clearance have been observed . Studies carried out at CJIL, Agra and elsewhere suggest an important role for these mycobacteria as immunotherapeutic agents . Other mycobacteria-M.habana, M.phlei, M.gordonae-have also been reported to be promising experimentally . In addition, various drugs such as levamisole, zinc and RACA 854 have been observed to have immunomodulatory role in leprosy cases . Other promising immunomudlators include transfer factor, interferon gamma, interleukin 2 and acetoacetylated M.leprae . The progress achieved shows that immunotherapy may be considered as adjunct to chemotherapy to enhance bacterial killing as well as bacterial clearance and thus may be recommended to shorten the treatment period, specially in bacilliferous leprosy cases.

Lett Appl Microbiol, 1996 Oct, 23(4), 249 - 52
Characterization of novel non-toxic Bacillus thuringiensis isolates from Korea; Roh JY et al.; Four Bacillus Thuringiensis isolates from soil samples produced parasporal inclusions which were non-toxic to insects . The isolates were named B . thuringiensis NTB-1, NTB-2, NTB-3 and NTB-4 . The parasporal inclusions were shown to be ovoid by phase contrast and scanning electron microscopy . The serotypes of the four isolates were determined by agglutination using 33 antisera; NTB-1 and NTB-4 seemed to be subsp . israelensis, and NTB-2 seemed to be subsp . pondicheriensis . NTB-3 did not react with the 33 antisera . However, comparison of parasporal protein and plasmid DNA patterns of the four isolates with those of 15 known non-toxic B . thuringiensis strains demonstrated that the four isolates are novel.

Biosci Biotechnol Biochem, 1996 Oct, 60(10), 1705 - 8
Chitinous component of the cell wall of Fusarium oxysporum, its structure deduced from chitosanase digestion; Fukamizo T et al.; The cell of Fusarium oxysporum was digested with commercial Bacillus chitosanase . The chitosanase produced low molecular weight heterooligosaccharides consisting of GlcN and GlcNAc from the cell wall . A main component of the digestion products was identified as 2-amino-2-deoxy-beta-D-glucopyranosyl- (1-->4)-2-acetamido-2-deoxy-D-glucopyranose . The chitosanase appeared to be more effective than Streptomyces griseus chitinase for cell wall digestion . Moreover, maltose was unexpectedly found in the digestion products, indicating that the cell wall contains alpha-1,4-linked glucan chain as a polysaccharide component.

Biosci Biotechnol Biochem, 1996 Oct, 60(10), 1655 - 9
Molecular cloning and expression of the pyrimidine nucleoside phosphorylase gene from Bacillus stearothermophilus TH 6-2; Okuyama K et al.; The pyrimidine nucleoside phosphorylase (Py-NPase) of Bacillus stearothermophilus TH 6-2 is a dimer of 46-kDa subunits and catalyzes the reversible phosphorolysis of uridine and thymidine . The gene encoding this pyrimidine nucleoside phosphorylase (pyn gene) has been cloned and sequenced from B . stearothermophilus TH 6-2 . The pyn gene corresponded to an open reading frame of 1299 nucleotides that translates into a putative 433 amino acid protein with a molecular weight of 46,271 . The deduced amino terminal sequence of Py-NPase coincided with that previously found for the purified enzyme . The deduced amino acid sequence of Py-NPase shared significant similarity with those of human and Escherichia coli thymidine phosphorylases . The cloned pyn gene was overexpressed in E . coli cells to produce an active enzyme in large quantities that accounted for approximately 20% of the total protein.

Biosci Biotechnol Biochem, 1996 Oct, 60(10), 1633 - 6
Molecular cloning and nucleotide sequence of the groEL gene from the alkaliphilic Bacillus sp . strain C-125 and reactivation of thermally inactivated alpha-glucosidase by recombinant GroEL; Xu Y et al.; The groEL gene of the alkaliphilic Bacillus sp . strain C-125 was cloned in Escherichia coli and sequenced . The groEL gene encoded a polypeptide of 544 amino acids and was preceded by the incomplete groES gene, lacking its 5'-end . The sequence of the derived amino acids was 87.5% identical to that of B . subtilis, 85.4% identical to that of B . stearothemophilus, and 60.9% identical to that of E . coli . The GroEL protein was expressed in E . coli . Purified GroEL protected yeast alpha-glucosidase from irreversible aggregation at a high temperature and the addition of Mg-ATP was essential for reactivation of the alpha-glucosidase . The addition of E . coli GroES increased recovery of the enzyme activity, indicating that C-125 GroEL could function in coordination with E . coli GroES.

Enzyme Microb Technol, 1996 Oct, 19(5), 367 - 73
Comparison of the properties of native and pentaammineruthenium(III)-modified xylanase; Evans BR et al.; Two xylanases, xynA of Bacillus pumilus and xyn II of Trichoderma reesei, were purified and then modified by the attachment of pentaammineruthenium, thereby resulting in the generation of a xylanase with veratryl alcohol oxidase activity . Hydrolytic activity of T . reesei xyn II on soluble xylans was unchanged by modification with pentaammineruthenium; however, modification of B . pumilus xynA greatly reduced xylan hydrolysis unless the active site of the xylanase was protected with xylose during the modification . The presence of histidine, cysteine, or reduced glutathione during xylan hydrolysis greatly increased the xylanase activity of the pentaammineruthenium-modified B . pumilus xylanase . Glycine, glutamic acid, methionine, or oxidized glutathione had no effect on xylanase activity.

Aust N Z J Public Health, 1996 Oct, 20(5), 521 - 4
BCG vaccination practices in Victoria; Altmann AE et al.; To determine the patterns of usage of bacille Calmette-Guerin (BCG) vaccine in Victoria and assess whether the vaccine was being administered to those in high-risk groups, as identified by the National Health and Medical Research Council, an audit of BCG vaccine unit sales and expenditure, over the past four years was conducted . A postal survey covering all registered Victorian BCG vaccinators inquired about BCG vaccination practices during 1993 . Vaccine sales and expenditure had nearly doubled since 1991 . The number of registered vaccinators had also increased . The survey response rate was 77 per cent, (228 of 295) . Half of the vaccinators were working in general practice, 11 per cent of vaccinators used no set guideline for client selection, 69 per cent vaccinated fewer than 25 people in 1993, 26 per cent had vaccinated neonates (mainly southeast Asian), with few of these vaccinations being carried out in maternity hospitals . Tertiary students and ethnic groups were the most commonly vaccinated groups . Only small amounts of BCG were being given to people outside risk groups, mainly travellers and anxious public . There has been significant increase in numbers of registered vaccinators and use of BCG with much wastage . Application of guidelines was inconsistent and coverage of high-risk groups varied . Despite some selection for vaccination by personal choice, little vaccine appeared to be used in nonrecommended groups . Subsequent changes in practice have resulted, including publicising and clarifying guidelines, reduction in the number of vaccinators, vaccinator upgrading courses, and restructuring of the vaccine ordering system.

