Bacillus

J Biol Chem, 1992 Nov 5, 267(31), 22108 - 14
Catalytic properties of the cloned amylase from Bacillus licheniformis; Kim IC et al.; A gene encoding a new amylolytic enzyme of Bacillus licheniformis (BLMA) has been cloned, and we characterized the enzyme expressed in Escherichia coli . The genomic DNA of B . licheniformis was double-digested with EcoRI and BamHI and ligated the pBR322 . The transformed E . coli was selected by its amylolytic activity, which carries the recombinant plasmid pIJ322 containing a 3.5-kilobase fragment of B . licheniformis DNA . The purified enzyme encoded by pIJ322 was capable of hydrolyzing pullulan and cyclodextrin as well as starch . It was active over a pH range of 6-8 and its optimum temperature was 50 degrees C . The molecular weight of the enzyme was 64,000, and the isoelectric point was 5.4 . It degraded soluble starch by cleaving maltose units preferentially but did not attack alpha-1,6-linkage . The enzyme also hydrolyzed pullulan to panose units exclusively . In the presence of glucose, however, it transferred the panosyl moiety to glucose with the formation of alpha-1,6-linkage . The specificity of transferring activity is evident from the result of the maltosyl-transferring reaction which produces isopanose from maltotriose and glucose . The molecular structure of the enzyme deduced from the nucleotide sequence of the clone maintains limited similarity in the conserved regions to the other amylolytic enzymes.

Microbiologia, 1992 Nov, 8(2), 115 - 8
{Detection of Bacillus larvae in mixed populations of bacterial spores from larval remains}; Alippi AM; An accurate laboratory technique for the detection of Bacillus larvae from larval remains of Apis mellifera with mixed bacterial spore populations was developed . The incorporation of nalidixic acid to the culture medium (3 micrograms/ml) was a satisfactory procedure for the separation of Bacillus larvae strains from Bacillus alvei motile colonies.

Mol Biol (Mosk), 1992 Nov-Dec, 26(6), 1338 - 49
{Extracellular alkaline ribonuclease of Bacillus thuringiensis var . subtoxicus}; Dement'ev AA et al.; Intraspecific selection of Bacillus thuringiensis strains producing extracellular alkaline ribonucleases was carried out . Subtoxicus subspecies with increased expression of the enzyme was detected . A method was developed to isolate preparative amounts of homogeneous extracellular RNase of B . thuringiensis var . subtoxicus . The physico-chemical and catalytic properties of the enzyme was studied and compared with extracellular RNases of others Bacillus species . The conclusion about the structural and evolutional conservation of Bacillus extracellular RNases was drawn.

Immunobiology, 1992 Nov, 186(3-4), 230 - 40
Induction of an increased number of dendritic cells in the peritoneal cavity of rats by intraperitoneal administration of Bacillus Calmette-Guérin; van Vugt E et al.; Recently we described the presence of a small number of DC among the peritoneal cells of steady state rats . These DC had the same morphological characteristics and a similar antigen-presenting capacity as DC isolated from the spleen . This study shows that in the peritoneal cavity, which is a non-lymphoid microenvironment, the number of DC increases after i.p . administration of BCG . Next to this relatively small influx of DC, the approximately three-fold increase of the total number of cells is predominantly caused by an enormous influx of neutrophilic granulocytes, and to a lesser extent by an influx of macrophages . The phenotype and the antigen-presenting capacity of peritoneal DC has not changed, while the number of Ia-positive M phi has increased . Nevertheless, due to a suppressive effect of the peritoneal M phi, the total peritoneal cell suspension is no longer capable of presenting antigen.

Appl Environ Microbiol, 1992 Nov, 58(11), 3779 - 83
Alteration of specific activity and stability of thermostable neutral protease by site-directed mutagenesis; Kubo M et al.; On the basis of three-dimensional information, many amino acid substitutions were introduced in the thermostable neutral protease (NprM) of Bacillus stearothermophilus MK232 by site-directed mutagenesis . When Glu at position 143 (Glu-143), which is one of the proposed active sites, was substituted for by Gln and Asp, the proteolytic activity disappeared . F114A (Phe-114 to Ala), Y110W (Tyr-110 to Trp), and Y211W (Tyr-211 to Trp) mutant enzymes had higher activity (1.3- to 1.6-fold) than the wild-type enzyme . When an autolysis site, Tyr-93, was replaced by Gly and Ser, the remaining activities of those mutant enzymes were higher than that of the wild-type enzyme.

Clin Infect Dis, 1992 Nov, 15 Suppl 1, S268 - 73
Evaluation of new anti-infective drugs for the treatment of gastritis and peptic ulcer disease associated with infection by Helicobacter pylori . Infectious Diseases Society of America and the Food and Drug Administration; Murray DM et al.; Helicobacter pylori is a gram-negative, microaerophilic, spiral bacillus . Infection by this organism is currently believed to be the major cause of type B gastritis . Inflammation and infection may persist for years in the absence of therapeutic intervention . There is currently no approved antimicrobial therapy for gastritis . Clinical investigations have shown that combination regimens including bismuth salts and antimicrobial drugs result in the relief of symptoms, the resolution of histologic evidence of gastritis, the eradication of H . pylori, high rates of ulcer healing, and lower rates of ulcer relapse than have been found with other therapies (antacids and H2 antagonists) . A randomized, double-blind, placebo-controlled study design is recommended for evaluation of new therapies . Study participants should have their progress monitored by endoscopy performed at enrollment, at completion of therapy, and 3 months thereafter . Assessment of microbiological outcome is paramount for final evaluation of the patient.

J Parenter Sci Technol, 1992 Nov-Dec, 46(6), 215 - 25
Vaporized hydrogen peroxide sterilization of freeze dryers; Johnson JW et al.; The feasibility of using vapor hydrogen peroxide (VHP) as an alternative to steam sterilization has been examined using a pilot plant freeze dryer equipped with a prototype vapor generator . Specific objectives of the study discussed in this presentation were to: 1 . Identify critical process variables affecting the lethality of VHP to Bacillus stearothermophilus spores, particularly within dead legs in the system . 2 . Measure the efficacy of system degassing after sterilization . 3 . Determine the effect of repeated sterilization cycles on the integrity of elastomeric components of the freeze dryer . Penetration of adequate concentrations of hydrogen peroxide vapor into small diameter piping, such as tubing connected to pressure gauges, is the most challenging aspect of VHP sterilization of freeze dryers . Prior to equipment modifications, spore strips placed within such dead legs remained positive irrespective of the number of gas/degas pulses and system pressure . Equipment modifications necessary to effect complete kill of biological indicators placed in system dead legs is discussed . Results of this study support the conclusion that vaporized hydrogen peroxide shows promise as an alternative sterilization method for freeze dryers.

J Infect, 1992 Nov, 25(3), 291 - 7
Bacillus cereus meningitis in two neurosurgical patients: an investigation into the source of the organism; Barrie D et al.; Two patients developed Bacillus cereus meningitis following neurosurgery . During the subsequent investigation into the source of the organism, linen was discovered to be heavily contaminated with B . cereus . No other prolific source of the organism was found . It seems probable that lint from contaminated fabric was the vehicle of transmission of the organism during extended surgery . Linen should be considered as a possible source of B . cereus infection.

Mol Reprod Dev, 1992 Nov, 33(3), 235 - 42
Genome exclusion and gametic DAPI-DNA content in the hybridogenetic Bacillus rossius-grandii benazzii complex (Insecta Phasmatodea); Tinti F et al.; Among Sicilian stick insects, two hybridogenetic complexes have been discovered: Bacillus rossius-grandii benazzii and B . rossius-grandii grandii, which also produce androgenetic offspring . The egg maturation of the former is analyzed here through DAPI fluorometry, which, besides the assessment of the meiotic stages, also allows their DNA measurements and the analysis of sperm-head evolution into male pronuclei in these polyspermic eggs . Hybridogenetic eggs undergo an extrasynthesis of chromosomes, because two groups of n autobivalents (4C each) are segregated at metaphase 1st; the two groups must correspond to the pure parental species haplosets . Then the grandii chromosomes degenerate (1st polar body), while the rossius chromosomes divide further to produce two groups of n autodiads (2C each); one of them degenerates (2nd polar body), and the other is ready to perform syngamy (female pronucleus) . Meanwhile, several B . grandii sperm evolve into male pronuclei by doubling their DNA (from 1C to 2C content) and assuming an interphase nucleus appearance . If regular mixis occurs, the F1 hybrid constitution is restored but, if it fails, a fusion between two sperms may occur, originating fully paternal descendants (natural androgenesis) . The genome exclusion mechanism of stick-insect hybridogens appears to be more primitive than those observed in the already known hybridogenetic complexes of Poeciliopsis and Rana esculenta . Unfertilized eggs of hybridogens are capable of self-activation, but the cytology of the related clonally reproducing B . whitei indicates that its parthenogenetic mechanism stems from the hybridization event (hybrid theory) rather than from tychoparthenogenetic potentialities (spontaneous theory).

Ultrastruct Pathol, 1992 Nov-Dec, 16(6), 629 - 40
Immunohistochemical and electron microscopic profiles of cutaneous Kaposi's sarcoma and bacillary angiomatosis; Kostianovsky M et al.; Thirty cases of cutaneous Kaposi's sarcoma (KS) were evaluated and compared with eight cases of acquired immunodeficiency syndrome (AIDS)-related bacillary angiomatosis (BA) . The morphologic features of both lesions were studied by light and electron microscopy and by immunohistochemistry with monoclonal endothelial antibodies against CD34, BNH9, and factor VIII-related antigen as well as the lectins Ulex europaeus 1 and Psophocarpus tetragonolobus . Macrophage/monocyte markers used were alpha 1-antitrypsin, lysosome, Kp1 (CD68), and polyclonal factor XIIIa . Electron microscopic studies demonstrated that most of the spindle cells in KS showed a paucity of cell organelles and an absence of Weibel-Palade bodies (WPB), whereas the cells in BA showed activated endothelial cells with WPB . By immunohistochemistry the spindle cells in KS were consistently positive for CD34 only, whereas proliferating cells in BA expressed all endothelial markers used . Numerous cells expressing macrophage/monocyte markers were present surrounding both KS and BA, and a small number of similar cells were entrapped within both lesions . The results demonstrated a restricted immunohistochemical profile for endothelial cell markers in spindle cells of KS (CD34+) distinct from that of endothelial cells in BA . These findings suggest that the spindle cells in KS are poorly differentiated endothelial cells or that they belong to an endothelial cell subset with partial expression of endothelial phenotype.

Clin Infect Dis, 1992 Nov, 15(5), 855 - 7
Significant infections due to Bacillus species following abrasions associated with motor vehicle-related trauma; Wong MT et al.; Non-anthracis Bacillus species are ubiquitous gram-positive spore-forming organisms that were once believed to be nonpathogenic but are now recognized as causing a variety of infections . We report a new aspect of trauma associated with bacillus infection: clinically significant infection by Bacillus species in patients who are involved in motor vehicle accidents and sustain injury related to road contact . Cases were evaluated retrospectively from May 1990 through December 1991 . Four patients who had documented infections with Bacillus species and who were involved in motor vehicle accidents associated with road trauma were identified during this period . The antibiotic susceptibility profile of the Bacillus species consistently demonstrated resistance to beta-lactam antibiotics . This series of cases illustrates an additional aspect of disease associated with bacteria of the Bacillus species that should be considered for patients who have sustained injuries from motor vehicle accidents associated with road trauma.

Biochem J, 1992 Nov 1, 287 ( Pt 3), 971 - 7
Purification and properties of DNA polymerase from Bacillus caldotenax; Burrows JA et al.; A thermostable DNA polymerase was prepared from Bacillus caldotenax by using a four-step chromatography procedure . The protein exists as a monomer of M(r) 94,000, has a pI of 4.9 and has no associated 3'-5' or 5'-3'-exonuclease activities or endonuclease activity . The temperature optimum of the enzyme was about 70 degrees C and the pH for maximum activity was about 7.5 . The enzyme has an absolute requirement for a bivalent cation, and maximum activity was obtained at the unusually high concentration of 70 mM-MgCl2 . Mg2+ could be replaced by MnCl2 or CoCl2, with decreased activity, at the lower optimal concentrations of 1 mM and 2.5 mM respectively . Enzyme activity was inhibited in the presence of 2',3'-dideoxy-TTP, arabinosyl-CTP and aphidicolin . Enzyme activity was stimulated with KCl concentrations of about 100 mM, and concentrations of univalent salts above about 150 mM inhibited activity . The enzyme could use activated calf thymus DNA, poly(dA).p(dT)10 or primed single-stranded phage M13 DNA as a template and maximum activity was obtained with poly(dA).p(dT)10 . The enzyme was inactive on unprimed single-stranded DNA, double-stranded DNA and polyribonucleotide template/primer . The apparent Km values for individual dNTPs, determined with the other dNTPs at saturating concentrations, were 5.7 microM (dCTP), 6.3 microM (dATP, dGTP) and 6.4 microM (dTTP) . The Km value for the overall incorporation of each dNTP from an equimolar mixture of all four dNTPs was 24.7 microM . The kcat . value was about 1.05 s-1 . The kcat./Km value was 0.16-0.18 M-1.s-1 for individual dNTPs and 0.04 for the incorporation of an equimolar mixture of all four dNTPs . Some of the properties of the enzyme show it may be classified as an alpha-Type DNA polymerase.

Biochem J, 1992 Nov 1, 287 ( Pt 3), 685 - 90
Investigation of the first step of biotin biosynthesis in Bacillus sphaericus . Purification and characterization of the pimeloyl-CoA synthase, and uptake of pimelate; Ploux O et al.; The pimeloyl-CoA synthase from Bacillus sphaericus has been purified to homogeneity from an overproducing strain of Escherichia coli . The purification yielded milligram quantities of the synthase with a specific activity of 1 unit/mg of protein . Analysis of the products showed that this enzyme catalysed the transformation of pimelate into pimeloyl-CoA with concomitant hydrolysis of ATP to AMP . Using a continuous spectrophotometric assay, we have examined the catalytic properties of the pure enzyme . The pH profile under Vmax . conditions showed a maximum around 8.5 . Apparent Km values for pimelate, CoASH, ATP.Mg2- and Mg2+ were respectively 145 microM, 33 microM, 170 microM and 2.3 mM . The enzyme was inhibited by Mg2+ above 10 mM . This acid-CoA ligase exhibited a very sharp substrate specificity, e.g . neither GTP nor pimelate analogues (di- or mono-carboxylic acids) were processed . The bivalent metal ion requirement was also investigated: Mn2+ (73%) and Co2+ (32%) but not Ca2+ could replace Mg2+ . The enzyme was inhibited by metal chelators such as 1,10-phenanthroline and EDTA . The synthase was a homodimer with a 28,000-M(r) subunit . N-Terminal sequencing definitely proved that this enzyme was encoded by the bioW gene . A careful study of pimelate uptake by B . sphaericus, E . coli and Pseudomonas dentrificans showed that this metabolite crossed the membrane of these microorganisms by passive diffusion, ruling out the involvement of the bioX gene product as pimelate carrier.

Am Rev Respir Dis, 1992 Nov, 146(5 Pt 1), 1330 - 3
Miliary Mycobacterium bovis induced by intravesical bacille Calmette-Guérin immunotherapy; McParland C et al.; Intravesical instillation of bacille Calmette-Guerin (BCG), an attenuated strain of Mycobacterium bovis, is the treatment of choice for many patients with bladder cancer . In a small percentage, this therapy is associated with systemic side effects including pneumonitis . It is uncertain whether these systemic manifestations are due to dissemination of infection or due to hypersensitivity, an etiologic distinction that has important therapeutic implications . We report the first case in which miliary M . bovis was proven to be the responsible mechanism, by culture of M . bovis biovar BCG from a transbronchial lung biopsy and complete resolution on anti-tuberculous chemotherapy.

