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 nu