Tuber Lung Dis, 1996 Oct, 77(5), 476 - 81
BCG vaccination in low birth weight newborns: analysis of lymphocyte proliferation, IL-2 generation and intradermal reaction to PPD; Ferreira AA et al.; OBJECTIVE: To study vaccinal scar formation and post-vaccinal immune response in newborns with birth weight ranging from 2000 to 2499 g vaccinated in the first week of life with intradermal bacille Calmette-Guerin (BCG) (Moreau-Rio de Janeiro strain) . METHOD: Specific immune response to PPD was assessed in 30 low birth weight newborns (mean birth weight = 2311.7 +/- 122.1 g; mean gestational age = 38.1 +/- 1.8 weeks) in comparison to 56 control infants (mean birth weight = 3198.9 +/- 267.2 g; mean gestational age = 38.5 +/- 1.2 weeks . RESULTS: Low birth weight infants have an efficient immune response to vaccinal stimulus when compared to control infants as judged by specific in vitro lymphocyte proliferation (mean SI = 9.7 +/- 12.9 vs SI = 8.8 +/- 10.0, P = 0.72) and IL-2 production (mean SI = 3.1 +/- 3.4 vs SI = 2.6 +/- 2.0, P = 0.38) . Intradermal reaction to PPD was also comparable in both groups (mean induration diameter = 9.5 +/- 5.1 mm vs 9.6 +/- 5.0 mm, P = 0.94) . CONCLUSION: These data suggest that low birth weight newborns show a good immune response to BCG, thus reinforcing the inclusion of such infants in regular vaccination programs with intradermal BCG.

Tuber Lung Dis, 1996 Oct, 77(5), 437 - 43
Effects of the human immunodeficiency virus on tuberculosis in children; Jeena PM et al.; SETTING: Human immunodeficiency virus (HIV) infection has altered the epidemiological and clinical profile of tuberculosis (TB) worldwide . In children however, unlike in adults, very little has been documented about the interaction between the two diseases . OBJECTIVE: To examine the clinical features and response to TB treatment in children with TB and HIV, and compare them with those with TB alone . DESIGN: A prospectively enrolled case study with systematically selected controls was conducted between 1992 and 1994 at King George V tuberculosis hospital, in Durban . Forty children with TB and HIV (Group A) were compared with 40 children with TB alone (Group B) . The diagnosis of TB was made in accordance with established criteria . Measures of comparison between the groups included: history of contact with a TB case, clinical presentation on admission, presence of bacille Calmette-Guerin (BCG) scar, reaction to tuberculin test, clinical response to anti-tuberculosis treatment (mean weight gain per month, improved appetite, resolution of chest signs, decreasing size of visceromegaly), radiological response to treatment (assessed according to an objective score on admission, at 6 months and on discharge), other associated diseases, nosocomial infections and survival . RESULTS: The mean age of the children in Group A was 25 months and in Group B 31 months . The clearest differences between the groups on admission were clinical features and response to tuberculin testing . Group A were more frequently anergic to tuberculin testing (P < 0.0001) and more often had symptoms and signs suggestive of TB (P = 0.002) . Clinical response to treatment on discharge was worse in Group A than in Group B (P = 0.005) . Radiological response to treatment at six months and on discharge was poorer in Group A than in Group B (P = 0.46; P = 0.006, respectively) . Six children in group A and none in group B died (P = 0.012) . The mean duration of treatment (and therefore period until discharge) was 8.9 months in Group B and 8.5 months in Group A for those who survived . History of contact, evidence of BCG inoculation and nosocomial infections were similar in both groups . CONCLUSION: HIV infection adversely affects the outcome of TB in children as assessed by response to treatment and survival.

Tuber Lung Dis, 1996 Oct, 77(5), 414 - 9
Tuberculosis risk after exposure on airplanes; Miller MA et al.; SETTING: Domestic and international air-flights . OBJECTIVE: To estimate the risk of tuberculosis (TB) transmission aboard aircraft . DESIGN: A contact investigation of passengers and crew from two flights was conducted following identification of a fellow passenger with pulmonary TB . Immediate post-exposure and follow-up tuberculin skin tests (TSTs) were obtained . RESULTS: Of 120 contacts, 86 (72%) had a negative TST (< 5 mm); 29 (24%) a positive TST (> or = 5 mm), and 5 (4%) a TST conversion . Of the 29 persons with a positive TST, 27 had other identified risk factors for TB . Risk factors for positive TST included non-US birth (Relative Risk (RR) 9.7 P < 0.01) or history of Bacille Calmette-Guerin (BCG) vaccination (RR undefined; P < 0.01) . Risk was not associated with specific aircraft or seat relative to the index case for US-born contacts . All five TST converters were born in countries where BCG vaccine is routinely given . CONCLUSION: The positive TST reactions and conversions suggest boosting from BCG vaccination or prior exposure in TB-endemic countries . Since two positive contacts had no other identified risk factor, TB transmission on board the aircraft could not be excluded . Contact investigation of exposed aircraft passengers should be considered on a case-by-case basis, with consideration of the infectiousness of the ill passenger and the flight circumstances.

Nihon Kyobu Shikkan Gakkai Zasshi, 1996 Oct, 34(10), 1140 - 4
{Infection with Mycobacterium avium presenting as polypoid lesions in left upper-lobe bronchus of an immunocompetent host}; Watanabe A et al.; In April 1993, a 51-year-old woman had a fever, and an infiltrative shadow was seen in the left upper lobe on a chest X-ray film . Repeated sputum cultures were positive for Mycobacterium avium complex . She underwent antituberculosis therapy consisting of pyrazinamide, ofloxacin, and streptomycin . Her symptom disappeared and the abnormal shadow resolved . In January 1994, she was admitted to the hospital because of bloody sputum and abnormal chest X-ray findings consisting of a left hilar mass and atelectasis of the left upper lobe . Bronchoscopy revealed multiple polypoid lesions without necrosis in the left upper-lobe bronchus . Histological examination showed that the tumor consisted of an aggregation of lymphocytes and plasma cells, and was positive for Ziehl-Neelsen stain . The acid-fast bacillus was identified as Mycobacterium avium by the DNA probe method . Anti-tuberculosis treatment was given: rifampicin, isoniazid, sparfloxacin, and clarithromycin . Three months later, the atelectasis and the polypoid mass in the left upper-lobe bronchus had disappeared . We believe that the polypoid lesions in the left upper-lobe bronchus were due to infection by Mycobacterium avium . The patient was HIV-negative and immunocompetent . Such endobronchial lesions caused by Mycobacterium avium are rare in HIV-negative hosts.

J Biochem (Tokyo), 1996 Oct, 120(4), 851 - 5
Convenient synthesis of beta-(1-->3)-galactosyl disaccharide alpha-glycoside and its analogs as mimic units of mucin-type carbohydrate; Murata T et al.; beta-Galactosidase from porcine testes induced regioselective transglycosylation from lactose to the 3-position of 2-acetamido glycosides . When alpha-D-GalNAc-OC6H4NO2-p was used as an acceptor, the enzyme synthesized mainly beta-D-Gal-(1-->3)-alpha-D-GalNAc-OC6H4NO2-p with its (1-->6) linked isomer . The use of an inclusion complex of the glycoside acceptor with beta-CD increased the efficiency of transglycosylation by increasing the solubility of the acceptor . In the same way, the use of beta-D-GalNAc-OC6H4NO2-p as acceptor led to the preferential synthesis of beta-D-Gal-(1-->3)-beta-D-GalNAc-OC6H4NO2-p over that of its (1-->6) linked isomer . beta-D-Gal-(1.3)-beta-D-GlcNAc-OC6H4NO2-p was also synthesized with beta-D-GlcNAc-OC6H4NO2-p acceptor by the consecutive use of beta-D-galactosidases from porcine testes and Bacillus circulans . These enzyme reactions are efficient enough to allow the one-pot preparation of the desired disaccharide glycosides.