J Urol, 1992 Nov, 148(5), 1583 - 6
Expression of adhesion molecules by bladder cancer cells: modulation by interferon-gamma and tumour necrosis factor-alpha; Jackson AM et al.; The constitutive expression by eight human bladder cancer cell lines of the cell adhesion molecules intercellular adhesion molecule-1 and intercellular adhesion molecule-2 was studied using monoclonal antibody probes in conjunction with flow-cytometry . Tumour lines of low grade (G1) did not constitutively express intercellular adhesion molecule-1, rather they were found to express intercellular adhesion molecule-2 . The G2 cells expressed no intercellular adhesion molecule-2, however, a low percentage did express intercellular adhesion molecule-1 . High grade cells (G3) only expressed intercellular adhesion molecule-1 on their cell surface but at higher levels than the G2 cell line . Exposure of the bladder cancer cell lines to interferon-gamma induced and augmented the expression of intercellular adhesion molecule-1 by all except one of the cell lines (UMUC3) . Intercellular adhesion molecule-2 expression remained unaltered . The modulation of intercellular adhesion molecule-1 expression was dependent on the concentration of interferon-gamma and the duration of stimulus . De novo intercellular adhesion molecule-1 expression, induced by interferon-gamma, was rapid (< 4 hours) with only a short period of stimulation being required (< 10 seconds) . The rapid increase in expression of intercellular adhesion molecule-1 required de novo protein synthesis and was not the result of release of intercellular adhesion molecule-1 from an intracellular pool . Interferon-gamma and tumour necrosis factor-alpha were found to act synergistically in the induction and augmentation of intercellular adhesion molecule-1 expression . Optimal induction occurred with 10 Uml-1 of both molecules . These results suggest a correlation between constitutive adhesion molecule expression and the histopathological grade of the tumour . The implications of these findings for Bacillus Calmette Guerin and interferon-gamma immunotherapy of bladder cancer is discussed.

J Urol, 1992 Nov, 148(5), 1534 - 5
Epididymo-orchitis developing as a late manifestation of intravesical bacillus Calmette-Guerin therapy and masquerading as a primary testicular malignancy: a report of 2 cases; Truelson T et al.; In 2 cases epididymo-orchitis, indistinguishable from a testicular tumor, developed as a late (15 and 34 months, respectively) complication following use of Tice strain bacillus Calmette-Guerin for treatment of superficial bladder carcinoma . In each instance the lesion was asymptomatic and ultrasonography demonstrated a complex scrotal mass . Inguinal orchiectomy was performed for diagnosis and therapy . The importance of obtaining a mycobacterial culture for further therapy planning is stressed.

J Lab Clin Med, 1992 Nov, 120(5), 740 - 5
Respiratory epithelial carbohydrate levels of rats with gram-negative bacillary colonization; Mason CM et al.; One mechanism by which severe illness or stress might facilitate adherence and colonization of GNB to respiratory epithelium is by altering epithelial cell surface carbohydrates . To investigate this possibility we used radiolabeled lectins to quantitate carbohydrate levels on intact buccal and tracheal epithelium . A rat model of GNB colonization, in which renal infarction was performed to produce colonization, was used . Buccal and tracheal epithelial surface carbohydrate levels from normal rats and rats 48 hours after renal infarction were compared . Buccal and tracheal epithelium from the renal infarction animals had decreased amounts of sialic acid and fucose, and decreased levels of these sugars occurred at the same time that heavy oropharyngeal GNB colonization developed . Tracheas obtained from the infarcted animals bound three times more Type 1 piliated GNB than normal tracheas . Sialic acid and fucose levels are decreased early after stress, and we speculate that altered epithelial carbohydrates may predispose to GNB colonization by exposing binding sites for GNB.

J Histochem Cytochem, 1992 Nov, 40(11), 1779 - 88
Nature and distribution of mineral-binding, keratan sulfate-containing glycoconjugates in rat and rabbit bone; Maeno M et al.; The presence of keratan sulfate (KS) and KS proteoglycans in bone has been demonstrated in birds and rabbits but comparison with other animal species has not been investigated . The nature and distribution of mineral-binding, KS-containing glycoconjugates in rat and rabbit bone were investigated with a monoclonal antibody (MAb 5D4) specific for KS . Mineral-binding proteins were extracted from the mineralized bone with 0.4 M EDTA without guanidine-HCl (E-extract) . On Western blot analysis of SDS-polyacrylamide gel electrophoresis, rat E-extract gave a weak 5D4-reactive band, M(r) 66,000-68,000, whereas rabbit E-extract produced two major reactive populations of small and large molecular size; one population consisted of two closely spaced bands at M(r) 61,000-63,000 and 66,000-68,000, and the other population consisted of one band at approximately M(r) 200,000 . The identity of KS chains was further established by the sensitivity of these bands to keratanase II (Bacillus sp . Ks 36) and endo-beta-galactosidase . Immunocytochemistry with MAb 5D4 showed that, in rat bone, staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas the remainder of the mineralized matrix lacked staining . In contrast, in rabbit bone the staining was distributed over the entire portion of the mineralized matrix with focal accumulation of staining in the wall of the lacunocanalicular system . These results indicate that rat bone contains a mineral-binding, KS-containing glycoconjugate with preferential localization in the wall of the lacunocanalicular system, whereas rabbit bone contains at least two or possibly three types of KS-containing glycoconjugates distributed over the entire portion of the mineralized matrix.

J Bacteriol, 1992 Nov, 174(22), 7478 - 81
Analysis of mutations in cyclodextrin glucanotransferase from Bacillus stearothermophilus which affect cyclization characteristics and thermostability; Fujiwara S et al.; Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin from starch . The CGTase molecule is composed of four globular domains, A, B, C, and D . In order to gain better understanding of the amylolytic and cyclization mechanisms of CGTase, mutant CGTases were constructed from a CGTase gene (cgt1) of Bacillus stearothermophilus NO2 . Cgt1-F191Y (Phe at position 191 was replaced by Tyr), Cgt1-F191Y-F255Y, Cgt1-W254V-F255I, Cgt1-W254V, and Cgt1-F255I were constructed for the analysis of the NH2-terminal region . It was revealed that amino acids surrounding a spiral amylose are important for cyclization characteristics and that hydrophobic amino acids just after the Glu catalytic site play an important role in the hydrolysis characteristics of the enzyme . Mutant CGTases Cgt1-T591F and Cgt1-W629F were also constructed to study the role of a second substrate-binding site in domain D, and it was suggested that substrate binding at both domains A and D stabilized the enzyme and optimized cyclodextrin production.

Infect Immun, 1992 Nov, 60(11), 4452 - 9
Mycobacterium leprae produces extracellular homologs of the antigen 85 complex; Pessolani MC et al.; The antigen 85 complex is a set of at least three closely related secreted proteins (85A, 85B, and 85C) of 30 to 32 kDa produced by Mycobacterium tuberculosis and other mycobacteria . Their prominence in Mycobacterium leprae, the one obligate intracellular pathogen of the genus, had been assumed on the basis of immunological evidence and proof of the existence of the gene encoding the 85B protein of the complex . We have now observed the production of this family of proteins by M . leprae through analysis of various fractions by Western blotting (immunoblotting) with monospecific rabbit antisera raised against the individual Mycobacterium bovis BCG 85A, 85B, and 85C proteins . A predominant cross-reactive band with an apparent molecular mass of 30 kDa was detected in extracts of nondisrupted whole M . leprae and in soluble fractions prepared from the tissues of M . leprae-infected armadillos . Further studies of the subcellular distribution of this protein within the bacterium confirmed that it is secreted by the organism, an observation that explains past difficulties in detecting the antigen 85 complex in M . leprae . Confirmation that the M . leprae product is a member of the antigen 85 complex was obtained by comparison of peptide fingerprints with those from the BCG product . The pattern of reactivity of the M . leprae antigen 85 complex with anti-M . bovis BCG 85B serum, as well as two-dimensional electrophoresis, established that the 85B component was the predominant member of the complex in M . leprae . The fibronectin-binding capacity of the M . leprae and BCG 85 complexes was reinvestigated by new approaches and is questioned . Nevertheless, the results obtained with the native proteins reinforce previous reports, derived primarily from the use of homologous proteins, that the antigen 85 complex is one of the dominant protein immunogens of the leprosy bacillus.

Appl Microbiol Biotechnol, 1992 Nov, 38(2), 243 - 7
Cloning and expression of a thermostable exo-alpha-1,4-glucosidase gene from Bacillus stearothermophilus ATCC12016 in Escherichia coli; Takii Y et al.; The gene coding for a thermostable exo-alpha-1,4-glucosidase (alpha-glucoside glucohydrolase: EC 3.2.1.20) of Bacillus stearothermophilus ATCC 12016 was cloned within a 2.8-kb AvaI fragment of DNA using the plasmid pUC19 as a vector and Escherichia coli JM109 as a host . E . coli with the hybrid plasmid accumulated exo-alpha-1,4-glucosidase mainly in the cytoplasm . The level of enzyme production was about sevenfold higher than that observed for B . stearothermophilus . The cloned enzyme coincided absolutely with the B . stearothermophilus enzyme in its relative molecular mass (62,000), isoelectric point (5.0), amino-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-Ala-Tyr-Gln-Ile-Tyr-), the temperature dependency of its activity and stability, and its antigenic determinants.

Appl Microbiol Biotechnol, 1992 Nov, 38(2), 147 - 51
Introduction of sulphhydryl groups into the crystalline bacterial cell surface layer protein from Bacillus stearothermophilus PV72 and its application as an immobilization matrix; Sara M et al.; The crystalline cell surface layer (S-layer) from Bacillus stearothermophilus PV72 was used as a matrix for reversible immobilization of beta-D-galactosidase via disulphide bonds . In order to obtain an immobilization matrix stable towards acid, alkali and reducing agents such as dithiothreitol (DTT), the S-layer subunits were first cross-linked with glutaraldehyde . This was done in a way whereby 75% of the free amino groups remained unmodified, and then could be completely converted into sulphhydryl groups upon reaction with the monofunctional imidoester iminothiolane . After activation of the sulphhydryl groups with 2,2'-dipyridyldisulphide, 550 micrograms beta-D-galactosidase could be immobilized per milligram of S-layer protein, which corresponds to one beta-D-galactosidase molecule {relative molecular mass (M(r)), 116,000} per two S-layer subunits (M(r), 130,000) . At least 90% of the sulphhydryl groups from the S-layer protein could be regenerated for further activation by cleaving the disulphide bonds with DTT . In comparative studies beta-D-galactosidase was linked to carbodiimide-activated carboxyl groups of the S-layer protein.

Biosci Biotechnol Biochem, 1992 Nov, 56(11), 1792 - 6
Purification and some properties of a Haim-sensitive alpha-amylase from newly isolated Bacillus sp . No . 195; Kawaguchi T et al.; Newly isolated Bacillus sp . No . 195 produced an extracellular alpha-amylase sensitive to Haim which was found to inhibit specifically animal alpha-amylases . The enzyme was purified easily by two steps of starch adsorption and gel filtration using Sephacryl S-200 . The purified enzyme, which showed a single band on native-PAGE or SDS-PAGE, had a molecular weight of 60,000 as judged on SDS-PAGE . The optimum pH value for activity and the isoelectric point were around 7.0 and 4.5, respectively . The sensitivity of the amylase to Haim was similar to that of animal amylase rather than bacterial amylase . It was suggested that a Haim-amylase complex might be formed at the molar ratio of 1:1 . The amino acid sequence F-S-W similar to the triplet F-E-W highly conserved among alpha-amylases sensitive to proteinaceous inhibitors, such as Hoe 467-A or Haim, was found in the amino-terminal part of the No . 195 amylase.

Biull Eksp Biol Med, 1992 Nov, 114(11), 510 - 2
{The vitamin D endocrine system and bone tissue mineral metabolism in rats with adjuvant arthritis: the effect of 1,25-dihydroxyvitamin D3}; Sergeev IN et al.; Adjuvant arthritis was induced in male rats by injecting bacillus Calmette-Guerin in mineral oil in a hindpaw . A decrease in bone density, calcium and phosphorus content due to polyarthritis was found in the tibia of the noninjected hind leg . Arthritic rats demonstrated serum 1,25-dihydroxyvitamin D deficiency along with constant level of 25-hydroxyvitamin D . The disease caused a significant expression of 1,25-dihydroxyvitamin D3 receptors in lymphocytes . Arthritic rats were treated with 1,25-dihydroxyvitamin D3 (0.15 mg/kg/day orally) for 35 days . The treatment prevented the development of osteoporosis and a decrease of 1,25-dihydroxyvitamin D levels as well as reduced the expression of 1,25-dihydroxyvitamin D receptors in lymphocytes.

Nippon Rai Gakkai Zasshi, 1992 Nov, 61(3), 165 - 74
Demonstration of PGL-I & LAM-B antigens in paraffin sections of leprosy skin lesions; Wang T et al.; An investigation on the demonstration of PGL-I and LAM-B antigens in thirty-four paraffin embedded skin biopsies taken from leprosy patients who covered the whole spectrum of the disease and in four control specimens was carried out . Neither the PGL-I antigen nor the LAM-B antigen was demonstrated in the normal skin specimens that were used as negative control; and only the LAM-B antigen appeared in the tuberculosis specimens in which the PGL-I antigen was negative . The PGL-I antigen was demonstrated on thirty-three leprosy samples except one TT sample and the LAM-B antigen, on all samples by immunochemical staining technique . The antigens were identified as intracytoplasmic bacillary staining, in solitary, granular as well as debris patterns; and as soluble antigenic staining, in vacuolar or amorphous pattern . In LL and BL cases, the antigens were detected predominantly from macrophages and peripheral nerves in all five staining patterns; in BB cases, from macrophages mostly in the granular as well as debris patterns, from the nerves in the vacuolar pattern; while in TT and the majority of BT cases, they were mainly from nerve remnants inside the granuloma in the vacuolar or amorphous staining pattern . In addition, it is interesting to note that the immunochemical staining was able to differentiate the foamy change from the hydropic degeneration . We also found that the antigens distributed in arrector pili muscles and the walls of muscular vessels were obviously related to the unmyelinated nerve fibers innervating the smooth muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Clin Microbiol Infect Dis, 1992 Nov, 11(11), 1058 - 63
Clinical importance of Bilophila wadsworthia; Finegold S et al.; Bilophila wadsworthia is an anaerobic, gram-negative, asaccharolytic, urease-positive, bile-resistant, catalase-positive bacillus, originally recovered from infections in patients with gangrenous and perforated appendicitis . Additional isolations from clinical specimens, including pleural fluid, joint fluid, blood and pus from a scrotal abscess, mandibular osteomyelitis and axillary hidradenitis suppurativa are described here . Bilophila is found as normal flora in feces and, occasionally, in saliva and in the vagina . Isolates from humans are usually beta-lactamase positive and therefore resistant to certain beta-lactam antibiotics . Two percent of strains are also resistant to clindamycin.

Trans R Soc Trop Med Hyg, 1992 Nov-Dec, 86(6), 686 - 92
Does antibody to mycobacterial antigens, including lipoarabinomannan, limit dissemination in childhood tuberculosis?
Costello AM, Kumar A, Narayan V, Akbar MS, Ahmed S, Abou-Zeid C, Rook GA, Stanford J, Moreno C.
Serum immunoglobulin (Ig) G responses to a variety of mycobacterial antigens were measured in children from the UK, in children with tuberculosis from Hyderabad, India and Dhaka, Bangladesh, classified according to whether the disease was disseminated or localized, and in non-tuberculous controls . Anti-lipoarabinomannan (LAM) IgG responses in UK children showed a marked trough between 6 months and 3 years coincident with the reported peak incidence of disseminated tuberculosis . Geometric mean IgG responses to sonicates of slow-growing mycobacteria (rich in LAM) in 36 children with disseminated tuberculosis were markedly lower than in 99 children with localized tuberculous lesions (for Mycobacterium scrofulaceum P < 0.01, for M . tuberculosis P < 0.01, and for M . vaccae P < 0.01) . Responses to purified LAM were also lower in the disseminated tuberculosis group (P < 0.05) but there was no difference between the groups in their response to mycobacterial 65 kDa protein . Multiple regression analysis showed that the reduced response to sonicated mycobacterial antigens and to LAM in children with disseminated disease was independent of age, nutritional status, skin test reactivity, duration of previous symptoms, and city of origin . There was no evidence for sequestration of antibody to immune complexes . These findings are compatible with the hypothesis that children with low levels of antibody to sonicated mycobacterial antigen and to LAM, or those who cannot mount an antibody response, are predisposed to dissemination . A role for antibody in preventing disseminated forms of tuberculosis in childhood has implications for the development of improved vaccines and for the optimum timing of vaccination with bacille Calmette-Guerin.