J Vet Med Sci, 1996 Oct, 58(10), 1027 - 9
Mouse lethal activity of a HEp-2 vacuolation factor, cereulide, produced by Bacillus cereus isolated from vomiting-type food poisoning; Shinagawa K et al.; The HEp-2 vacuolation factor (or cereulide) produced by Bacillus cereus isolated from vomiting-type food poisoning, which is supposed to induce emesis, was found to give mouse and suncus lethality after intravenous and intraperitoneal administration . The emetic activity of the factor was also found to be resistant to heating at 121 degrees C for 15 min, exposure to pH 2 and 11, and to digestion with proteolytic enzymes such as pepsin and trypsin . These findings suggest that the cereulide produced by B . cereus is stable in the digestive tracts, induce emesis, and show lethal activity leading to cellular damage.

Immunol Cell Biol, 1996 Oct, 74(5), 401 - 7
Regulation of autoimmune diabetes: characteristics of non-islet-antigen specific therapies; Gazda LS et al.; Non-islet-antigen specific treatments have been shown to alter the natural history of insulin dependent diabetes in both the non-obese diabetic (NOD) mouse and in recently diagnosed patients . However concerns have been raised regarding the possibility that non-islet-antigen specific therapy may trade cell mediated autoimmunity for antibody dependent autoimmunity . Female NOD mice at approximately 70 days of age were treated with the non-islet-antigen specific agents complete Freund's adjuvant (CFA) and Bacillus Calmette-Guerin (BCG) and assayed for the development of antibody mediated autoimmunity at 300 days of age . Autoantibodies to red cells were not detected in any of the BCG (n = 19) or CFA (n = 15) treated animals, while 2 of 13 age-matched NOD animals had autoantibodies to red cells, shown by a positive direct Coomb's test . Anti-nuclear autoantibodies and complement deposition in the renal glomeruli were not significantly increased in the treated animals as compared to age-matched non-diabetic mice . The relative effectiveness of CFA and BCG treatment was examined in terms of the ability of these agents to preserve insulin containing islets . Complete Freund's adjuvant treatment was found to be more effective in preserving insulin containing islets when compared to BCG treatment . This study demonstrates that it is possible to inhibit the development of autoimmune diabetes without increasing the probability that treated animals will develop antibody dependent autoimmunity.

Int J Biol Macromol, 1996 Oct, 19(3), 165 - 9
Electron microscopic investigation of the diffusion of Bacillus licheniformis alpha-amylase into corn starch granules; Helbert W et al.; A method for the direct electron microscopic observation of amylases in interaction with starch granules is presented . The technique involves immuno-gold labeling of the enzymes and cross-sectioning of hydrated starch granules . This approach allows the analysis of the internal degradation of starch with a concomitant visualization of enzymes at the sites of hydrolysis . The visualization of enzymes at the surface, inside the channel and inside the core of the degraded granules shows that the alpha-amylase molecules first proceed from the surface toward the center (centripetal hydrolysis) . Then the core is completely degraded from within by erosion of its periphery (centrifugal hydrolysis) . In the first case (centripetal hydrolysis), the enzymes act by progressing along the polysaccharide chains . By contrast, the centrifugal hydrolysis leads to even erosion, indicative of a more diffusive motion of the enzymes.

Vet Hum Toxicol, 1996 Oct, 38(5), 333 - 6
Immunosuppressant activity of aflatoxin ingestion in rabbits measured by response to Mycobacterium bovis antigen I . Cell mediated immune response measured by skin test reaction; Dimitri RA et al.; Thirty New Zealand 2-mo-old rabbits were divided into 5 groups . Groups 1 and 2 were fed 2 ppm aflatoxin B1 daily for 5 w before sensitization and continued for another 7 w . Groups 1 and 3 were infected with Bacillus of Calmette and Guerin while groups 2 and 4 were sensitized with killed cells of Mycobacterium bovis . Group 5 did not receive any treatment (control) . Citrated whole blood samples were collected from all groups before treatment and at 5, 8 and 12 w during the experiment . The lymphoblastogensis assay was done presensitization and at 3 and 7 w post-sensitization for all groups . The lymphocyte stimulation indices and diameter of skin reactions were significantly reduced in the aflatoxin-treated groups (P < 0.001) . In addition, 3/12 rabbits (25%) from the aflatoxin groups (groups 1 and 2) failed to produce any detectable response to the tuberculin test . Aflatoxin inhibited lymphocyte proliferation and negatively influenced the tuberculin skin test.

J Gen Virol, 1996 Oct, 77 ( Pt 10), 2637 - 44
The nucleotide sequence of RNA1 and RNA2 of olive latent virus 2 and its relationships in the family Bromoviridae; Grieco F et al.; The complete nucleotide sequence of RNA1 and RNA2 of olive latent virus 2 (OLV-2), a virus with quasi-spherical to bacilliform particles and a non-polyadenylated tripartite ssRNA genome, was determined . RNA1 consists of 3126 nucleotides and contains a single open reading frame (ORF) coding for a polypeptide with a molecular mass of 102689 Da (p1a) . RNA2 is also a monocistronic molecule, 2734 nt in length, coding for a polypeptide with a molecular mass of 90631 Da (p2a) . The translation products of RNA1 and RNA2 possess the motifs proper to helicase, methyltransferase (RNA1) and RNA polymerase (RNA2), suggesting that both are involved in the replication of the viral RNA . The similarities found between OLV-2 and members of the Bromoviridae in some properties and in the sequences of all genomic products (including p1a and p2a) are strongly indicative that it belongs in this family . OLV-2, however, did not show a direct relationship with any of the current genera in the family . Rather, it revealed homologies in diverging directions with one or other of the Bromoviridae genus, thus qualifying as the possible representative of a new taxon in this family.

Br J Rheumatol, 1996 Oct, 35(10), 1008 - 10
Mycobacterium xenopi--an unusual presentation as tenosynovitis of the wrist in an immunocompetent patient; Coombes GM et al.; Mycobacterium xenopi is an atypical acid-fast bacillus which may colonize tap water supplies . It typically causes pulmonary infection, particularly in patients with pre-existing lung damage, and non-pulmonary involvement is rare . We describe the first reported case of tenosynovitis due to this organism in an immunocompetent male patient.

J Clin Oncol, 1996 Oct, 14(10), 2646 - 52
Overexpression of p53 protein in a high-risk population of patients with superficial bladder cancer before and after bacillus Calmette-Guérin therapy: correlation to clinical outcome; Lacombe L et al.; PURPOSE: We have previously demonstrated that p53 overexpression is predictive of disease progression and survival in Ta, Tis, and T1 tumors . Instillation of Bacillus Calmette-Guerin (BCG) is now accepted to be the most efficient adjuvant therapy for superficial bladder carcinoma . The aim of this study was to determine if p53 status, assessed before and after intravesical BCG therapy, can predict clinical outcome in a high-risk population of patients with superficial bladder carcinoma . MATERIALS AND METHODS: We examined 196 tissue specimens from 98 patients, obtained immediately before and after intravesical BCG therapy . The pretherapy population was composed of 22 Ta, 57 Tis, and 19 T1 tumors . After BCG, 66 specimens were TO and 32 had residual tumors . Nuclear p53 overexpression was analyzed in relation to time to disease progression and disease-specific survival . RESULTS: The median follow-up duration was 44 months . The detection of nuclear p53 overexpression before BCG therapy did not predict response to BCG therapy . Pre-BCG p53 protein overexpression, response to BCG therapy, and pre-BCG stage were all independent markers of disease progression . In patients with residual disease after BCG therapy (nonresponders), multivariate analysis confirmed that posttherapy p53 overexpression was the only independent marker of disease progression . CONCLUSION: In this high-risk population of patients with superficial bladder tumors, patients who have p53 nuclear overexpression in the tumor and stage T1 disease before BCG therapy are at high risk of disease progression . Furthermore, in the group of patients with residual disease after BCG therapy, p53 status is a better predictor of disease progression than post-BCG stage.

Am J Gastroenterol, 1996 Oct, 91(10), 2220 - 3
Bacillary angiomatosis associated with extensive esophageal polyposis: a new mucocutaneous manifestation of acquired immunodeficiency disease (AIDS); Chang AD et al.; Bacillary angiomatosis is a rare infection that has been associated with human immunodeficiency virus infection . The causative organism is Rochalimaea henselae and contact with cats is a risk factor . We present a case of a 37-yr-old man who had recent prolonged exposure to a cat and presented with fever, iron deficiency anemia, and guaiac-positive stools who had biopsy-proven bacillary angiomatosis skin lesions and on esophagogastroduodenoscopy had multiple, diffuse, friable, polypoid lesions in the esophagus . The histology of the esophageal polyps was identical to the skin lesions, and the polyps disappeared after treatment with erythromycin . Bacillary angiomatosis should be included in the differential diagnosis of infectious upper gastrointestinal manifestations associated with AIDS.

Obstet Gynecol, 1996 Oct, 88(4 Pt 2), 709 - 11
Bacillary angiomatosis of the cervix and vulva in a patient with AIDS; Long SR et al.; BACKGROUND: Bacillary angiomatosis is a clinicopathologic entity that most often is identified in the skin of patients with AIDS . This report presents an example of bacillary angiomatosis of the female genital tract . CASE: Bacillary angiomatosis presented as red-purple nodules of the vulva and cervix in a 32-year-old woman with AIDS . Histologic examination revealed the lobular epithelioid vascular proliferation and hazy clumps of bacteria that characterize bacillary angiomatosis . The diagnosis was confirmed on Warthin-Starry-stained issue and by blood cultures, which were positive for Bartonella (Rochalimaea) henselae . CONCLUSION: Accurate diagnosis of this infection is important because 1) bacillary angiomatosis is commonly mistaken for Kaposi sarcoma, 2) it is effectively treated with inexpensive antibiotics, and 3) undiagnosed and/or untreated bacillary angiomatosis may lead to overwhelming disseminated infection and death.

Biochemistry, 1996 Oct 1, 35(39), 12970 - 7
Expression and site-directed mutagenesis of the phosphatidylcholine-preferring phospholipase C of Bacillus cereus: probing the role of the active site Glu146; Martin SF et al.; A series of site-specific mutants of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) was prepared in which the glutamic acid residue at position 146 was replaced with glutamine, aspartic acid, histidine, and leucine to elucidate what role Glu146 might play in catalysis . An expression system for the native enzyme in Escherichia coli was first developed to provide PLCBc that was fused via an intervening factor Xa protease recognition sequence at its N-terminus to maltose binding protein (MBP) . This MBP-PLCBc fusion protein was isolated at levels of 50-70 mg/L of culture; selective trypsin digestion of the MBP-PLCBc fusion protein followed by chromatographic purification yielded recombinant PLCBc at levels of ca . 10 mg/L . Polymerase chain reaction (PCR) mutagenesis on the PLCBc gene (plc) was then used to replace the Glu146 codon with those for glutamine (E146Q), aspartic acid (E146D), histidine (E146H), and leucine (E146L) . The catalytic efficiency of the E146Q mutant was 1.6% that of native PLCBc, while the other mutants each possessed activities of 0.2-0.3% of the wild type . The kcat/Km vs pH profiles for both E146Q and native PLCBc have ascending acidic limbs, suggesting that Glu146 does not serve as the general base in the hydrolysis reaction . As measured by circular dichroism, all of the mutant proteins contained less helical structure and underwent denaturation at lower temperatures than the wild type in the order: wild type > E146Q > E146D approximately E146H approximately E146L . Atomic absorption analyses indicated that the mutant proteins also exhibited lower Zn2+ content than the wild type . Thus, the Glu146 residue in PLCBc stabilizes the secondary and tertiary structure of the enzyme and serves as a critical ligand for Zn2, but it does not appear to have any specific catalytic role.

J Bacteriol, 1996 Oct, 178(19), 5602 - 9
Evidence that an N-terminal S-layer protein fragment triggers the release of a cell-associated high-molecular-weight amylase in Bacillus stearothermophilus ATCC 12980; Egelseer EM et al.; During growth on starch medium, the S-layer-carrying Bacillus stearothermophilus ATCC 12980 and an S-layer-deficient variant each secreted three amylases, with identical molecular weights of 58,000, 122,000, and 184,000, into the culture fluid . Only the high-molecular-weight amylase (hmwA) was also identified as cell associated . Extraction and reassociation experiments showed that the hmwA had a high-level affinity to the peptidoglycan-containing layer and to the S-layer surface, but the interactions with the peptidoglycan-containing layer were stronger than those with the S-layer surface . For the S-layer-deficient variant, no changes in the amount of cell-associated and free hmwA could be observed during growth on starch medium, while for the S-layer-carrying strain, cell association of the hmwA strongly depended on the growth phase of the cells . The maximum amount of cell-associated hmwA was observed 3 h after inoculation, which corresponded to early exponential growth . The steady decrease in cell-associated hmwA during continued growth correlated with the appearance and the increasing intensity of a protein with an apparent molecular weight of 60,000 on sodium dodecyl sulfate gels . This protein had a high-level affinity to the peptidoglycan-containing layer and was identified as an N-terminal S-layer protein fragment which did not result from proteolytic cleavage of the whole S-layer protein but seems to be a truncated copy of the S-layer protein which is coexpressed with the hmwA under certain culture conditions . During growth on starch medium, the N-terminal S-layer protein fragment was integrated into the S-layer lattice, which led to the loss of its regular structure over a wide range and to the loss of amylase binding sites . Results obtained in the present study provide evidence that the N-terminal part of the S-layer protein is responsible for the anchoring of the subunits to the peptidoglycan-containing layer, while the surface-located C-terminal half could function as a binding site for the hmwA.