Plant Mol Biol, 1992 Nov, 20(3), 539 - 48
Expression of a chimeric CaMV 35S Bacillus thuringiensis insecticidal protein gene in transgenic tobacco; Carozzi NB et al.; Insecticidal transgenic tobacco plants containing a truncated Bacillus thuringiensis cryIA(b) crystal protein (ICP) gene expressed from the CaMV 35S promoter were analyzed for ICP gene expression under field and greenhouse conditions over the course of a growing season . We present new information on temporal and tissue-specific expression of a CaMV 35S/cryIA(b) gene . Levels of cryIA(b) protein and mRNA were compared in both homozygous and hemizygous lines throughout plant development . Levels of ICP mRNA and protein increased during plant development with a pronounced rise in expression at the time of flowering . Homozygous ICP lines produced higher levels of ICP than the corresponding hemizygous lines . ELISA analysis of different tissues in the tobacco plant showed ICP gene expression in most tissues with a predominance of ICP in older tissue . All transgenic ICP tobacco lines which were studied in the field and greenhouse contained 400 ng to 1 microgram ICP per gram fresh weight in leaves from the mid-section of the plant at flowering . The amounts of ICP produced by field lines were directly comparable to levels observed in greenhouse-grown plants.

Appl Microbiol Biotechnol, 1992 Nov, 38(2), 173 - 8
Isolation of a gramicidin S hyperproducing strain of Bacillus brevis by use of a fluorescence activated cell sorting system; Azuma T et al.; A gramicidin S (GS) hyperproducing mutant of Bacillus brevis was isolated by using a protein-staining fluorescence dye (fluorescein isothiocyanate, FITC), and a fluorescence-activated cell sorting system (FACS) . By flow cytometry (FCM) analysis after staining with FITC, higher producing cells of the wild-type had higher fluorescence signals than cells with low productivity or cells from a GS non-producing mutant . Staining with FITC did not affect the viability of cells under the conditions chosen for FCM analysis . This enabled us to recover viable cells after sorting . After wild-type cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, mutants with higher fluorescence than the parental strain were obtained by cell sorting . Among them, strain 18 was chosen as a GS hyperproducer; it produced 590 micrograms GS/ml compared to 350 micrograms/ml by the wild-type strain . This method has the advantage of being able to screen large numbers of cells in a short time . Furthermore, use of the fluorescence dye technique will expand the use of FACS to the improvement of other cultures that produce metabolites that do not have a specific fluorescence or strong enough fluorescence for normal cell sorting.

J Mol Biol, 1992 Oct 20, 227(4), 1263 - 4
Purification, crystallization and preliminary X-ray analysis of the 3-phosphoglycerate kinase from Bacillus stearothermophilus; Davies GJ et al.; As part of a programme investigating the molecular basis of thermal stability in proteins we have isolated and characterized the thermally stable 3-phosphoglycerate kinase (PGK) from Bacillus stearothermophilus NCA 1503 . The B . stearothermophilus PGK has been crystallized in a form suitable for X-ray diffraction analysis . Crystals which diffract to greater than 1.8 A resolution have been grown in the presence of the nucleotide substrate, MgATP, using polyethylene glycol (PEG 600) as a precipitant . The best crystals have been obtained using "seeding" techniques and are monoclinic, space group P2(1), with cell dimensions a = 40.5 A, b = 74.0 A, c = 68.5 A and beta = 99.8 degrees.

Biochim Biophys Acta, 1992 Oct 19, 1111(1), 27 - 34
Vasopressin stimulation of vanadate-sensitive Na+ transport by liver plasma membrane vesicles . Evidence for regulation via phospholipase C and protein kinase C activities; Piec G et al.; The rate of vanadate-sensitive 22Na+ uptake by isolated liver membrane vesicles, reflecting transport by Na+/K(+)-ATPase, was measured to study the role played by phospholipase C and protein kinase C in the regulation of this process by vasopressin . Na+ uptake was enhanced 2-3-fold by 100 nM {Arg8}vasopressin and the hormone effect was mimicked by 0.1 microM inositol 1,4,5-trisphosphate as well as by 1.0 microM myo-inositol . The stimulation by vasopressin was potentiated by phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis (5-10 mU/ml) . No effect of the bacterial enzyme was observed in the absence of the hormone . Phorbol myristate acetate (0.5-1 microM) suppressed the stimulation by vasopressin but had no effect in the absence of the hormone . High concentrations of bacterial phosphatidylinositol-specific phospholipase C (50-100 mU/ml) also antagonized the hormone stimulation . Staurosporine (50-100 nM) prevented the antagonistic effect of bacterial phospholipase C (50 mU/ml) and EGTA (1 mM) partially protected the hormonal stimulation in the presence of phorbol myristate acetate . Our results suggest that the stimulatory effect of vasopressin on Na+ transport is mediated by phospholipase C and products derived from the inositol moiety of membrane phospholipids . Membrane-associated protein kinase C appears to be at least partially responsible for the desensitization to stimulation by vasopressin.

Presse Med, 1992 Oct 17, 21(34), 1625 - 30
{Bacillary angiomatosis}; Robert C et al.; Bacillary angiomatosis (BA) is a recently described infection usually found in patients with human immunodeficiency virus disease . BA is caused by a Gram-negative coccobacillus . This organism is primarily responsible for skin lesions of the pseudo-botryomycoma type or inflammatory nodules, but it also produces fever, degradation of the general condition and visceral lesions involving the lymph nodes, the liver, the spleen and the bones . Histology shows vascular proliferation with turgid endothelial cells and mostly neutrophilic inflammatory infiltrates . BA is susceptible to many antibiotics . The authors describe the history of the disease and its clinical and histological features, discuss its differential diagnosis and principally deal with the relationship between BA and cat-scratch disease and between BA and verruca peruana . They also present the molecular biology technique which enables a genotypic diagnosis of the disease to be made, replacing a deficient phenotype.

Cancer Res, 1992 Oct 15, 52(20), 5663 - 7
Immunization of colorectal cancer patients with modified ovine submaxillary gland mucin and adjuvants induces IgM and IgG antibodies to sialylated Tn; O'Boyle KP et al.; Tn and sialylated Tn (sTn) are blood group-related epitopes expressed on mucins of colon carcinoma and other epithelial tumors and are, therefore, potential targets for immunological control . We have immunized 20 colorectal cancer patients at high risk for recurrence with a vaccine consisting of partially desialylated ovine submaxillary gland mucin (modified OSM) which contains both Tn and sTn determinants . Six patients were treated with modified OSM alone (group 1), eight patients were treated with modified OSM and the immunological adjuvant DETOX (group 2), and six patients were treated with modified OSM and Bacillus Calmette-Guerin (group 3) . Pre- and postvaccination sera were tested by enzyme-linked immunosorbent assay and dot blot immune stains for antibodies reactive with modified OSM . Antibody titers increased in 4 of 8 patients immunized with modified OSM and DETOX, in 5 of 6 patients immunized with modified OSM and B . Calmette-Guerin, and in 0 of 6 patients receiving modified OSM without adjuvant . The specificity of induced IgM and IgG antibodies was confirmed by demonstrating reactivity with OSM, bovine submaxillary mucin, and synthetic glycoconjugates sTn-human serum albumin (HSA) and Tn-HSA in enzyme-linked immunosorbent assay and immune stains . Median IgM pre-postvaccination reciprocal titers were 20/80 for Tn-HSA and 10/320 for sTn-HSA . Low level IgG antibody titers against sTn-HSA were detected after vaccination in 7 patients . Toxicity was limited to inflammatory skin reactions at the site of vaccination resulting from the adjuvants . No inflammatory infiltrates were seen in the skin when the modified OSM vaccine was administered in the absence of an immunological adjuvant . These results demonstrate that sTn and Tn can be recognized by the human immune system and that vaccines containing these structures can be administered safely with immunological adjuvants . Attempts to augment the immunogenicity of these carbohydrate antigens by covalent attachment to immunogenic carrier proteins and the use of more potent immunological adjuvants are now being pursued.

J Biol Chem, 1992 Oct 5, 267(28), 19919 - 23
Mammalian cells that express Bacillus cereus phosphatidylinositol-specific phospholipase C have increased levels of inositol cyclic 1:2-phosphate, inositol 1-phosphate, and inositol 2-phosphate; Ross TS et al.; Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol . NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated . Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold) . The increases correlated with the levels of enzyme expression obtained in each cell type . The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection . The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered . Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.

J Infect Dis, 1992 Oct, 166(4), 874 - 84
The 38-kDa protein of Mycobacterium tuberculosis: a review; Harboe M et al.; This review illustrates that the 38-kDa protein is one of the most important antigens of Mycobacterium tuberculosis . It is actively secreted but partly attached to the surface of the mycobacterial cell by a lipid tail that may also be responsible for binding of carbohydrate to the protein . It is a major constituent of M . tuberculosis culture fluid after growth on the synthetic Sauton medium and occurs in bacille Calmette-Guerin in far lower concentrations . The protein induces B and T cell responses with high specificity for infection with M . tuberculosis and is a prime candidate for development of new diagnostic reagents for tuberculosis.

Plant Mol Biol, 1992 Oct, 20(1), 81 - 93
Arabidopsis thaliana small subunit leader and transit peptide enhance the expression of Bacillus thuringiensis proteins in transgenic plants; Wong EY et al.; The expression of the modified gene for a truncated form of the cryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein from Bacillus thuringiensis var . kurstaki (B.t.k.) HD73, under control of the Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunit ats1A promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants . Examination of leaf tissue revealed that the ats1A promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10-fold to 20-fold increase in cryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-En35S) was used to express the same cryIA(c) gene . Transient expression assays in tobacco protoplasts and the whole plant results support the conclusion that the transit peptide plus untranslated sequences upstream of that region are both required for the increase in expression of the CryIA(c) protein . Furthermore, the CaMV-En35S promoter can be used with the Arabidopsis ats1A untranslated leader and transit peptide to increase expression of this protein . While subcellular fractionation revealed that the truncated CryIA(c) protein fused to the ats1A transit peptide is located in the chloroplast, the increase in gene expression is independent of targeting of the CryIA(c) protein to the chloroplast . The results reported here provide new insight into the role of 5' untranslated leader sequences and translational fusions to increase heterologous gene expression, and they demonstrate the utility of this approach in the development of insect-resistant crops.

J Biochem (Tokyo), 1992 Oct, 112(4), 488 - 91
Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47, and its purification; Tamura H et al.; Sphingomyelinase (sphingomyelin cholinephosphohydrolase) {EC 3.1.4.12} of Bacillus cereus was overproduced in a protein-hyperproducing strain, B . brevis 47, by cloning the gene into an expression vector pNU211, which has been developed to express a foreign gene utilizing a promoter and a signal sequence of an outer cell wall protein gene . From 1 liter of culture, about 10 mg of protein was purified to near-homogeneity by two steps of column chromatography; this is almost 500 times higher production compared to the conventional preparation from the original strain, B . cereus IAM 1208 . The N-terminal amino acid sequence of the secreted enzyme was identical to that of the authentic enzyme, indicating that the signal sequence for secretion of B . cereus was processed properly in B . brevis 47.

Int J Food Microbiol, 1992 Oct, 17(2), 85 - 99
Media for the detection and enumeration of Bacillus cereus in foods: a review; van Netten P et al.; Bacillus cereus is an established cause of food poisoning in addition to being a troublesome and persistent contaminant, responsible for a variety of spoilage defects in processed foods and dairy products . A range of diagnostic and selective media has been developed to facilitate the detection and enumeration of B . cereus in routine surveillance situations and food poisoning investigations . These media are reviewed with respect to the selective and diagnostic systems they employ, their ability to recover and differentiate the target organism, and their advantages and limitations in particular applications.

Wei Sheng Wu Xue Bao, 1992 Oct, 32(5), 314 - 9
{Molecular cloning and identification of 130kd mosquitocidal protein gene of Bacillus thuringiensis var . israelensis (Bti)}; Hua X et al.; The location of 130kd mosquitocidal protein gene of Bti 4Q5 strain on its 75Md plasmid was confirmed by southern hybridization using a 18-base oligonucleotide probe . The crystal protein containing the component of 130kd toxic protein was purified . The crystal protein exhibiting the mosquitocidal activity against larvae of Aedes aegypti was shown by bioassay . The purified 75Md plasmid DNA of Bti 4Q5 strain was completely digested with HindIII restriction enzyme, ligated with the vector pUC18 and transformed into the recipient cells of E . coli TG1 . From Apr transformants, four clones with HindIII restriction fragment inserts highly homologous to the 18-base oligonucleotide probe were obtained by in situ hybridization and southern hybridization . The 5.2kb HindIII restriction fragment insert was obtained in clone pFH2 and clone pFH4, and 2.3kb HindIII restriction fragment insert in clone pFH1 and pFH3 . For pFH2 and pFH4, the 5.2kb fragment was inserted in pUC18 in opposite orientation . It contained 130kd mosquitocidal protein gene (type I) identified by restriction enzyme map analysis . The 2.3kb HindIII fragment insert in other two clones (pFH1 and pFH3) harbored a part of the type II mosquitocidal protein gene which can be used as a probe for cloning of the type II mosquitocidal protein gene.

Protein Eng, 1992 Oct, 5(7), 693 - 701
Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases; Juteau JM et al.; Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding . Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin . In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation . Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins . Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate . Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase . Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field . Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral . Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes . The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins . This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.

Protein Eng, 1992 Oct, 5(7), 611 - 5
The structural consequences of exchanging tryptophan and tyrosine residues in B . stearothermophilus lactate dehydrogenase; Roper DI et al.; A mutant Bacillus stearothermophilus lactate dehydrogenase has been prepared in which all three tryptophan residues in the wild-type enzyme have been replaced by tyrosines . In addition, a tyrosine residue has been mutated to a tryptophan, which acts as a fluorescence probe to monitor protein folding . The mutant enzyme crystallizes in the same crystal form as the wild-type . The crystal structure of the mutant has been determined at 2.8 A resolution . Solution studies have suggested that there is little effect upon the mutant enzyme as judged by its kinetic properties . Comparison of the crystal structures of the mutant and wild-type enzymes confirms this conclusion, and reveals that alterations in structure in the region of these mutations are of a similar magnitude to those observed throughout the structure, and are not significant when compared with the errors in atomic positions expected for a structure at this resolution.

Immunol Cell Biol, 1992 Oct, 70 ( Pt 5), 295 - 300
Antibodies to 65 kDa and 70 kDa heat shock proteins in rheumatoid arthritis and systemic lupus erythematosus; Panchapakesan J et al.; To test the potential role of autoimmunity to the highly conserved heat shock proteins (HSP) in immune arthritides, the sera from 99 patients with rheumatoid arthritis (RA), 48 patients with systemic lupus erythematosus (SLE) and 65 normal controls were examined by ELISA for IgG and IgM antibodies to the 65 kDa and 70 kDa heat shock proteins from Mycobacterium bovis (Bacille Calmette-Guerin; BCG) . In RA sera there are significant numbers of individuals with increased IgM anti-65 kDa and anti-BCG reactivity as well as IgG anti-70 kDa when compared with controls . In SLE both IgM and IgG anti-BCG, together with IgM anti-65 kDa, differed significantly from controls . The results were compared with previous reports in similar groups of patients, and it is clear that no consistent pattern of reactivity emerges . While further work may be justified looking carefully at the disease duration and other subsets of both RA and SLE, it is difficult at this stage to conclude that antibodies to autologous HSP that cross-react with mycobacterial HSP play a major role in disease pathogenesis.

Planta Med, 1992 Oct, 58(5), 405 - 9
Effects of Phaeodactylum tricornutum and Dunaliella tertiolecta extracts on the central nervous system; Villar R et al.; Continuing work on the effects of aqueous extracts of the microalgae Phaeodactylum tricornutum Bohlin (Bacillariophyceae) and Dunaliella tertiolecta Butcher (Chlorophyceae) on the central nervous system, we report their effects on spontaneous motor activity, rectal temperature, exploratory behaviour, muscle relaxation, catalepsy, and conditioned avoidance responses . Both extracts showed activity as a CNS depressant and a potential muscle relaxant, the latter more marked in the case of P . tricornutum.

Planta Med, 1992 Oct, 58(5), 398 - 404
Effects of Skeletonema costatum extracts on the central nervous system; Villar R et al.; We report the effects of aqueous extracts of the microalga Skeletonema costatum Greve (Cleve) (Bacillariophyceae) on spontaneous motor activity, rectal temperature, motor coordination, amphetamine-induced hypermotility, exploratory behaviour, muscle relaxation, catalepsy, conditioned avoidance responses, oxotremorin-induced cholinergic syndrome, and pentylenetetrazole-induced convulsions . The S . costatum extract at dosages of 310 and 620 mg/kg works like an antidopaminergic drug with anticholinergic properties, that does not induce catalepsy and with a notable muscle relaxing activity.

Lab Anim Sci, 1992 Oct, 42(5), 449 - 53
Correlation between megaloileitis and antibodies to Bacillus piliformis in laboratory rat colonies; Hansen AK et al.; Rat colonies in which antibodies to Bacillus piliformis were detected in animals examined at the age of 8 to 15 weeks were compared with rat colonies where no such antibodies were present . The seropositive colonies had a low incidence of megaloileitis in 5-week-old rats of Sprague-Dawley stock and some few inbred strains . In seronegative colonies, no megaloileitis was detected . In rats with megaloileitis, significantly high titers to B . piliformis were noted and the agents could be identified in the ileal mucosa by immunofluorescence technique.

Lab Anim Sci, 1992 Oct, 42(5), 444 - 8
Rederivation of rat colonies seropositive for Bacillus piliformis and the subsequent screening for antibodies; Hansen AK et al.; Latent infection of rats in a breeding colony with Bacillus piliformis detectable by antibodies to the agent in an immunofluorescence assay was eliminated by a combination of traditional rederivation techniques, using animal units not previously used for rat breeding, and the use of specific disinfection procedures . The success rate was apparently correlated with the use of peracetic acid instead of aldehyde products to decontaminate the animal unit.

Lab Anim Sci, 1992 Oct, 42(5), 439 - 43
Subclinical infection and transmission of Tyzzer's disease in rats; Motzel SL et al.; Two isolates of Bacillus piliformis originally obtained from rats from Japan and Indiana were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting . Protein and antigen profiles revealed heterogeneity between the two isolates, demonstrating that more than one isolate of B . piliformis is capable of infecting rats . Results of parallel infection and transmission studies with the two isolates were almost identical . Orally inoculated rats remained asymptomatic; however, enzyme-linked immunosorbent assay results revealed a significant increase in serum antibodies to B . piliformis . Formalin-killed B . piliformis elicited no serum antibody response among rats inoculated orally, indicating that viable organisms, capable of replicating within the host, are needed to induce a systemic humoral response . Naive rats and weanling gerbils were housed on soiled bedding from the experimentally infected, asymptomatic, seropositive rats . Although gerbils showed no clinical signs or histopathologic evidence of Tyzzer's disease, rats housed on bedding collected 1 or 2 weeks postinoculation seroconverted and remained seropositive but asymptomatic throughout the study . These results demonstrate that subclinically infected rats are capable of transmitting B . piliformis to naive rats and suggest that the histopathologic evaluation of sentinel gerbils may not be an effective method for detecting all strains of B . piliformis.

Biochem Int, 1992 Oct, 28(1), 97 - 103
Purification and properties of 2,3-dihydroxy-p-cumate-3,4-dioxygenase from Bacillus species; Ninnekar HZ; 2,3-Dihydroxy-p-cumate-3,4-dioxygenase, an enzyme involved in the catabolism of p-cymene, was purified to homogeneity from Bacillus species by affinity chromatography . Purification of the dioxygenase allowed the observation of the immediate ring cleavage product of 2,3-dihydroxy-p-cumate . The enzyme was optimally active at pH 8.2 and at 35 degrees C . The Km value for 2,3-dihydroxy-p-cumate was 32 microM . The enzyme had a broad substrate specificity for 3-substituted catechols . The activity of the enzyme was inhibited by heavy metals, sulphydryl inhibitors, iron-chelating agents, and substrate analogues . Fe2+ was suggested as a cofactor.

Appl Environ Microbiol, 1992 Oct, 58(10), 3343 - 6
Evaluation of synergism among Bacillus thuringiensis toxins; Tabashnik BE; A simple test for synergism among toxins is described and applied to previously reported data on independent and joint toxicities of insecticidal proteins from Bacillus thuringiensis . The analysis shows synergism between a 27-kDa (CytA) toxin and 130- or 65-kDa (CryIV) toxins from B . thuringiensis subsp . israelensis against Aedes aegypti larvae . No positive synergism between 130- and 65-kDa toxins or among three CryIA toxins tested against seven species of Lepidoptera occurred . Comparisons with the original interpretations of these data show one case in which synergism occurred but was reported previously as absent and two cases that were not synergistic but were reported previously as suggestive of synergism . These results show that lack of an appropriate test for synergism can produce misleading conclusions . The methods described here can be used to test for synergistic effects of any poisons.

Mol Gen Genet, 1992 Oct, 235(1), 147 - 52
Cloning and molecular characterization of the secY genes from Bacillus licheniformis and Staphylococcus carnosus: comparative analysis of nine members of the SecY family; Tschauder S et al.; SecY is a central component of the export machinery that mediates the translocation of secretory proteins across the plasma membrane of Escherichia coli . We have cloned and sequenced the secY genes from Bacillus licheniformis and Staphylococcus carnosus . The deduced amino acid sequences are highly homologous to those of other known SecY polypeptides, all having the potential to form 10 transmembrane segments . Comparative analysis of 9 SecY polypeptides, derived from different bacteria, revealed that 14 amino acid positions (2.7%) are identical in all SecY proteins and 89 (16.9%) show conservative changes . Clusters of conserved amino acid residues were found in 4 of the 10 transmembrane segments and 2 of the 6 cytoplasmic domains . It is suggested that the conserved regions might be involved in the translocation activity of SecY or might be required for the correct interaction of SecY with other components of the secretion apparatus.

Mikrobiyol Bul, 1992 Oct, 26(4), 355 - 8
{The growth of some Bradyrhizobium strains in uremic conditions in the presence of antimicrobial substances}; Ayhan K et al.; The growth rate of Bradyrhizobium japonicum USDA 110 and its STRR and AMPR mutant strains (resistant to streptomycin and ampicillin respectively), as well as TAL 945 and TAL 946, TAL 947 (mutants of USDA 110 and USDa 138 respectively) on yeast extract mannitol agar (YEMA) containing different concentrations of Crystal Violet and Brilliant Green . According to our results only the growth of Bacillus megaterium B17 was effected from Crystal Violet while the others were not effected from both of dyes in any concentration.

Nippon Hinyokika Gakkai Zasshi, 1992 Oct, 83(10), 1689 - 95
{Prophylaxis of superficial bladder tumor with the intravesical instillation of bacillus Calmette-Guerin or anti-cancer agents . A retrospective study}; Suzuki T et al.; It was retrospectively analyzed whether intravesical instillation of bacillus Calmette-Guerin (BCG) or anti-cancer agents had prophylactic effect or not after removal of superficial bladder tumors . The results over a follow-up period ranging from 6 to 40 months showed 23.8 per cent recurrence in group 1 patients treated with BCG (21 patients), 46.7 per cent recurrence in group 2 treated with anti-cancer agents (45 patients) and 52.3 per cent recurrence in the group 3 (127 patients) which were not received any intravesical drugs . Long-term results among the 3 groups calculated with Kaplan-Meier method demonstrated that the instillation of BCG or of anti-cancer agents was more useful for prophylaxis, compared with the actuarial non-recurrence rates of group 3 . The instillation of BCG showed good prophylactic effects especially in recurrent, grade 2 and pTa bladder tumors . The instillation of anti-cancer agents showed to provide prolonged protection from recurrence, but the instillation of BCG did not.

Proteins, 1992 Oct, 14(2), 224 - 36
Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases; Eijsink VG et al.; Variants of the thermolabile neutral protease (Npr) of B . subtilis (Npr-sub) and the thermostable neutral protease of B . stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined . Mutations were designed to alter the interaction between the middle and C-terminal subdomain of these enzymes . In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction . In thermostable Nprs (like Npr-ste) a 10 residue beta-hairpin, covering the domain interface, makes an additional contribution . The hydrophobic residue at position 315 was replaced by smaller amino acids . In addition, the beta-hairpin was deleted from Npr-ste and inserted into Npr-sub . The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the beta-hairpin for the structural integrity of Nprs . Combined mutants showed that the effects of individual mutations affecting the interaction between the subdomains were not additive . The effects on thermostability decreased as the strength of the subdomain interaction increased . The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability . The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis.

J Parasitol, 1992 Oct, 78(5), 800 - 4
Tikusnema javaense n . gen., n . sp . (Nematoda: Acuarioidea) and other nematodes from Rattus argentiventer collected in west Java, Indonesia; Hasegawa H et al.; Nematodes collected from the ricefield rat, Rattus argentiventer (Rodentia: Muridae), in Pusakanagara and Sukamandi, West Java, Indonesia, are reported . Tikusnema javaense n . gen., n . sp . (Nematoda:Acuariidae:Seuratiinae) is described from the small intestine . This new genus is distinguished readily from other genera of the subfamily Seuratiinae in having 4 strongly protruded cuticular leaves in the posterior cephalic portion and in having a pair of prominent cuticular ornamentations posterior to deirids . Besides T . javaense, Eucoleus bacillatus, Strongyloides ratti, Nippostrongylus brasiliensis, Syphacia muris, and Physaloptera sp . were detected.

J Pediatr, 1992 Oct, 121(4), 574 - 8
Bacillary angiomatosis in a child undergoing chemotherapy; Myers SA et al.; Bacillary angiomatosis is an infectious disease of the skin and viscera characterized by vascular lesions, originally described in patients with human immunodeficiency virus infection . There are also case reports of bacillary angiomatosis occurring in immunocompetent patients and in noninfected patients with suppressed immune function . We report a case of bacillary angiomatosis in a child undergoing chemotherapy for acute leukemia.

J Bacteriol, 1992 Oct, 174(19), 6171 - 8
Structure, function, and fate of the BlaR signal transducer involved in induction of beta-lactamase in Bacillus licheniformis; Zhu Y et al.; The membrane-spanning protein BlaR is essential for the induction of beta-lactamase in Bacillus licheniformis . Its nature and location were confirmed by the use of an antiserum specific for its carboxy-terminal penicillin sensor, its function was studied by genetic dissection, and the structure of the penicillin sensor was derived from hydrophobic cluster analysis of the amino acid sequence by using, as a reference, the class A beta-lactamases with known three-dimensional structures . During the first 2 h after the addition of the beta-lactam inducer, full-size BlaR, bound to the plasma membrane, is produced, and then beta-lactamase is produced . By 2 h after induction, BlaR is present in various (membrane-bound and cytosolic) forms, and there is a gradual decrease in beta-lactamase production . The penicillin sensors of BlaR and the class D beta-lactamases show strong similarities in primary structures . They appear to have the same basic spatial disposition of secondary structures as that of the class A beta-lactamases, except that they lack several alpha helices and, therefore, have a partially uncovered five-stranded beta sheet and a more readily accessible active site . Alterations of BlaR affecting conserved secondary structures of the penicillin sensor and specific sites of the transducer annihilate beta-lactamase inducibility.

Infect Immun, 1992 Oct, 60(10), 4051 - 8
Adhesion to and invasion of cultured human cells by Bartonella bacilliformis; Hill EM et al.; Bartonella bacilliformis was tested for its ability to adhere to and invade tissue culture cell monolayers . The parasite was able to efficiently bind and penetrate human dermal fibroblasts, human laryngeal epithelium, and human umbilical vein endothelial cells . Exposure of the organism to immune serum prepared against a crude Bartonella extract containing cell wall and membranous material resulted in decreased ability of the parasite to invade host cells . There was also an overall reduction in the invasiveness of bartonellae and total host cell association when human laryngeal epithelial cells and human umbilical vein endothelial cells were preexposed to cytochalasin D, indicating an active involvement of host cells in the uptake of bartonellae . Transmission electron microscopy revealed the presence of bartonellae inside and outside intracellular vacuoles . These data suggest that a surface-associated factor is involved in the invasion process and that internalization of the parasite by host cells involves a microfilament-dependent process similar to phagocytosis.

Biosci Biotechnol Biochem, 1992 Oct, 56(10), 1552 - 6
Enzymatic synthesis of 2-chloro-4-nitrophenyl 4,6-O-3-ketobutylidene beta-maltopentaoside, a substrate for alpha-amylase; Ishimaru K et al.; A transglycosylation reaction with 2-chloro-4-nitrophenyl beta-maltoside as an acceptor was done with 4,6-O-3-ketobutylidene maltopentaose and Bacillus macerans cyclodextrin glucanotransferase in an aqueous solution containing 50% n-propanol, and there were two main transglycosylation products . They were identified as 2-chloro-4-nitrophenyl 4,6-O-3-ketobutylidene beta-maltopentaoside and 2-chloro-4-nitrophenyl 4,6-O-3-ketobutylidene beta-maltohexaoside, and their yields were 30% and 21% respectively on the basis of the decrease of 4,6-O-3-ketobutylidene maltopentaose . For the production of 2-chloro-4-nitrophenyl 4,6-O-3-ketobutylidene beta-maltopentaoside at high substrates concentrations, the addition of n-propanol in this reaction not only increased the solubility of 2-chloro-4-nitrophenyl beta-maltoside sufficiently but also suppressed side reactions.

Indian J Exp Biol, 1992 Oct, 30(10), 915 - 7
Sensitivity of mosquito-pathogenic bacterial strains to various antibodies; Gupta DK et al.; Four strains of Bacillus sphaericus, 1593, 2362, 9001 and 9002, B . thuringiensis H-14 and B . thuringiensis neoleonensis were tested for sensitivity against 18 antibiotics . The results revealed that all the four strains of B . sphaericus are resistant to colistin, nalidixic acid, polymyxin B and streptomycin . However, B . thuringiensis H-14 was resistant to 9 antibiotics, viz . ampicillin, cephalexin, carbenicillin, co-trimoxazole, colistin, cloxacillin, penicillin, nitrofurantoin and polymyxin B whereas B . thuringiensis neoleonensis was found to be resistant to 8 antibiotics . These results may help in isolation of potential and resistant mosquito pathogenic bacteria.