Radiology, 1996 Oct, 201(1), 43 - 4
Miliary lung disease after intravesical bacillus Calmette-Guérin immunotherapy; Jasmer RM et al.; Clinical and radiologic findings in a 73-year-old man who developed a systemic illness while receiving intravesical bacillus Calmette-Guerin (BCG) immunotherapy for bladder cancer are presented . Thin-section chest computed tomographic findings included a diffuse pattern of small nodules consistent with miliary disease . Potential mechanisms explaining the pulmonary disease resulting from intravesical BCG treatment include a hypersensitivity reaction or actual BCG infection of the lungs.

J Biol Chem, 1996 Sep 27, 271(39), 24075 - 83
Amino acid sequence and molecular structure of an alkaline amylopullulanase from Bacillus that hydrolyzes alpha-1,4 and alpha-1,6 linkages in polysaccharides at different active sites; Hatada Y et al.; An amylopullulanase from alkalophilic Bacillus sp . KSM-1378 hydrolyzes both alpha-1,6 linkages in pullulan and alpha-1,4 linkages in other polysaccharides, with maximum activity in each case at an alkaline pH, to generate oligosaccharides (Ara, K., Saeki, K., Igarashi, K., Takaiwa, M., Uemura, T., Hagihara, H., Kawai, S., and Ito, S . (1995) Biochim . Biophys . Acta 1243, 315-324) . Here, we report the molecular cloning and sequencing of the gene for and the structure of this enzyme and show that its dual hydrolytic activities are associated with two independent active sites . The structural gene contained a single, long open reading frame of 5,814 base pairs, corresponding to 1,938 amino acids that included a signal peptide of 32 amino acids . The molecular mass of the extracellular mature enzyme (Glu33 through Leu1938) was calculated to be 211,450 Da, a value close to the 210 kDa determined for the amylopullulanase produced by Bacillus sp . KSM-1378 . The amylase and the pullulanase domains were located in the amino-terminal half and in the carboxyl-terminal half of the enzyme, respectively, being separated by a tandem repeat of a sequence of 35 amino acids . Four regions, designated I, II, III, and IV, were highly conserved in each catalytic domain, and they included a putative catalytic triad Asp550-Glu579-Asp645 for the amylase activity and Asp1464-Glu1493-Asp1581 for the pullulanase activity . The purified enzyme was rotary shadowed at a low angle and observed by transmission electron microscopy; it appeared to be a "castanet-like" or "bent dumbbell-like" molecule with a diameter of approximately 25 nm.

Biochemistry, 1996 Sep 24, 35(38), 12549 - 59
Effects of NAD+ binding on the luminescence of tryptophans 84 and 310 of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus; Gabellieri E et al.; The individual fluorescence and phosphorescence properties of W84 and W310 in Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase were identified through the construction of a single tryptophan mutant (W84F) and by comparison of the emission between mutant and wild-type enzymes . The results show that the luminescence of W310 is red-shifted and substantially quenched relative to that of W84 . It displays an average subnanosecond fluorescence lifetime (tau F) and a very short, 50 microseconds, room-temperature phosphorescence (RTP) lifetime (tau P) . The perturbation of W310 luminescence is believed to arise from a stacking interaction with Y283 . In contrast, W84 exhibits a fluorescence lifetime tau F of several nanoseconds and a long-lived phosphorescence lifetime tau P, typical of buried, unperturbed TrP residues . NAD+ binding to the tetrameric enzyme causes a 55% reduction of W310 fluorescence intensity together with a nearly complete quenching of its low-temperature phosphorescence . W84, which is located far from the nicotinamide moiety of NAD+, is much less affected by the binding of the coenzyme; the reduction in fluorescence intensity is 35%, and its phosphorescence intensity is unchanged . Another consequence of NAD+ binding is a significant decrease of the RTP lifetime tau P of W84, manifesting thereby a conformational change in the region of the coenzyme-binding domain . However, no change is observed in the RTP lifetime tau P of W310 located in the catalytic domain . These findings and those obtained at partial coenzyme saturation support the conclusions derived from high-resolution crystallographic structures {Skarzynski, T., & Wonacott, A . J., (1988) J . Mol . Biol . 203, 1097-1118} that the NAD(+)-induced conformational change is sequential and that subtle rearrangement in the structure of unligated subunits might be responsible for the negative cooperative behavior of NAD+ binding.

Nucleic Acids Res, 1996 Sep 15, 24(18), 3590 - 2
BaeI, another unusual BcgI-like restriction endonuclease; Sears LE et al.; BcgI and BcgI-like restriction endonucleases have a very distinct characteristic which causes them to differ from the other classified restriction enzymes; they all cleave double-stranded DNA specifically on both sides of the recognition sequence to excise a short DNA fragment including the recognition sites . Here we report a new BcgI-like restriction endonuclease, BaeI, isolated from Bacillus sphaericus . Like BcgI, BaeI also cleaves double-stranded DNA on both strands upstream and downstream of its recognition sequence (10/15)ACNNNNGTAYC(12/7) . There are two dominant polypeptides in the final preparation of BaeI with molecular masses of approximately 80 and 55 kDa . Both are slightly larger than the two BcgI subunits . BaeI requires both Mg2+ and AdoMet to cleave DNA . Accompanying bilateral cleavage activity, the heteromeric BaeI also has an N6-adenine methyltransferase activity which modifies the symmetrically located adenines within its recognition sequence.

J Immunol Methods, 1996 Sep 13, 196(1), 33 - 9
Development of an immunoradiometric assay for quantitative determination of CrylA(b) protein in transgenic sugarcane plants; Vazquez RI et al.; An immunoradiometric assay (IRMA) system was performed to quantify the recombinant CrylA(b) protein produced by transgenic sugarcane lines . The method allowed detection of 0.1-1 ng CrylA(b) per 25 micrograms of soluble protein in leaf extracts from plants transformed with an expression vector containing a truncated version of the CrylA(b) gene from Bacillus thuringiensis . The technique was based upon the use of radioiodinated immunopurified antibodies specific to natural CrylA proteins in a one-step sandwich procedure by direct simultaneous incubation of the leaf extracts with the detecting antibody solution . This IRMA system provides a simple routine method to quantify the CrylA proteins in transgenic plants with different expression levels . We suggest that the methodology presented herein may become an efficient tool to quantify heterologous or native plant proteins, present at low levels in tissue extracts.