Pneumoftiziologia, 1992 Oct-Dec, 41(4), 199 - 205
{The association of tuberculosis with HIV/AIDS infection in children in Romania}; Mihailescu P et al.; The great number of AIDS cases in children in Romania, together with the high annual risk of Tb infection, created the premises for the occurrence of a relatively great number of disease cases through HIV infection/AIDS + tuberculosis, particularly in the age-group "0-5 years" . Serum positive HIV children were considered as AIDS cases when tuberculosis was also associated . Twelve cases in which the infections were concomitant, transmitted through injections, constituted an exception to the point . The 12 children serum positive for HIV showed a primary musculo-cutaneous complex on their thighs, at the very place of injections . A proportion of 50% of them showed a favourable evolution under anti-Tb treatment . Most children developed primary tuberculosis aerogenically acquired, associated with AIDS . A proportion of 59.5% of them evoluted towards severe disseminated forms (milliaria, meningitis), with many deaths, and 37.8% only showed a favorable evolution under anti-tuberculosis treatment . HIV infection in children took place predominantly between 1987-1989 . Tuberculosis was associated 1-2 years later, when the switching from bacillary infection into active tuberculosis was facilitated by the progressive immunodepression which is specific for AIDS . The tuberculin test with 2 IU-PPD was positive in less advanced AIDS cases but faded in children in the final stage of the syndrome or in those with severe forms of tuberculosis . Tuberculosis finding out in children with HIV infection/AIDS is however possible; therefore, skin test reaction is compulsory in all children in this category . In children with a tuberculosis cured through specific treatment in their histories, the association of HIV infection reaching AIDS stage can lead to a Tb relapse.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteins . 1992 Oct;14(2):324.
Purification and crystallization of insecticidal delta-endotoxin CryIIIB2 from Bacillus thuringiensis; Cody V et al.; CryIIIB2, an insecticidal protein from Bacillus thuringiensis has been crystallized from 0.6 M NaBr and HEPES buffer at pH 7.0 and X-ray diffraction data collected on a native crystal to 2.4 A . The insecticidal protein was obtained from a Bacillus thuringiensis (Bt) strain EG7231 . Crystals of the endotoxin are orthorhombic, space group C2221, with unit cell dimensions of a = 122.44, b = 131.81, and c = 105.37 A . A unit cell contains one molecule of the 67,000 Da endotoxin per asymmetric unit.

Infect Agents Dis, 1992 Oct, 1(5), 245 - 53
Identification of uncultured microorganisms: expanding the spectrum of characterized microbial pathogens; Relman DA et al.; The combination of enzymatic nucleic acid amplification techniques with 16S rRNA-based molecular phylogeny has brought about a new approach to the identification of microbial pathogens that can not be cultivated in the laboratory . The applications of this experimental approach to bacillary angiomatosis and to Whipple's disease have revealed the presence of two previously uncharacterized organisms . These results suggest the existence of a far greater microbial diversity among human pathogens than has been so far appreciated with culture-dependent methods . PCR-based studies of aquatic environmental microbial communities have already reached similar conclusions . As a result, new and provocative questions are raised concerning the association of amplified 16S rRNA sequences with diseased tissue . The answers must await the results of further investigations and the expansion of sequence data bases.

FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 143 - 8
Mode of replication, size and distribution of naturally occurring plasmids in Bacillus thuringiensis; Baum JA et al.; Cloned replication origin regions, derived from both small (4.9-7.5 MDa) and large (43-60 MDa) plasmids of Bacillus thuringiensis subspecies kurstaki strains HD73 and HD263 were used as hybridization probes in a Southern-blot analysis to assess both the size and horizontal distribution of native plasmid replicon groups among different subspecies of B . thuringiensis . In general, resident plasmids hybridizing to the replication origin regions derived from strains HD263 and HD73 were more commonly found in kurstaki strains than in non-kurstaki strains, suggesting a non-random distribution of plasmid incompatibility groups . Replication origin regions derived from the large HD263 plasmids (43-60 MDa) hybridized almost exclusively with large plasmids (greater than 30 MDa) of widely varying sizes . In contrast, replication origin regions derived from small plasmids hybridized exclusively with small plasmids (less than 10 MDa) showing little size variation . These results are consistent with previous observations concerning the relationship between plasmid size, mode of replication, and structural stability.

J Biol Chem, 1992 Sep 25, 267(27), 19388 - 95
Molecular cloning and sequencing of the gene for mycocerosic acid synthase, a novel fatty acid elongating multifunctional enzyme, from Mycobacterium tuberculosis var . bovis Bacillus Calmette-Guerin; Mathur M et al.; Mycocerosyl lipids are found uniquely in the cell walls of pathogenic mycobacteria . Mycocerosic acid synthase (MAS) is a multifunctional protein which catalyzes elongation of n-fatty acyl-CoA with methylamalonyl-CoA as the elongating agent (Rainwater, D . L., and Kolattukudy, P . E . (1985) J . Biol . Chem . 260, 616-623) . To understand how the various domains that catalyze the reactions involved in chain elongation are organized, mas gene from Mycobacterium tuberculosis bovis BCG was cloned . A lambda gt11 library of AluI partially digested genomic DNA from the organism was screened with an oligonucleotide probe designed from the N-terminal amino acid sequence of purified MAS . Using terminal segments of inserts from positive clones as the probe, the library was rescreened and the process was repeated . Sequencing of four overlapping clones revealed a contiguous sequence of 9699 base pair(s) (bp) of mycobacterial genome containing a 6330-bp open reading frame that could code for a protein of 2100 amino acids with a molecular mass of 225,437 daltons . The authenticity of the open reading frame as that of MAS was verified by correspondence of the amino acid sequences deduced from the gene with the directly determined amino acid sequences of the N terminus and three different internal peptide fragments . By comparing the MAS amino acid sequence with the sequences in the active site regions of known fatty acid synthases and polyketide synthases the functional domains in MAS were identified . This analysis showed that the domains were organized in the following order: beta-ketoacyl synthase, acyl transferase, dehydratase-enoyl reductase, beta-ketoreductase, acyl carrier protein; no thioesterase-like domain could be found . These results establish MAS as the first case of an elongating multifunctional enzyme composed of two identical subunits that resemble the vertebrate fatty acid synthase in size, subunit structure, and linear organization of functional domains . Southern and Western blot analyses showed absence of mas gene and encoded proteins in Mycobacterium smegmatis and Escherichia coli . This result is consistent with the report that mycocerosic acid is present only in pathogenic mycobacteria.

J Biol Chem, 1992 Sep 25, 267(27), 19278 - 90
Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin; Fernandez-Moreno MA et al.; A 5.3-kb region of the Streptomyces coelicolor actinorhodin gene cluster, including the genes for polyketide biosynthesis, was sequenced . Six identified open reading frames (ORF1-6) were related to genetically characterized mutations of classes actI, VII, IV, and VB by complementation analysis . ORF1-6 run divergently from the adjacent actIII gene, which encodes the polyketide synthase (PKS) ketoreductase, and appear to form an operon . The deduced gene products of ORF1-3 are similar to fatty acid synthases (FAS) of different organisms and PKS genes from other polyketide producers . The predicted ORF5 gene product is similar to type II beta-lactamases of Bacillus cereus and Bacteroides fragilis . The ORF6 product does not resemble other known proteins . Combining the genetical, biochemical, and similarity data, the potential activities of the products of the six genes can be postulated as: 1) condensing enzyme/acyl transferase (ORF1 + ORF2); 2) acyl carrier protein (ORF3); 3) putative cyclase/dehydrase (ORF4); 4) dehydrase (ORF5); and 5) "dimerase" (ORF6) . The data show that the actinorhodin PKS consists of discrete monofunctional components, like that of the Escherichia coli (Type II) FAS, rather than the multifunctional polypeptides for the macrolide PKSs and vertebrate FASs (Type I).

Biochim Biophys Acta, 1992 Sep 23, 1159(2), 185 - 92
Effects of pH on conformational properties related to the toxicity of Bacillus thuringiensis delta-endotoxin; Venugopal MG et al.; The delta-endotoxin of Bacillus thuringiensis subspecies kurstaki is an intracellular crystalline proteinaceous inclusion which, upon ingestion, is toxic to lepidopteran insects . Upon dissolution at pH > 9 it yields a protein subunit called protoxin . Under appropriate conditions, protoxin is hydrolyzed to a toxin molecule, which is responsible for killing the insect . It is known that this toxic activity decreases considerably above pH 10 . In this study, circular dichroism spectroscopy has been used to examine the secondary structures of the protoxin and toxin molecules at different pH values to determine if there are detectable conformational changes associated with their pH-dependent functional properties . At pH 10, where toxic activity is approximately maximal, both the protoxin and toxin molecules were found to assume a conformation that is on an average approx . 26% alpha-helix and approx . 45% beta-structure . As the pH was increased above 10, where the insecticidal activity decreases, the magnitude of the CD spectrum at 222 nm decreased for protoxin and the calculated alpha-helix contents of both protoxin and toxin molecules decreased . The net secondary structure did not change significantly at pH values below 10 . Significant conformational differences are observed between the secondary structure of the protoxin and toxin molecules at different pH values . The pH-dependent changes in secondary structure of the protoxin and toxin can be correlated with the effects of pH on the insecticidal activity of these proteins.

Biochemistry, 1992 Sep 22, 31(37), 8740 - 6
Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis; Klein C et al.; An X-ray structure analysis of a crystal of mutant Asp229----Ala of cyclodextrin glycosyltransferase from Bacillus circulans (Ec 2.4.1.19) that had been shortly exposed to beta-cyclodextrin showed density corresponding to a maltose bound at the catalytic center . The crystal structure was refined to an R-factor of 18.7% at 2.5-A resolution . The catalytic center is defined by homology with the structurally known alpha-amylases and by the observation that mutants Asp229----Ala and Asp328----Ala are almost inactive . By model building, the density-defined maltose was extended to a full beta-cyclodextrin, which then indicated the general locations of seven subsites for glucosyl units . The catalytically competent residues Asp229, Glu257, and Asp328 are at the reducing end of the density-defined maltose . In the unligated wild-type structure, Glu257 and Asp328 form a 2.6-A hydrogen bond between their carboxylates in an arrangement that resembles those of the catalytically competent carboxylates in acid proteases . Presumably, the first catalytic step is an attack of the proton between Glu257 and Asp328 on the oxygen of the glycosidic bond.

Biochemistry, 1992 Sep 22, 31(37), 8978 - 83
Substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus examined using the resolved enantiomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate); Leigh AJ et al.; The substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus is examined using the resolved optical isomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate), a chromogenic substrate for the phospholipase . The synthetic route employs mild acid-labile protecting groups and separation of the substituted myo-inositol enantiomers as the (-)-camphanyl ester diastereomers . Measurements of the initial rates of cleavage of the D and L enantiomers of the nitrophenyl substrate by phosphatidylinositol-specific phospholipase C from B . cereus show that this enzyme is essentially stereospecific for the D enantiomer . Under identical conditions, the rate of cleavage of the L isomer is less than 0.2% of that observed for the D isomer . The same is observed for the highly homologous enzyme from Bacillus thuringiensis . There is no measurable inhibition by the L enantiomer of the B . cereus enzyme acting on the D enantiomer, even when the molar ratio of L:D is 5, indicating that binding of the L enantiomer to the phospholipase is negligible . Thus, the enzyme active site is exquisitely sensitive to the stereochemistry of the myo-inositol group of the substrate.

Gene, 1992 Sep 21, 119(1), 143 - 4
Sequence of the subtilisin-encoding gene from an antarctic psychrotroph Bacillus TA41; Davail S et al.; The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph, Bacillus TA41, was determined . The primary structure of the subtilisin precursor corresponds to a preproenzyme of 419 amino acids . Asp144, His181 and Ser359 are the proposed catalytic residues of the protease active site.

Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 641 - 7
Genomic amplification and expression of delta-endotoxin fragment of Bacillus thuringiensis; Roy P; delta-Endotoxin gene of Bacillus thuringiensis HD-1 var kurstaki codes for the insecticidal crystal protein (ICP) specific for lepidopteran insects . Since the N-terminal half of the toxin is sufficient both for insect specificity and toxicity, the coding sequence of this part of the gene CryIA(b) was amplified by PCR and cloned in pUC19 . As there was no expression of immunologically detectable delta-endotoxin in this clone in E . coli, the amplified ICP gene was transferred to an expression vector pGEx2T . Restriction mapping and immunoblotting confirmed the presence and expression of the CryIA(b) gene . This insert should be suitable for expression in plant system if it is mobilized into a plant binary vector.

Biochem J, 1992 Sep 15, 286 ( Pt 3), 857 - 62
Effect of side-chain amide thionation on turnover of beta-lactam substrates by beta-lactamases . Further evidence on the question of side-chain hydrogen-bonding in catalysis; Pratt RF et al.; Two side-chain-thionated beta-lactams, a penicillin and a cephalosporin, have been prepared and found to be not significantly poorer as substrates of typical serine (classes A and C) beta-lactamases than are their oxo analogues . This result is interpreted to mean that any hydrogen-bonding site on these enzymes for the beta-lactam side-chain amide carbonyl group must be flexible and is more likely to be a passive rather than active or essential feature of the active site . Previously, data from crystal structures and site-directed mutagenesis had suggested that the side chain of Asn-132 of class-A beta-lactamases, a component of the conserved SDN loop, forms a hydrogen bond with the side-chain carbonyl of the beta-lactam substrate and may provide significant transition-state stabilization during catalysis . The thionocephalosporin was also equally as good as its oxo analogue as a substrate of the class-B beta-lactamase II of Bacillus cereus and not significantly less effective as an inhibitor of the Streptomyces R61 DD-peptidase; a tight hydrogen-bond donor site for the beta-lactam side-chain amide is apparently not present in these enzymes either.

FEMS Microbiol Lett, 1992 Sep 15, 75(2-3), 187 - 92
Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species; Jahns T; A nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.3) inactivated by incubation at low temperatures was detected in several species of the genus Bacillus, including strains of B . cereus, B . laterosporus, B . lentus, B . panthotenicus, B . pasteurii, B . sphaericus, B . stearothermophilus, B . subtilis and B . thuringiensis . Incubation of cell-free extracts of these strains at 0 degrees C resulted in an 80-100% inactivation of NAD-GluDH activity within 120 min . The addition of 20% glycerol protected the enzyme from this inactivation in the cold . Strains of B . fastidiosus, B . licheniformis, B . macerans, B . megaterium and B . pumilus were found to lack NAD-GluDH activity.

Appl Environ Microbiol, 1992 Sep, 58(9), 2820 - 6
Immunological demonstration of a unique 3,4-dihydroxyphenylacetate 2,3-dioxygenase in soil Arthrobacter strains; Olson PE et al.; Many bacteria biosynthesize 3,4-dihydroxyphenylacetate 2,3-dioxygenases for growth on aromatic acids, but gram-negative organisms have been most extensively studied . A gram-positive strain containing 2,3-dioxygenase activity was identified as Arthrobacter strain Mn-1 . The 2,3-dioxygenase from strain Mn-1 was purified to homogeneity by fast protein liquid chromatography with a Mono Q anion-exchange column . Rabbit polyclonal antidioxygenase antibodies were prepared . Ouchterlony double-diffusion and Western blotting (immunoblotting) protocols were used to probe the distribution of the Mn-1 dioxygenase antigen in soil bacteria . Fourteen 2,3-dioxygenase-containing Bacillus and Pseudomonas strains did not contain immunologically cross-reactive proteins . Six of eight Arthrobacter strains contained 2,3-dioxygenase activity, and all of them produced cross-reactive proteins . The data presented here suggest that a unique type of dioxygenase is geographically widespread but is taxonomically confined to Arthrobacter soil bacteria.

JAMA, 1992 Sep 9, 268(10), 1280 - 6
Hospital outbreak of multidrug-resistant Mycobacterium tuberculosis infections . Factors in transmission to staff and HIV-infected patients; Beck-Sague C et al.; OBJECTIVE--To describe transmission of multidrug-resistant (MDR) Mycobacterium tuberculosis infection among patients and health care workers (HCWs) in a ward and clinic for human immunodeficiency virus (HIV)-infected patients in a hospital, four studies were conducted . METHODS--Case patients and control patients were persons who had been treated in the HIV ward or clinic, whose clinical course was consistent with tuberculosis and who had at least one positive culture for M tuberculosis between January 1, 1988, and January 31, 1990, resistant to at least isoniazid and rifampin (case patients), or whose isolates were susceptible to all drugs tested (control patients) . In the first study, case patients and control patients were compared to identify risk factors for MDR tuberculosis . In the second study, inpatient and outpatient days of MDR tuberculosis case patients were compared to determine whether acid-fast bacillus (AFB) smear-positivity or aerosolized pentamidine use was associated with higher numbers of subsequent MDR tuberculosis cases among exposed patients . In the third study, restriction fragment length polymorphism analysis was performed on available MDR and sensitive M tuberculosis isolates . In the fourth study, skin test conversion rates among HCWs in the HIV ward and clinic were compared with those of HCWs in another ward, and the strength of the associations between skin test conversions among HCWs on the HIV ward and the number of person-days that AFB smear-positive case patients and control patients were on this ward was estimated . RESULTS--Case patients were more likely than control patients to have been exposed on the HIV ward or clinic to an AFB smear-positive case patient (P less than .001) . Inpatient and outpatient days of MDR tuberculosis case patients were associated with more subsequent cases of MDR tuberculosis if exposing case patients were smear-positive or if they received aerosolized pentamidine (P less than or equal to .01) . Of 13 MDR isolates, all had one of two restriction fragment length polymorphism patterns; 10 sensitive isolates had restriction fragment length polymorphism patterns that were different from each other . The HCW skin test conversion rate was higher on the HIV ward and clinic than on the comparison ward (P less than .01) . The risk of occupational acquisition of infection increased in direct proportion to the number of person-days that AFB smear-positive case patients were on the HIV ward (r = .75; P = .005), but did not increase in proportion to the number of person-days that AFB smear-positive control patients were there (r = -.36; P = NS) . After isolation measures for AFB smear-positive tuberculosis patients were improved, MDR tuberculosis cases decreased to seven of 214 tuberculosis patients . CONCLUSIONS--Nosocomial transmission of MDR M tuberculosis infection to patients and HCWs occurred on the HIV ward and clinic . Infectiousness of MDR tuberculosis case patients was associated with AFB sputum-smear positivity . Case patients with MDR tuberculosis created a greater risk of skin test conversion for HCWs on the HIV ward than drug-susceptible control patients.

Biochemistry, 1992 Sep 8, 31(35), 8307 - 14
Construction of a stable dimer of Bacillus stearothermophilus lactate dehydrogenase; Jackson RM et al.; A molecular graphics analysis of the features which prevent cytosolic malate dehydrogenase dimers from forming tetramers was evaluated by its success in predicting the synthesis of a version of the LDH framework which is a stable dimer . Surface residues responsible for malate dehydrogenases being dimers were revealed by superimposing the structures of two dimers of pig cytosolic malate dehydrogenase on one homologous tetramer of L-lactate dehydrogenase from Bacillus stearothermophilus . Four regions were identified as composing the P-axis dimer-dimer interface . Two regions of the dimer were surface loops that collided when built as a tetramer: a large loop (residues 203-207, KNOBI) and a small loop (residues 264-269, KNOBII), and these were candidates to explain the dimeric character of malate dehydrogenase . The analysis was tested by constructing a synthetic B . stearothermophilus lactate dehydrogenase (KNOBI) containing the large malate dehydrogenase loop (residues 203-207 being AYIKLQAKE, and extra four amino acids) . The new construct was thermotolerant (90 degrees C) and enzymically active with kcat and KM (pyruvate) values similar to those of the wild-type enzyme . However, whereas the allosteric activator fructose 1,6-bisphosphate decreased KM 100 times for wild type, it had no influence on KNOBI . The molecular volumes of 1-120 microM concentrations of the construct were measured by time-resolved decay of tryptophan fluorescence anisotropy and by gel filtration . Both methods showed the molecular weight of wild type increased from dimer to tetramer with Kd about 20 microM dimer . KNOBI remained a dimer under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

J Leukoc Biol, 1992 Sep, 52(3), 303 - 6
Macrophage inflammatory proteins MIP-1 and MIP-2 are involved in T cell-mediated neutrophil recruitment; Appelberg R; Mice intraperitoneally (i.p.) infected with Mycobacterium bovis bacille Calmette-Guerin (BCG) respond to an i.p . challenge with mycobacterial antigen with an acute and extensive accumulation of neutrophils . This influx was not mimicked by the inoculation of recombinant interferon-gamma (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) . The antigen-induced recruitment of neutrophils was not affected by coinoculation of anti-IFN-gamma antibodies, was enhanced by anti-TNF-alpha antisera, and was significantly reduced by antisera against macrophage inflammatory proteins MIP-1 and MIP-2 . The latter two sera had no additive effects . We hypothesize that mycobacteria-specific T cells are triggered by antigen to secrete MIP-1 and MIP-2, which directly mediate, at least partly, the influx of neutrophils that takes place in this model.

Am Rev Respir Dis, 1992 Sep, 146(3), 752 - 6
Factors associated with tuberculin reactivity among the foreign-born in Montreal; Menzies R et al.; Because tuberculosis among the foreign-born is of increasing importance in North America, it has recently been recommended that newly arriving immigrants be tuberculin tested and preventive therapy given to all those with significant reactions . The factors affecting the prevalence of tuberculin reactions were assessed in a community-based tuberculin survey among foreign-born schoolchildren and young adults . Of 1,198 foreign-born who were tuberculin tested, 32.4% had significant tuberculin reactions . False-positive tuberculin reactions due to sensitivity to purified protein derivative (PPD)-B (for Mycobacterium avium) were uncommon and those due to BCG vaccination of importance only among immigrants from countries with low tuberculosis rates . Tuberculin reactions of 10+ mm were associated with tuberculosis rates in the country of origin (p less than or equal to 0.001), age when immigrated (p less than or equal to 0.001), bacillus Calmette-Guerin (BCG) vaccination (p less than or equal to 0.01), and residence in poorer neighborhoods in Montreal (p less than or equal to 0.001), but not with number of years resident in Canada . The booster phenomenon, seen in 16% of those undergoing two-step testing, was most strongly associated with prior BCG vaccination (p less than or equal to 0.001) and also with tuberculosis rates in the country of origin (p less than or equal to 0.08), age of immigration (p less than or equal to 0.01), and number of years resident in Canada (p less than or equal to 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Chest, 1992 Sep, 102(3), 972 - 4
Polypoid endobronchial lesions . A manifestation of bacillary angiomatosis; Slater LN et al.; Polypoid endobronchial lesions occurred in a patient with acquired immunodeficiency syndrome (AIDS) with recent fever, skin lesions, lymphadenopathy, lung infiltrates, and pleural effusions . His condition improved with antimicrobials and vincristine . After therapy ceased, skin lesions recurred and gastroesophageal mucosal lesions developed . Bacillary angiomatosis was identified during retrospective analysis of skin and endobronchial biopsy specimens.

Clin Exp Immunol, 1992 Sep, 89(3), 402 - 6
The pattern of mycobacterial antigen recognition in sera from Mantoux-negative individuals is essentially unaffected by bacille Calmette-Guérin (BCG) vaccination in either south India or London; Das S et al.; Paired sera were obtained before and 8 weeks after routine BCG vaccination from 20 PPD-S Mantoux-negative individuals who were living adjacent to the Chingleput BCG vaccine trial area in Tamil Nadu and from seven Mantoux-negative school-children in London, UK . Most subjects became Mantoux-positive after vaccination . In ELISA tests against soluble extracts of BCG or Mycobacterium tuberculosis H37Rv or against PPD-S, pre-vaccination antibody titres of South Indian subjects were about twice those of British subjects but there was no increase in titre of antibodies after vaccination of either population . Western blotting showed that even before vaccination, and even in British subjects, antibodies were present that recognized numerous antigenic components in extracts of BCG and M . tuberculosis . There was no consistent difference between band patterns with South Indian and British subjects and any effect of vaccination on the patterns was minimal.

J Urol, 1992 Sep, 148(3), 900 - 5
Bacillus Calmette-Guerin abrogates in vitro invasion and motility of human bladder tumor cells via fibronectin interaction; Garden RJ et al.; Intravesical bacillus Calmette-Guerin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma of the bladder (TCC) . The mechanisms by which BCG limits tumor cell activity have thus far been unclear . We investigated the interaction between BCG and invasive human TCC cell line EJ in an in vitro invasion assay . We observed that BCG inhibited the invasion of EJ cells through an artificial basement membrane . In terms of the steps involved in tumor cell invasion, i.e . attachment, proteolysis, and motility, BCG was found to limit tumor cell motility . Attachment and proliferation of tumor cells were not affected by BCG . The effects of BCG on tumor cell migration were mediated by fibronectin (FN), a basement membrane glycoprotein component . Abrogation of BCG-FN-tumor cell interactions with anti-FN antibodies eliminated the ability of BCG to block tumor cell invasion . Fibronectin appears to link BCG and tumor cells via independent FN binding receptors to separate domains of the FN molecule . The molecular mechanism by which BCG may limit tumor cell motility may be its ability to protect against the formation of specific FN sequences as a result of protease cathepsin B digestion . A 31 kD and 27 kD FN band were absent from purified or tumor cell associated cathepsin B digestion when incubated in the presence of BCG, but present in the absence of BCG . Furthermore when purified from SDS polyacrylamide gel electrophoresis, the fragments were shown to have motility stimulating activity for the invasive EJ cells . These findings suggest that BCG functions as a potent inhibitor of tumor cell invasion . We conclude that BCG-fibronectin-tumor cell interactions may have a direct influence on the invasive mechanisms, such as motility, of tumor cells.

J Urol, 1992 Sep, 148(3), 797 - 801
Management of stage T1 superficial bladder cancer with intravesical bacillus Calmette-Guerin therapy; Cookson MS et al.; We reviewed our results with 86 patients who had a pretreatment history of a stage T1 tumor . All patients were treated with transurethral resection of all visible tumor followed by intravesical bacillus Calmette-Guerin (BCG) and many patients received additional maintenance therapy . Local recurrences were treated with repeat transurethral resection followed by additional BCG . Median followup was 59 months, with a range of 9 to 149 months . Overall, 78 of 86 patients (91%) were free of tumor recurrence with BCG therapy . This result includes 69% of the patients who responded to the initial transurethral resection and intravesical BCG, and 22% who ceased having tumors after additional treatments for local recurrences . Only 7% of the patients had progression to stage T2 tumors after BCG therapy . Grade of the stage T1 tumor, concurrent carcinoma in situ and tumor multiplicity before BCG did not predict tumor recurrence or progression . Of patients with recurrences after BCG therapy, those with stage T1 tumors had a higher rate of progression compared to those with stage Ta tumors but the difference was not statistically significant (p = 0.086) . These data clearly support the efficacy of transurethral resection plus intravesical BCG immunotherapy in the treatment of stage T1 tumors as well as in the prevention of disease progression.

Biochemistry, 1992 Sep 1, 31(34), 7787 - 95
Evidence for temperature-dependent conformational changes in the L-lactate dehydrogenase from Bacillus stearothermophilus; Kotik M et al.; L-Lactate dehydrogenase from Bacillus stearothermophilus (BSLDH) has been shown to change its conformation in a temperature-dependent manner in the temperature range between 25 and 70 degrees C . To provide a more detailed understanding of this reversible structural reorganization of the tetrameric form of BSLDH, we have determined in the presence of 5 mM fructose, 1,6-bisphosphate (FBP) the effect of temperature on far-UV and near-UV circular dichroism (CD), Nile red-binding to the enzyme surface, NADH binding, fluorescence polarization of fluorescamine-labeled protein, and hydrogen-deuterium exchange . In addition, we have analyzed the temperature dependence of the dimer-tetramer equilibrium of this protein by steady-state enzyme kinetics in the absence of FBP . The results obtained from these measurements at various temperatures can be summarized as follows . No changes in the secondary-structure distribution are detectable from far-UV CD measurements . On the other hand, near-UV CD data reveal that changes in the arrangements of aromatic side chains do occur . With increasing temperature, the asymmetry of the environment around aromatic residues decreases with a small change at 45 degrees C and a more pronounced change at 65 degrees C . Nile red-binding data suggest that the BSLDH surface hydrophobicity changes with temperature . It appears that decreasing the surface hydrophobicity may be a strategy to increase the protein stability of the active enzyme . We have noted significant alterations in the thermodynamic binding parameters of NADH above 45 degrees C, indicating a conformational change in the active site at 45 degrees C . The hydrodynamic volume of BSLDH is also temperature dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioorg Khim, 1992 Sep, 18(9), 1186 - 9
{Site-specific endonuclease BstM6I from Bacillus stearothermophilus M6}; Shapovalova NI et al.; A site-specific endonuclease activity was found in extract of Bacillus stearothermophilus M6 isolated from molasses . The functionally pure enzyme designated as BstM6I was obtained by consecutive chromatographies on blue sepharose, hydroxyapatite, and heparin-sepharose . The endonuclease recognizes the nucleotide sequence CC decreases WGG in double-stranded DNA and cleaves it as indicated by the arrow to give one-nucleotide 5'-protruding ends . Consequently, the site-specific endonuclease BstM6I is an isoshizomer of BstNI.

Trop Med Parasitol, 1992 Sep, 43(3), 191 - 4
Detection of Bartonella bacilliformis in cultures, blood, and formalin preserved skin biopsies by use of the polymerase chain reaction; Maass M et al.; A polymerase chain reaction (PCR) is described for the detection of Bartonella bacilliformis, the etiologic agent of bartonellosis, which cannot be identified biochemically . Amplification of a genomic 231 bp Bartonella DNA sequence permitted specific identification of 12 Bartonella isolates from Peruvian bartonellosis patients as well as detection of Bartonella DNA in blood samples and formaldehyde preserved skin biopsies . Specificity of amplification products was confirmed by restriction fragment analysis . No positive results were obtained with Brucella abortus, phylogenetically closely related to B . bacilliformis, and several other bacterial, fungal, and protozoal species . PCR appears as a promising technique for specific identification of B . bacilliformis in cultures and in clinical materials with further applications in taxonomic studies and in the investigation of Bartonella-like isolates obtained outside South America.

Chem Pharm Bull (Tokyo), 1992 Sep, 40(9), 2478 - 82
Changes in enzymatic and membrane-adsorbing activities of sphingomyelinase from Bacillus cereus by modification with a polyethylene glycol derivative; Matsuyama H et al.; Sphingomyelinase (SMPLC) from Bacillus cereus was modified with a polyethylene glycol (PEG) derivative, methoxypolyethylene glycol-succinimidyl succinate (ss-PEG) . The molecular weight of the ss-PEG-modified SMPLC was calculated to be approx . 150 kDa by gel-filtration whereas that of the native enzyme, was 25 kDa . By this modification, the enzyme increased its thermostability and retained its hydrolytic activity toward 2-(N-hexadecanoylamino)-4-nitrophenylphosphocholine (HNP) and sphingomyelin (SM) in the mixed micelles with the surfactants such as Triton X-100 and sodium deoxycholate (SDC) . However, the activity toward liposomal SM was significantly decreased, and all the enzyme activities toward bovine erythrocytes, including membraneous SM-hydrolyzing and hemolytic activities as well as the enzyme adsorption onto the erythrocyte membranes, were completely lost.

Br J Urol, 1992 Sep, 70(3), 271 - 5
Outcome in carcinoma in situ of bladder treated with intravesical bacille Calmette-Guérin; Harland SJ et al.; Fifty-three patients with carcinoma in situ of the bladder were treated with Evans strain BCG given intravesically . Complete remission was achieved after either one or two 6-weekly courses in 53% of patients . The median duration of remission was 32 months . Treatment-related bladder symptoms were minor during the first course, more severe during the second . There was no relation between severity of symptoms and likelihood of response . With a median follow-up of 32 months, disease progression has occurred in 10% of complete responders, whereas failure to respond on either cystoscopic, histological or cytological grounds was associated with a 48% progression rate . Although intravesical BCG produces impressive responses in carcinoma in situ of the bladder, managed conservatively the condition remains a dangerous one.

Hinyokika Kiyo, 1992 Sep, 38(9), 1063 - 5
{Multiorgan failure following intravesical bacillus Calmette-Guerin administration: a case report}; Baba Y et al.; We describe a case of multiorgan failure after intravesical bacillus Carmette-Guern (BCG) immunotherapy for bladder cancer . A 58-year-old man with superficial transitional cell carcinoma of the bladder was initially treated by transurethral resection and intravenous chemotherapy, and then administered 11 sessions of BCG intravesically . He was administered BCG intravesically after cystoscopic examination . The next day he complained of nausea and malaise . He became hypotensive . The symptom progressed with multiorgan failure, disseminated intravascular coagulation and respiratory failure . The patient gradually improved with administration of antibiotics and corticosteroid, and hemodialysis, without antituberculous antibiotics . Intravesical instillation of BCG should not be carried out immediately after cystoscopic examination.