Biochem Biophys Res Commun, 1996 Sep 4, 226(1), 8 - 14
Substitution of residues on the proximal side of Cry1A Bacillus thuringiensis delta-endotoxins affects irreversible binding to Manduca sexta midgut membrane; Hussain SR et al.; Substitution of a positively charged residue (R93F) or addition of a negatively charged residue (A92D) at the N-terminal of alpha 3 helix of domain I of the Cry1Ac delta-endotoxin resulted in a substantial reduction in toxicity against Manduca sexta . The N-terminal residues of helix 3 are considered to be on the same (proximal) surface of the toxin as the loops in domain II which are involved in the binding of the toxin to the receptor . The loss of toxicity was not caused by a decrease in the initial binding but rather by reduced irreversible binding . Only 65 and 75% of the A92D and R93F mutant toxin, respectively, bound to midgut vesicles irreversibly, compared to 94% of the wild type toxin . On the other hand, replacing A119 in a loop on the distal side of the helices with negatively charged residues (A119D or A119E) did not affect toxicity or irreversible binding.

Biochemistry, 1996 Sep 3, 35(35), 11355 - 60
Membrane permeabilization induced by cytolytic delta-endotoxin CytA from Bacillus thuringiensis var . israelensis; Butko P et al.; CytA is a member of a functionally defined family of insecticidal delta-endotoxins occurring in parasporal crystals of Bacillus thuringiensis var . israelensis . We investigated the ability of CytA to permeabilize the membrane and release fluorescence marker molecules from unilamellar lipid vesicles . Both protoxin (27 kDa) and proteolytically activated toxin (24 kDa) were very effective in permeabilization of unilamellar lipid vesicles: concentrations as low as several nanomolar produced a significant effect . The toxin was about 2-3 times more effective than the protoxin . The concentration of CytA required for the same extent of calcein release in large unilamellar vesicles (LUV) was 5-10 times lower than that in small unilamellar vesicles (SUV) . Both small (calcein) and large (fluorescein-dextrans, MW 3000 and 10 000) molecules were released from the vesicles by CytA with comparable single-exponential kinetics . The release was an all-or-none event, i.e., each vesicle either released all of its contents or remained completely intact . Binding of CytA to lipid membranes did not show appreciable cooperativity, the apparent binding constant (Kapp) being on the order of 10(5) M-1 . The plots of kinetics of release vs bound protein/ lipid ratio and the differential effects of CytA on LUV vs SUV indicate that at least 140 toxin molecules or 311 protoxin molecules must bind to an LUV before the latter starts losing its integrity . The necessity of adsorption of this relatively large number of toxin molecules to trigger permeabilization, together with the lack of discrimination in the size of the released marker molecules, suggests that the effect of CytA is a general, detergent-like, perturbation of the membrane rather than creation of small, well-defined, proteinaceous channels.

Southeast Asian J Trop Med Public Health, 1996 Sep, 27(3), 628 - 32
Microdroplet application of mosquitocidal Bacillus thuringiensis using ultra-low-volume generator for the control of mosquitos; Seleena P et al.; In an effort to develop a more effective technique in dispersing a microbial control agent, Bacillus thuringiensis (Bt), a truck-mounted ultra low volume (ULV) generator (Scorpion) was used to disperse B . thuringiensis israelensis (Bti) and Bti with malathion . Complete larval and adult mortalities for all tested mosquito species within the first 70-80 feet from the ULV generator were achieved . Beyond that distance less than 50% mortality was achieved as insufficient sprayed particles reached the area . A minimum of 10(3) Bti colony forming units per ml is required to cause 100% larval mortality . The sprayed Bti larvicidal toxins were persistent in the test water 7 days post ULV . The effectiveness of B . thuringiensis jegathesan (Btj), a new mosquitocidal Bt serotype was also evaluated . Similar mortality results as Bti were achieved except that the Btj toxins underwent degradation in the test water, since less than 50% less in larval mortality was observed in 7 days post ULV samples . This ULV method has the potential to disperse Bt and malathion effectively for a simultaneous control of mosquito adults and larvae.

Southeast Asian J Trop Med Public Health, 1996 Sep, 27(3), 622 - 7
Efficacy of Bacillus sphaericus in different breeding habitats of Culex quinquefasciatus; Gunasekaran K et al.; 'Spherifix', an alginate based slow release formulation of Bacillus sphaericus was field tested in different types of breeding habitats of Culex quinquefasciatus at the dose of 15 kg ai/ha at bimonthly interval . The efficacy of the formulation was higher in most of the months except in rainy and post-rainy months . The mean percentage reduction +/-SD during the treatment phase of one year was 31.2 +/- 17.9, 50 +/- 29.4, 28.3 +/- 17.6, 30.3 +/- 21.1, 66 +/- 22.5 and 53 +/- 20.4 in larval density and 49 +/- 20.8, 65.1 +/- 26.1, 30.3 +/- 21.9, 59.8 +/- 22.6, 63.1 +/- 21.9 and 47.7 +/- 24.2 in pupal density respectively in cement tanks, cesspools, cesspits, disused wells, unlined drains and cement lined drains . The reduction in immature density was relatively higher in undisturbed, debris free and shallow habitats such as cesspools, unlined drains and cement lined drains . After withdrawal of treatment, the effect of the formulation could be seen for a period of four months.

Mikrobiologiia, 1996 Sep-Oct, 65(5), 599 - 606
{Effect of culture medium components on the accumulation of extracellular bacillary ribonucleases in culture fluid of recombinant strains of Escherichia coli}; Gabdrakhmanova LA et al.; The effect of the concentrations of peptone, yeast extract, and inorganic phosphate on the expression of genes of extracellular ribonucleases from Bacillus intermedius 7P (binase) and Bacillus amyloliquefaciens H2 (barnase) was studied in Escherichia coli cells transformed with plasmids containing the structural genes of binase or barnase under the control of their own or synthetic regulatory region or the structural binase gene under the control of the regulatory regions of the genes of barnase or Bacillus pumilus RNase . Inorganic phosphate inhibited the expression of the binase gene under the control of its own regulatory region or the regulatory region of the RNase Bp gene . In all other cases, inorganic phosphate produced no effect on the synthesis of RNases by E . coli cells . This difference in the effects of phosphate may be due to the presence of a nucleotide sequence similar to the E . coli Pho box in the promoters of the binase and RNase Bp genes and the absence of this sequence in the barnase gene promoter . It was shown that high peptone and yeast extract concentrations in the cultivation medium are required for good growth of the recombinant E . coli strains and the biosynthesis of RNases.

Rev Sci Tech, 1996 Sep, 15(3), 1061 - 73
Cat-scratch disease and bacillary angiomatosis; Chomel BB; Cat-scratch disease (CSD) was first described by Debre in 1950, yet the causative bacterial agent of CSD remained obscure until 1992, when Bartonella (formerly Rochalimaea) henselae was implicated in CSD by serological and microbiological studies . B . henselae had initially been linked to bacillary angiomatosis (BA), a vascular proliferative disease most commonly associated with long-standing human immunodeficiency virus (HIV) infection or other significant immunosuppression . B . henselae has also been associated with bacillary peliosis, relapsing bacteraemia and endocarditis in humans . Cats are healthy carriers of B . henselae, and can be bacteraemic for months or years . It has recently been demonstrated that B . henselae can be transmitted from cat to cat by the cat flea, but not by direct contact between animals . The author discusses the present state of knowledge on the aetiology, clinical features and epidemiological characteristics of cat-scratch disease and bacillary angiomatosis.