J Clin Microbiol, 1992 Sep, 30(9), 2502 - 3
Bilophila wadsworthia bacteremia in two patients with hepatic abscesses; Kasten MJ et al.; Bilophila wadsworthia, a recently described anaerobic gram-negative bacillus, was isolated from blood cultures of two patients with liver abscesses . These isolates exhibited the expected phenotypic characteristics, and identification was confirmed by cell wall fatty acid analysis by gas-liquid chromatography . This is the first documentation of bacteremia due to B . wadsworthia.

Biosci Biotechnol Biochem, 1992 Sep, 56(9), 1429 - 33
Effects of Bacillus thuringiensis var . israelensis 20-kDa protein on production of the Bti 130-kDa crystal protein in Escherichia coli; Yoshisue H et al.; A 20-kDa protein of Bacillus thuringiensis var . israelensis (Bti) has been shown to be necessary for the efficient expression of the 27-kDa mosquitocidal protein gene in Escherichia coli . We have investigated the effects of this 20-kDa protein on the expression of two 130-kDa mosquitocidal protein genes (cryIVA, cryIVB) in E . coli by supplying the 20-kDa protein gene in trans . When a recombinant plasmid, pLH4BX, which was constructed to express cryIVA under the E . coli lac promoter on pUC19, coexisted with the 20-kDa protein gene, a striking increase in production of the fused CryIVA was detected . This was not accompanied by an increase in the amount of intracellular mRNA, suggesting that 20-kDa protein exerts a posttranscriptional effect . We conclude that the 20-kDa protein also stimulates the production of 130-kDa protein in E . coli.

Biosci Biotechnol Biochem, 1992 Sep, 56(9), 1406 - 9
A novel and efficient method for enzymatic synthesis of high purity maltose using moranoline (1-deoxynojirimycin); Maruo S et al.; A transglycosylation reaction with moranoline (1-deoxynojirimycin) was done with soluble starch as the glucosyl donor and Bacillus macerans amylase as a cyclodextrin glycosyltransferase {EC 2.4.1.19} . The resultant transglycosylation products with moranoline, obtained by treating the reaction mixture with a strong cation exchange resin, were hydrolyzed by beta-amylase {EC 3.2.1.2} from sweet potatoes . The hydrolysate was treated with a strong cation exchange resin, and high purity maltose was obtained.

Biosci Biotechnol Biochem, 1992 Sep, 56(9), 1386 - 91
Acceptor specificity of cyclodextrin glycosyltransferase from Bacillus stearothermophilus and synthesis of alpha-D-glucosyl O-beta-D-galactosyl-(1----4)-beta-D-glucoside; Kitahata S et al.; Bacillus stearothermophilus CGTase had a wider acceptor specificity than Bacillus macerans CGTase did and produced large amounts of transfer products of various acceptors such as D-galactose, D-mannose, D-fructose, D- and L-arabinose, D- and L-fucose, L-rhamnose, D-glucosamine, and lactose, which were inefficient acceptors for B . macerans CGTase . The main component of the smallest transfer products of lactose was assumed to be alpha-D-glucosyl O-beta-D-galactosyl-(1----4)-beta-D-glucoside.

Immunobiology, 1992 Sep, 185(5), 466 - 74
T cell maturation stage-linked heterogeneity of the glycosylphosphatidylinositol membrane anchor of Thy-1; Nakashima I et al.; We showed that some of Thy-1 molecules on murine thymocytes are resistant to phosphatidylinositol-specific phospholipase C (PI-PLC) derived from Bacillus thuringiensis . Both immature thymocytes with low CD3 expression and mature thymic T lymphocytes with high CD3 expression carried the PI-PLC-resistant Thy-1, and the PI-PLC-sensitivity of Thy-1 extensively varied among thymocyte subpopulations . In contrast, the same PI-PLC fully hydrolysed the anchor of Thy-1 on peripheral T lymphocytes . When the latter cells were activated with mitogen in vitro, however, some Thy-1 on them became resistant to PI-PLC . We then found that virtually all Thy-1 molecules on thymocytes became sensitive to PI-PLC when they were treated with hydroxylamine that should cleave ester-linked lipids . The result ruled out the possibility that the PI-PLC-resistant Thy-1 had a transmembranous peptide sequence, and suggested the presence of an additional fatty acyl group on the inositol ring of the Thy-1 anchor . In addition, the molecular size of the PI-PLC-resistant membrane-bound Thy-1 was only marginally larger than that of the PI-PLC-sensitive solubilized Thy-1 in detergent-partitioning SDS-PAGE analysis.

J Am Mosq Control Assoc, 1992 Sep, 8(3), 285 - 9
Factors influencing the activity of Bacillus thuringiensis var . israelensis treatments; Becker N et al.; Environmental factors influence the effectiveness of microbial control agents in mosquito control programs . Four of these factors (water temperature, larval density, sunlight and the effect of associated filter feeders) were studied with Bacillus thuringiensis var . israelensis under laboratory and semifield conditions in Europe using different instars of Aedes vexans, Ae . aegypti and Culex pipiens . Bioassays conducted at a low temperature (5 degrees C) yielded 10-fold higher LC50 and LC90 values compared with those conducted at a high temperature (25 degrees C) . The efficacy of B.t.i . decreased in a linear manner with increasing larval density . Sunlight can reduce the effectiveness of B.t.i . by several times . Competition in food intake by filter feeding Daphnia resulted in lower mortality of mosquito larvae after B.t.i . applications.

Prikl Biokhim Mikrobiol, 1992 Sep-Oct, 28(5), 731 - 7
{Bacterial polysaccharides of polymyxan 88A . Basic characteristics and extent of possible uses}; Matora AV et al.; A new high-viscous polysaccharide polymyxan from Bacillus polymyxa 88A is described . Polymyxan consists of an acid high-viscous polysaccharide (Mw 1-10 MD) and a neutral low-viscous polysaccharide (Mw 100-300 kD), which is a glucomannan containing equal amounts of monosaccharides and traces of uronic acids . The acid high-viscous polysaccharide consists of 36% glucose, 36% mannose, 7% galactose and 21% glucuronic acid . Data are presented on the application of polymyxan in baking industry and for preparation of drilling muds.

J Invertebr Pathol, 1992 Sep, 60(2), 121 - 6
Processing of delta-endotoxin from Bacillus thuringiensis subsp . kurstaki HD-1 and HD-73 by gut juices of various insect larvae; Ogiwara K et al.; Midgut juices were prepared from Adoxophyes sp., smaller tea tortrix (STT); Bombyx mori, silkworm (SW); Spodoptera litura, common cutworm (CCW); Plutella xylostella, diamondback moth (DBM); and Musca domestica, housefly (HF) and immobilized onto Sepharose 4B . delta-Endotoxins (ICPs) from Bacillus thuringiensis subsp . kurstaki HD-1 and HD-73 were digested by these immobilized gut juice proteases . All gut juices tested derived relatively proteolytic resistant cores from ICP . The molecular sizes of these cores, about 55 kDa in SDS-PAGE, were resulted . In the case of CCW, however, digestion was very strong and only 1/20 concentration of core protein remained relative to other digests . The N-terminal amino acid sequencing of the core proteins showed that they were truncated at the very end of the N-terminus of protoxin, CryIA, at different sites . Although housefly larvae were completely insensitive to active toxin, the gut juice produced the core, suggesting that the housefly may lack the binding sites for the core-active toxin.

Biochem J, 1992 Sep 1, 286 ( Pt 2), 435 - 40
Characterization of a Zn(2+)-requiring glycerophosphocholine cholinephosphodiesterase possessing p-nitrophenylphosphocholine phosphodiesterase activity; Sok DE et al.; p-Nitrophenylphosphocholine phosphodiesterase activity was purified 5000-fold from mouse brain by treatment of membranes with Bacillus cereus phospholipase C preparation and sequential chromatographies on concanavalin A-Sepharose and CM-Sephadex columns . The phosphodiesterase (Zn(2+)-requiring) showed Km and Vmax . values of 5.5 microM and 4.2 mumol/min per mg respectively in the hydrolysis of p-nitrophenylphosphocholine, and possessed an optimum pH of 10.5 and a molecular mass of approx . 74 kDa . The purified enzyme was found to convert glycerophosphocholine into glycerol and phosphocholine, with Km and Vmax . of 48 microM and 5 mumol/min per mg respectively . In the hydrolysis of glycerophosphocholine the enzyme also exhibited a Zn2+ requirement and optimal pH at 10.5 . Additionally, the p-nitrophenylphosphocholine phosphodiesterase activity was competitively inhibited by glycerophosphocholine, with a Ki value of 50 microM . These observations, together with chromatographic behaviour and heat-denaturation analyses, indicate that both p-nitrophenylphosphocholine phosphodiesterase and glycerophosphocholine cholinephosphodiesterase activities reside in the same protein.

J Bacteriol, 1992 Sep, 174(18), 5936 - 40
Kinetic properties of electrogenic Na+/H+ antiport in membrane vesicles from an alkalophilic Bacillus sp; Kitada M et al.; The effects of imposed proton motive force on the kinetic properties of the alkalophilic Bacillus sp . strain N-6 Na+/H+ antiport system have been studied by looking at the effect of delta psi (membrane potential, interior negative) and/or delta pH (proton gradient, interior alkaline) on Na+ efflux or H+ influx in right-side-out membrane vesicles . Imposed delta psi increased the Na+ efflux rate (V) linearly, and the slope of V versus delta psi was higher at pH 9 than at pH 8 . Kinetic experiments indicated that the delta psi caused a pronounced increase in the Vmax for Na+ efflux, whereas the Km values for Na+ were unaffected by the delta psi . As the internal H+ concentration increased, the Na+ efflux reaction was inhibited . This inhibition resulted in an increase in the apparent Km of the Na+ efflux reaction . These results have also been observed in delta pH-driven Na+ efflux experiments . When Na(+)-loaded membrane vesicles were energized by means of a valinomycin-induced inside-negative K+ diffusion potential, the generated acidic-interior pH gradients could be detected by changes in 9-aminoacridine fluorescence . The results of H+ influx experiments showed a good coincidence with those of Na+ efflux . H+ influx was enhanced by an increase of delta psi or internal Na+ concentration and inhibited by high internal H+ concentration . These results are consistent with our previous contentions that the Na+/H+ antiport system of this strain operates electrogenically and plays a central role in pH homeostasis at the alkaline pH range.

Transgenic Res, 1992 Sep, 1(5), 228 - 36
Synthetic cryIIIA gene from Bacillus thuringiensis improved for high expression in plants; Sutton DW et al.; A 1974 bp synthetic gene was constructed from chemically synthesized oligonucleotides in order to improve transgenic protein expression of the cryIIIA gene from Bacillus thuringiensis var . tenebrionis in transgenic tobacco . The crystal toxin genes (cry) from B . thuringiensis are difficult to express in plants even when under the control of efficient plant regulatory sequences . We identified and eliminated five classes of sequence found throughout the cryIIIA gene that mimic eukaryotic processing signals and which may be responsible for the low levels of transcription and translation . Furthermore, the GC content of the gene was raised from 36% to 49% and the codon usage was changed to be more plant-like . When the synthetic gene was placed behind the cauliflower mosaic virus 35S promoter and the alfalfa mosaic virus translational enhancer, up to 0.6% of the total protein in transgenic tobacco plants was cryIIIA as measured from immunoblot analysis . Bioassay data using potato beetle larvae confirmed this estimate.

P N G Med J, 1992 Sep, 35(3), 179 - 85
Factors affecting the use of immunization among urban settlement dwellers in Papua New Guinea; Freeman PA et al.; This paper examines the importance of selected social factors in the acceptance of childhood immunization in urban settlements in Port Moresby, Papua New Guinea . The study found that the provision of information to mothers on when to start immunization and how often the child should be immunized were key factors in determining immunization status . Maternal education was found to be positively associated with knowledge of immunization, but was not significantly associated with actual immunization practice . Over 70% of the women studied found out when to attend for immunization from the local maternal and child health (MCH) staff, hence emphasizing their importance in disseminating information . If coverage is to be increased further in urban settlements with mobile populations, maximal use must be made of local community organizations to disseminate the key immunization information and for follow-up of newborns and infants new to the community . Efforts to encourage mothers to deliver under supervision should also continue as this is an important point of first contact for immunizationPIP: A community-based study was carried out between July 30 and August 13, 1990, in order to assess the level of immunization coverage in 11 Port Moresby urban settlements and to examine selected social factors in the completion of the immunization . The population comprised women living in urban settlements with children under the age of 2 years . Multistage sampling was used to select the sample . A sample size of 323 children was derived on an expectation of 70% coverage, with an allowance for 5% error . 345 mothers were interviewed in Melanesian Pidgin by trained interviewers and 345 children were included in the survey . Of the mothers, only 154 had ever attended school and only 128 (37%) had completed more than 4 years of school . Maternal education was positively associated with residence in planned settlements (p 0.01) . Only 189 (55%) of mothers were able to explain the preventive effects of immunization . 58% did not know when immunizations should commence and 48% did not know how many times the child should be immunized in the first year of life . When asked what prevented them from attending immunization clinics, lack of money to pay the bus fare was the most common reason given (n = 24), followed by indifference (n = 23) and sickness in the family (n = 8) . Longer maternal education was associated with knowledge of when to start childhood immunizations (p 0.001), and of the number of times that children should be immunized (p 0.001) . Mothers' education was also associated with the ability of mothers to name the diseases which immunization prevents (p 0.001) . Among 232 (79%) of 293 children more than 1 year old, the immunization coverage was 93.5% for bacillus Calmette-Guerin, 85.3% for third dose of tetanus and oral poliomyelitis virus, and 82.8% for measles vaccine . 77.6% of children had received all antigens . The knowledge of mothers of when to start immunization was positively associated with actual immunization practice (p 0.01), as was the knowledge of the number of times that a child should be immunized (p 0.006) .

Nippon Koshu Eisei Zasshi, 1992 Sep, 39(9), 721 - 8
{A study on the distortion of distributions of delays in detecting pulmonary tuberculosis}; Toyota M et al.; It is reported that a cumulated percentage of the interval between the onset of symptoms and the first visit to a doctor (patient delay) and that between the first visit and diagnosis (doctor's delay) among pulmonary tuberculosis (TB) patients demonstrate a straight line on the log-normal probability paper . Factors influencing the straight line nature of these delays were analyzed . The samples consist of 454 patients who were diagnosed between 1986 and 1988, from the area served by the Kochi Chuo Health Center . They were divided into two groups; 125 patients detected due to the manifestation of additional symptoms related to TB while being treated for other diseases (group A), and 329 patients detected only after the onset of TB symptoms (group B) . Results are as follows . 1) When considering all patients (group A+B), patient delay among cases found through positive examination of tubercle bacillus and the doctor's delay among cases whose bacillus examination was negative demonstrated a straight line . However patient delay among bacillus negative cases demonstrated a downward tendency and the doctor's delay among bacillus positive cases demonstrated a upward tendency on the log-normal probability paper . 2) In the B group, both the patient's and doctor's delay demonstrated virtually straight lines among both bacillus positive and negative cases . However, in the A group, a straight line among bacillus positive cases was demonstrated, while a downward tendency was revealed among the bacillus negative cases.(ABSTRACT TRUNCATED AT 250 WORDS)

J Int Med Res, 1992 Sep, 20(5), 406 - 21
Effects of biological response modifiers with different modes of action used separately and together on immune responses in mice with syngeneic tumours; Matsunaga K et al.; The effect of a protein-bound polysaccharide (PSK) obtained from cultured mycelia of the Basidiomycetes Coriolus versicolor on activities involved in the host defence mechanism of C57BL/6 mice bearing adenocarcinoma 755 was compared with that of live bacille Calmette-Guerin (BCG) . Delayed footpad reaction, the activity of splenic natural killer cells and interferon production induced by concanavalin A in splenic cells of healthy mice were little affected by PSK, but in mice bearing tumours PSK prevented the tumour-induced reduction in these activities . Live BCG augmented these activities in healthy mice but had little effect on the reduction of activities induced by a tumour . The immunosuppressive activity of the serum of tumour-bearing mice was reduced by PSK administration; live BCG did not have this effect . The combined use of live BCG and PSK improved these activities in the host, with synergistic increases in the antitumour effect . These results suggest that the combined use of live BCG and PSK, which have different modes of action, may be useful in the treatment of cancer.