J Indian Med Assoc, 1996 Sep, 94(9), 338 - 40
Effectiveness of bacillus of Calmette-Guerin (BCG) vaccination against tuberculous meningitis: a case-control study; Zodpey SP et al.; A hospital-based pair-matched case-control study was carried out at Government Medical College Hospital, Nagpur to estimate the effectiveness of bacillus of Calmette-Guerin (BCG) vaccination against tuberculous meningitis . The study included 92 cases of tuberculous meningitis in the age group of 0-12 years and equal number of controls, matched for age, sex and socio-economic status . The protective effectiveness and prevented fraction were higher for the subjects in the age group of 0-6 years, males and subjects from upper strata of socio-economic class . The overall vaccine effectiveness and prevented fraction were estimated to be 86.54% (70.38-93.88%) and 65.54% (39.22-80.64%) respectively . Results of this study thus indicated that BCG vaccination was highly effective against tuberculous meningitis and played significant role in its prevention, in this population.

J Indian Med Assoc, 1996 Sep, 94(9), 331 - 3
Detection of HIV infection in pulmonary tuberculosis patients; Samuel NM et al.; Well documented 112 pulmonary tuberculosis patients were studied for the prevalence of human immunodeficiency virus (HIV) seropositivity by using two antibody screening tests along with western blot test . Nineteen of the pulmonary tuberculosis patients were HIV seropositive, 12 were acid-fast bacillus smear positive; 12 patients were tuberculin skin test positive and 15 patients were culture positive . As the incidence of HIV infection is increasing in India, it is observed that patients co-infected with HIV and TB are also on the rise . Recognition of the dual infection and taking adequate steps to deal with this epidemic are neededPIP: Infection with HIV depresses cell-mediated immunity by selectively depleting the CD4 T cells . That process increases the HIV-infected individual's susceptibility to other infections such as Myco tuberculosis . The World Health Organization estimates that more than 4 million people worldwide have been infected with HIV and Myco tuberculosis . 35 patients newly diagnosed with pulmonary tuberculosis (TB) at the Government Thiruvotteswarar Hospital of Thoracic Medicine in Madras and 77 at the Institute of Thoracic Medicine were tested for infection with HIV using particle agglutination, ELISA, and Western blot . 19 were HIV positive, 12 were acid-fast bacillus smear positive, 12 were tuberculin skin test positive, and 15 were culture positive . As the incidence of HIV infection increases in India, so does the number of patients coinfected with HIV and TB . Health personnel need to recognize such dual infection and take the proper steps to manage the epidemic .

Zentralbl Veterinarmed B, 1996 Sep, 43(7), 421 - 8
{The detection of toxinogenic Bacillus cereus strains}; Seidel KE et al.; Investigations into optimal culture conditions for Bacillus cereus in order to detect bacterial toxins in a cell culture system showed that Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum is the medium best suited for this purpose . The highest toxicity levels were seen when bacteria were cultured at 21 degrees C . In 42 of 43 Bacillus cereus-strains isolated from milk and milk products, toxin formation was detected using the MTT-test . In 11 strains, toxin formation was even shown when bacteria were cultured at 4 degrees C . When culture supernatants were examined in a commercially available ELISA, all cytotoxic strains were shown to form diarrheal toxin.

Prikl Biokhim Mikrobiol, 1996 Sep-Oct, 32(5), 554 - 6
{Influence of Bacillus intermedius RNAse on growth-stimulating and antagonistic characteristics of Trichoderma harzianum}; Egorov SI et al.; Bacillus intermedius RNAase (with specific activity of 1,000,000 units per one mg of protein) at concentration of 1 x 10(-3) mg/ml was shown to increase antagonistic and growth-stimulating properties of Trichoderma harzianum . An application of trichodermin which was treated with an enzyme enhanced cucumber crop capacity by 15-18% in industrial conditions.

Appl Microbiol Biotechnol, 1996 Sep, 46(2), 149 - 55
A cellulase gene from a new alkalophilic Bacillus sp . (strain N186-1) . Its cloning, nucleotide sequence and expression in Escherichia coli; Sanchez-Torres J et al.; Several alkalophilic Bacillus spp . strains were selected for their capacity to produce alkaline cellulases . Culture supernatants of these strains showed optimal cellulase activities between pH 8 and 9 and they were stable from pH 6 to pH 12 . A cellulase gene (celB1) from the alkalophilic Bacillus sp . strain N186-1 was cloned in Escherichia coli using polymerase chain reaction techniques . The cloned gene was present in a 2.539-bp HindIII fragment and its nucleotide sequence was determined . The coding sequence showed an open-reading frame encoding 389 amino acids . The amino acid sequence, deduced from the nucleotide sequence, permitted us to include it in family 5 (or A) of the glycosyl hydrolases . The complete open-reading frame of celB1 was cloned in the plasmid pET-11d and expressed in E . coli BL21 (DE3), in which a protein of 39 kDa was obtained in the cytoplasm; however, no endoglucanase activity was detected . A second construction in pET-12a allowed the production of a 39-kDa protein located in the periplasmic space of E . coli that had endoglucanase activity . The protein produced has optimal activity at pH 7 and 50 degrees C and it retains more than 70% of its activity after incubation for 1 h at pH 12.

Biosci Biotechnol Biochem, 1996 Sep, 60(9), 1524 - 5
Simple detection of xylosidase activity in single colonies, using 1-naphthyl-beta-D-xylopyranoside; Taguchi H et al.; Since the xylosidase of Bacillus pumilus hydrolyzed 1-naphthyl-beta-D-xylopyranoside (naphthyl-X) to produce xylose and 1-naphthol and a chromogenic azo compound is produced by coupling 1-naphthol and Fast Blue Salt B, a simple method for detection of xylosidase activity in single colonies was studied . Escherichia coli JM109 carrying the xylosidase gene of B . pumilus was cultivated at 37 degrees C for 18 h on a LB plate containing 0.5 mg/ml naphthyl-X, and then the plate was overlaid with 3 ml of a top layer containing 24 mg of agar and 6 mg of Fast Blue Salt B . After incubation of the plate at 37 degrees C for 1 h, each colony became reddish-brown . Even a small colony with xylosidase on the plate was easily distinguished from colonies without the enzyme.

Biosci Biotechnol Biochem, 1996 Sep, 60(9), 1483 - 5
Expression of 135-kDa insecticidal protein gene from Bacillus thuringiensis in the yeast Saccharomyces cerevisiae; Watanabe K et al.; Bacillus thuringiensis subsp . aizawai produces 130-kDa and 135-kDa (CryIA(a)) insecticidal proteins . When Saccharomyces cerevisiae was transformed by the vector carrying a cryIA(a) gene, the gene expression could not be observed . When the 5'-upstream region from the initiation codon was removed using a synthetic oligonucleotide, the CryIA(a) protein was successfully synthesized in yeast . The yeast extract containing CryIA(a) protein had insecticidal activity against Plutella xylostella larvae.

Biosci Biotechnol Biochem, 1996 Sep, 60(9), 1410 - 2
Accumulation of benzoic acid in suspension cultured cells of Pinus thunbergii Parl . in response to phenylacetic acid administration; Kawazu K et al.; The generation and accumulation of both benzoic acid (BA) and its conjugates were induced in suspension cultured cells of Pinus thunbergii by administering either phenylacetic acid (PA), a toxic metabolite of Bacillus cereus (strain HY-3) accompanying the pine wood nematode, or a lyophilized culture supernatant of this bacterium . BA conjugates reached their maximal levels in quantity two days after the administration and then decreased gradually until the 14th day, while BA increased significantly throughout this period . This pattern is similar to that in 3-year-old pine trees treated with PA, suggesting that the pathological reaction of pine tissues to the PA toxin might be involved in the pathogenesis mechanism for the pine wilt disease.

PDA J Pharm Sci Technol, 1996 Sep-Oct, 50(5), 324 - 5
Antimicrobial resistance of three species of Bacillus to thirty various antimicrobial agents; Moldenhauer JE et al.; Three frequently used strains of Bacillus were tested using antimicrobial susceptibility test discs to determine their resistance to various antimicrobial drug agents . The organisms selected were B . subtilis ATCC 6633 (used for USP B and F tests), B . stearothermophilus ATCC 12980 (used as a biological indicator for steam sterilization) and B . coagulans ATCC 51232 (used as a biological indicator for steam sterilization) . The results indicated that for the thirty antimicrobial drug agents tested, the only organisms which exhibited any resistance to these agents was B . subtilis, and it only showed resistance to two drugs (Aztreonam and Bacitracin).

J Immunother Emphasis Tumor Immunol, 1996 Sep, 19(5), 364 - 74
A phase I randomized study of subcutaneous adjuvant IL-2 in combination with an autologous tumor vaccine in patients with advanced renal cell carcinoma; Fenton RG et al.; We performed a prospective, randomized study to determine whether subcutaneous administration of interleukin-2 (IL-2) in combination with an autologous renal cell vaccine is feasible and can potentiate antitumor immunity . Seventeen patients with metastatic renal cell carcinoma underwent surgical resection with preparation of an autologous tumor cell vaccine . Patients were vaccinated intradermally twice at weakly intervals with 10(7) irradiated tumor cells plus bacillus Calmette-Guerin, and once with 10(7) tumor cells alone . Patients were randomized to one of three groups: no adjuvant IL-2, low-dose IL-2 (1.2 x 10(6) IU/m2), or high-dose IL-2 (1.2 x 10(7) IU/m2) . IL-2 was administered subcutaneously on the day of vaccination and the subsequent 4 days . Immune response was monitored by delayed-type hypersensitivity (DTH) response to tumor cells as compared with normal autologous renal cells . Sixteen of 17 patients received vaccine therapy . Four patients developed cellular immunity specific for autologous tumor cells as measured by DTH responses; two had received no IL-2 and two had received high-dose IL-2 . There were two partial responses (PR) noted, both in patients who received high-dose IL-2 . One responding patient was DTH(+) and one was negative . A third patient who was DTH(+) after vaccination with no IL-2 had a dramatic PR after receiving IL-2 subcutaneously in a subsequent protocol . Prospective testing of response to recall antigens indicated that only 5 of 12 tested patients were positive, including both clinical responders . These data suggest that subcutaneously administered adjuvant IL-2 does not dramatically augment the immunologic response to autologous renal cell vaccines as determined by the development of tumor-specific DTH response.

Nippon Koshu Eisei Zasshi, 1996 Sep, 43(9), 815 - 23
{Characteristics of pulmonary tuberculosis patients by mode of detection--comparison of patients detected between 1993-94 with those detected between 1986-88}; Toyota M et al.; In a previous study it was concluded that pulmonary tuberculosis (TB) patients who were detected at a hospital, excluding those detected by mass screening (group A), should be classified into three distinct groups . Namely some patients were detected only after the onset of TB symptoms (group B), with the others being treated for other diseases before being diagnosed as having TB (group C) . In group C, some patients were detected because of manifesting additional symptoms related to TB such as cough (group C-1), while the remaining patients were detected by chance while being examined for other diseases (group C-2) . Four groups respectively had specific characteristics which differentiated each other . The aim of this study is to elucidate changes of proportion and characteristics of group C compared to the former results . Pulmonary TB patients, who were registered between 1993 and 1994, in the area served by Kochi Prefectural Chuo Health Center (N = 332) were compared with those registered between 1986 and 1988 in the area . The results were as follows . 1) The proportion of group C was significantly higher in the period of 1993-94 than during 1986-88 . The proportion of elderly people in group C was higher in the period of 1993-1994 than in 1986-88 . The proportion of group C is projected to continue increasing as the proportion of elderly among the population increases . 2) The proportion of smear positive cases by sputum was lower in group C-2 than in group B and group C-1 . The proportion of either inactive TB or non TB cases was estimated to be higher in group C-2 than in group B and group C-1 . 3) In group C-1, the proportion of bacillus positive cases was higher in the period of 1993-94 than 1986-88 . The elongation of the interval between the onset of symptoms and diagnosis (total delay) was related to the severity of bacillus finding.

J Assoc Nurses AIDS Care, 1996 Sep-Oct, 7(5), 37 - 42
Bartonella infections and HIV disease; Lindauer A; Successful assessment and treatment of Bartonella in HIV-seropositive people depends on nursing's fundamental role in the management of these bacterial infections . Bartonella species are responsible for a variety of infections, including cat scratch disease and bacillary angiomatosis, which can be debilitating to people living with AIDS . This paper provides an overview of the clinical presentation and nursing management of Bartonella infection in PLWAs . The author discusses common diagnostic procedures, treatment strategies, and the nurse's role in caring for patients with a Bartonella infection.

J Am Mosq Control Assoc, 1996 Sep, 12(3 Pt 1), 499 - 502
Effects of three larvicides on the production of Aedes albopictus based on removal of pupal exuviae; Becnel JJ et al.; The production of adult Aedes albopictus from tires in northcentral Florida was monitored for 169 days by the daily removal of pupal exuviae . More than twice as many adults emerged from tires located in the shade (1.74 adults/tire/day) compared to tires in the sun (0.64 adults/tire/day) . The effect of 3 larvicides on the production of adult Ae albopictus was evaluated . The fungal pathogen Lagenidium giganteum was ineffective . A liquid formulation of Bacillus thuringiensis israelensis (Acrobe) provided significant control for 47 days, whereas a slow-release pellet formulation of the insect growth regulator methoprene (Altosid) provided almost complete control for 116 days.

J Am Mosq Control Assoc, 1996 Sep, 12(3 Pt 1), 409 - 13
Control of Culex quinquefasciatus with Bacillus sphaericus in Vasco City, Goa; Kumar A et al.; In a locality (Sada) of Vasco City, Goa, India, that was highly infested with mosquitoes, weekly spraying of Bacillus sphaericus (strain 101, serotype H 5a 5b) at the rate of 1 g/m2 in polluted water habitats, viz, surface drains, cess pits, cess pools, and septic tanks, resulted in a sharp decline in the immature and adult populations of Culex quinquefasciatus . The per man-hour adult densities, percent habitat positivity, and immature densities were significantly lower (P < 0.001) in the treated area compared to the control area through out the study period.

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