Biochim Biophys Acta, 1992 Aug 21, 1122(3), 243 - 50
Proteolytic modification of raw-starch-digesting amylase from Bacillus circulans F-2 with subtilisin: separation of the substrate-hydrolytic domain and the raw substrate-adsorbable domain; Kim CH et al.; Raw starch-digesting amylase (BF-2A, 93,000 Da) from Bacillus circulans F-2 was converted into two components during digestion with subtilisin . The two components were separated and designated BF-2A' (63 kDa) and BF-2B (30 kDa), respectively . BF-2A' exhibited the same hydrolysis curve for soluble starch as the original amylase (BF-2A) . Moreover, the catalytic activities of original and modified enzymes were indistinguishable in Km, Vmax and in their specific activity for soluble starch hydrolysis . However, its absorbability and digestibility on raw starch was greatly decreased . Furthermore, the enzymatic action pattern on soluble starch was differed greatly from that of BF-2A . The stability of the enzymes decreased below pH 5.5 and at 50 degrees C, while it was quite stable even at pH 12 . On the other hand, the smaller peptide (BF-2B) could be adsorbed onto raw starch . From these results, it is suggested that the larger peptide (BF-2A') has a region responsible for the expression of the enzyme activity to hydrolyze soluble substrate, and the smaller peptide (BF-2B) plays a role on raw starch adsorption and also contributes to the original enzyme-to-enzyme stabilization . A proposed model of the raw-starch-digesting enzyme from this strain is extensively discussed.

FEBS Lett, 1992 Aug 17, 308(2), 141 - 5
Essential catalytic role of Glu134 in endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase from B . licheniformis as determined by site-directed mutagenesis; Planas A et al.; Site-directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase (beta-glucanase) have been performed . Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes . Four mutant enzymes were expressed and purified from recombinant E . coli and their kinetics analysed with barley beta-glucan . Replacement of Glu134 by Gln produced a mutant (E134Q) that retains less than 0.3% of the wild-type activity . The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild-type indicative of their participation in substrate binding and transition-state complex stabilization . Glu134 is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst . These results strongly support a lysozyme-like mechanism for this family of Bacillus beta-glucan hydrolases with Glu134 being the essential acid catalyst.

Anal Biochem, 1992 Aug 15, 205(1), 151 - 8
A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry: identification of the positions of carbohydrate-linked asparagine in recombinant alpha-amylase by treatment with peptide-N-glycosidase F in 18O-labeled water; Gonzalez J et al.; Previously, a combined use of fast atom bombardment (FAB) mass spectrometry and peptide N-glycosidase F, an enzyme that cleaves the beta-aspartylglycosylamine linkage of Asn-linked carbohydrates, was successfully applied to identification of N-glycosylation sites in a glycoprotein with the known or DNA-derived sequence (S . A . Carr and G . D . Roberts, 1986, Anal . Biochem . 157, 396-406) . Here, we extended the method for easier identification of N-glycosylation sites in a glycoprotein even with unknown sequence . The glycoprotein is digested with peptide-N-glycosidase F in buffer containing 40 at% H2 18O, to yield a deglycosylated protein whose carbohydrate-linked Asn residues are converted to Asp partly labeled with 18O at their beta-carboxyl group during this digestion . The deglycosylated protein is further digested with proteolytic enzymes in an appropriate buffer prepared with normal water, and then peptides are separated on a reversed-phase column by HPLC . Peptides in which carbohydrate-linked Asn has been converted to Asp show a pair of signals ({M + 1}+ and {M + 3}+) in FAB mass spectra due to the partial incorporation of 18O into the beta-carboxyl groups of Asp residues, while the other peptides show normal isotopic ion distributions . Thus, both formally N-glycosylated peptides and, using collision-induced dissociation analysis, N-glycosylation sites can be identified . The application of the present method to the determination of N-glycosylation sites in a recombinant glycoprotein, Bacillus licheniformis alpha-amylase, is described.

Appl Environ Microbiol, 1992 Aug, 58(8), 2571 - 8
Rapid in situ hybridization technique using 16S rRNA segments for detecting and differentiating the closely related gram-positive organisms Bacillus polymyxa and Bacillus macerans; Jurtshuk RJ et al.; A rapid, sensitive, inexpensive in situ hybridization technique, using 30-mer 16S rRNA probes, can specifically differentiate two closely related Bacillus spp., B . polymyxa and B . macerans . The 16S rRNA probes were labeled with a rhodamine derivative (Texas Red), and quantitative fluorescence measurements were made on individual bacterial cells . The microscopic fields analyzed were selected by phase-contrast microscopy, and the fluorescence imaging analyses were performed on 16 to 67 individual cells . The labeled 16S rRNA probe, POL, whose sequence was a 100% match with B . polymyxa 16S rRNA but only a 60% match with B . macerans 16S rRNA, gave quantitative fluorescence ratio measurements that were 34.8-fold higher for B . polymyxa cells than for B . macerans cells . Conversely, the labeled probe, MAC, which matched B . polymyxa 16S rRNA in 86.6% of its positions and B . macerans 16S rRNA in 100% of its positions, gave quantitative fluorescence measurements that were 59.3-fold higher in B . macerans cells than in B . polymyxa cells . Control probes, whose 16S rRNA sequence segment (P-M) was present in both B . polymyxa and B . macerans as well as a panprokaryotic probe (16S), having a 100% match with all known bacteria, hybridized equally well with both organisms . These latter hybridizations generated very high fluorescence signals, but their comparative fluorescence ratios (the differences between two organisms) were low . The control paneukaryotic probe (28S), which had less than 30% identity for both B . macerans and B . polymyxa, did not hybridize with either organism.

Carbohydr Res, 1992 Aug 3, 232(2), 197 - 205
The crystal structure and physicochemical properties of L-ascorbic acid 2-glucoside; Mandai T et al.; The stable L-ascorbic acid glucoside produced by the action of the cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) from Bacillus stearothermophilus was crystallized from an aqueous solution . Determination of the molecular structure by single crystal X-ray analysis showed the compound to be 2-O-alpha-D-glucopyranosyl-L-ascorbic acid (AA-2G) . The crystals are orthorhombic, space group P2(1)2(1)2(1), with unit-cell dimensions a = 11.929 A, b = 24.351 A, and c = 4.864 A . The D-glucopyranose residue has the 4C1 conformation . These conclusions are in good agreement with those based on the 13C-NMR spectrum . The general physicochemical properties of crystalline AA-2G are reported.

J Bacteriol, 1992 Aug, 174(16), 5346 - 53
Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene; de Vries GE et al.; The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced . The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes . Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence . After purification of MDH from E . coli, the kinetic and biochemical properties of the enzyme were investigated . The physiological effect of MDH synthesis in E . coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed.

Gene, 1992 Aug 1, 117(1), 99 - 101
Bca77I: a new type-II restriction endonuclease from Bacillus caldolyticus cutting at the (A/T) increases CCGG(A/T) site; Schoenfeld T et al.; We describe a new restriction enzyme recognizing a degenerate hexanucleotide sequence and cleaving the 5'-W increases CCGGW site (W = A or T) . This enzyme cuts with high efficiency all four permutations of this sequence; ACCGGA (TCCGGT on the opposite strand), ACCGGT and TCCGGA . Methylation of the external cytosine completely blocks restriction while methylation of the internal cytosine only decreases the rate of restriction activity.

Cancer Res, 1992 Aug 1, 52(15), 4286 - 90
Murine bladder carcinoma cells present antigen to BCG-specific CD4+ T-cells; Lattime EC et al.; Intravesical administration of Bacillus Calmette-Guerin (BCG) is the most effective therapy for superficial transitional cell carcinoma of the bladder although its mechanism of action is not known . To determine if bladder tumors are capable of antigen presentation and thus might interact directly with BCG-specific T-cells, we studied the murine bladder tumor MB49 . MB49 (MHC Class II negative) (IA-), when induced to express IA with interferon, presented BCG to specific CD4+ T-cells obtained from bladder-draining lymph nodes following intravesical BCG administration . This interaction resulted in antigen- and IA-dependent interleukin 2 and tumor necrosis factor production . Interferon also induced MB49 IA expression in vivo . This first demonstration of antigen presentation by epithelial tumors supports new approaches to immunotherapy of these malignancies.

Urol Clin North Am, 1992 Aug, 19(3), 565 - 72
Complications of bacillus Calmette-Guérin immunotherapy; Lamm DL; Knowing when to give and when to withhold BCG will prevent most complications, but even when all precautions are taken, some complications will occur . The initial step in the treatment of infectious complications is the use of isoniazid . Routine prophylactic isoniazid should not be given, as animal studies have confirmed that immune stimulation, and presumably antitumor activity, can be inhibited by isoniazid prophylaxis . However, when cystitis persists more than 2 days or is so severe that it does not respond to symptomatic treatment, isoniazid 300 mg daily is used to control the symptoms, prevent progressive infection, and avoid the overgrowth of BCG, which can result in excessive organisms and suppression of the immune response . If symptoms progress despite isoniazid treatment or do not begin to abate within 1 to 2 weeks, rifampicin 600 mg daily is added . Rifampicin is given from the beginning in patients with potentially severe extravesical BCG infection such as pneumonitis, hepatitis, or nephritis . In patients with symptoms such as fever, malaise, or bladder irritation that respond within a few days, it generally is necessary to continue antitubercular antibiotics for only 2 weeks . Those with extravesical infection and those who do not respond promptly to treatment are treated for 3 months, and those with severe or deep-seated infection are treated for 6 months . The current recommendation for the treatment of sepsis after BCG administration, based on limited clinical experience and controlled animal experimentation, is to use isoniazid and rifampicin plus prednisolone 40 mg daily.(ABSTRACT TRUNCATED AT 250 WORDS)

Urol Clin North Am, 1992 Aug, 19(3), 557 - 64
Bacillus Calmette-Guérin immunotherapy . Techniques and results; Brosman SA; Intravesical instillation of bacillus Calmette-Guerin (BCG) has become an important adjunct in the management of patients with stages Ta and T1 transitional-cell carcinoma of the bladder and carcinoma in situ (CIS) . For BCG to be effective, patients should be assigned to the appropriate protocol . With proper treatment, tumor recurrences can be prevented in as many as 80% of patients, residual tumor eradicated in 60%, and CIS eliminated in 70% of appropriately selected patients.

J Bacteriol, 1992 Aug, 174(15), 5013 - 20
Cloning, nucleotide sequences, and enzymatic properties of glucose dehydrogenase isozymes from Bacillus megaterium IAM1030; Nagao T et al.; Bacillus megaterium is known to have several genes that code for isozymes of glucose dehydrogenase . Two of them, gdhI and gdhII, were cloned from B . megaterium IAM1030 in our previous work (T . Mitamura, R . V . Evora, T . Nakai, Y . Makino, S . Negoro, I . Urabe, and H . Okada, J . Ferment . Bioeng . 70:363-369, 1990) . In the present study, two new genes, gdhIII and gdhIV, were isolated from the same strain and their nucleotide sequences were identified . Each gene has an open reading frame of 783 bp available to encode a peptide of 261 amino acids . Thus, a total of four glucose dehydrogenase genes have been cloned from B . megaterium IAM1030 . In addition, this strain does not seem to have other glucose dehydrogenase genes that can be distinguished from the four cloned genes so far examined by Southern hybridization analysis . The two newly cloned genes were expressed in Escherichia coli cells, and the products, GlcDH-III and GlcDH-IV, were purified and characterized and compared with the other isozymes, GlcDH-I and GlcDH-II, encoded by gdhI and gdhII, respectively . These isozymes showed different mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (GlcDH-I greater than GlcDH-III = GlcDH-IV greater than GlcDH-II), although they have the same number of amino acid residues . Double-immunodiffusion tests showed that GlcDH-I is immunologically different from the other isozymes and that GlcDH-III and GlcDH-IV are identical to one another but a little different from GlcDH-II . These glucose dehydrogenases were stabilized in the presence of 2 M NaCl . The effect of NaCl was especially large for GlcDH-III, which is most unstable enzyme . Kinetic studies showed that these isozymes are divided into two groups with respect to coenzyme specificity, although they can utilize both NAD and NADP: GlcDH-III and GlcDH-IV prefer NAD, and GlcDH-I and GlcDH-II prefer NADP . The phylogenic relationship of these glucose dehydrogenase genes is also discussed.

Toxicon, 1992 Aug, 30(8), 801 - 13
Effect of 22R-hydroxycholesterol on the action of sphingomyelinase from Bacillus cereus toward bovine erythrocytes; Tomita M et al.; The incorporation of 22R-hydroxycholesterol {(22R)-5-cholestene-3 beta,22-diol} into the bovine erythrocyte membranes remarkably enhanced the degradation of sphingomyelin in erythrocyte membranes by the action of sphingomyelinase from Bacillus cereus, causing much faster hemolysis of erythrocytes . The stimulative effect of 22R-hydroxycholesterol on the breakdown of sphingomyelin was maximal in the presence of Mg2+ . On the other hand, in spite of the presence of 22R-hydroxycholesterol, the breakdown of sphingomyelin was inhibited by increasing concentrations of Ca2+ . Also, the incorporation of 22R-hydroxycholesterol into the erythrocyte membranes facilitated the specific adsorption of the enzyme onto the surface of the erythrocyte membranes . The specific adsorption of sphingomyelinase amounted to 20-40% of the total activity in the presence of Mg2+ and the absence of divalent metal ions . In the presence of Ca2+, the incorporation of 22R-hydroxycholesterol enhanced the enzyme adsorption, exceeding more than 90% of the total activity . Therefore, the incorporation of 22R-hydroxycholesterol into bovine erythrocyte membranes remarkably accelerates the breakdown of sphingomyelin in the presence of Mg2+, and the specific adsorption of sphingomyelinase onto erythrocytes in the presence of Ca2+.

Int J Epidemiol, 1992 Aug, 21(4), 807 - 11
Epidemiological features of an outbreak of diphtheria and its control with diphtheria toxoid immunization; Youwang Y et al.; An outbreak of diphtheria primarily involving adults occurred in seven townships and one farm in Jiangling county, Hubei, China from September 1988 to January 1989 . Of the 103 reported cases, 80 were aged over 16 years, and there were two deaths . One hundred cases were reported in Jingzhou and its surrounding townships, only three occurred in townships distant from Jingzhou . The incidence and case fatality rates were 11.54/100,000 and 1.94% respectively . Twenty-one strains of diphtheria bacillus were isolated from 68 patients . A special immunization programme using adsorbed diphtheria toxoid was conducted 1 month into the outbreak, following which the incidence declined rapidly . Five vaccinees subsequently developed diphtheria.

Appl Environ Microbiol, 1992 Aug, 58(8), 2536 - 42
Novel Bacillus thuringiensis insecticidal crystal protein with a silent activity against coleopteran larvae; Lambert B et al.; A novel Bacillus thuringiensis crystal protein with a silent activity against the Colorado potato beetle is described . The crystal proteins are produced as bipyramidal crystals . These crystals contain a protein of 129 kDa with a trypsin-resistant core fragment of 72 kDa . Neither a spore-crystal mixture nor in vitro-solubilized crystals are toxic to any of several Lepidoptera and Coleoptera species tested . In contrast, a trypsin-treated solution containing the 72-kDa tryptic core fragment of the protoxin is highly toxic to Colorado potato beetle larvae . The crystal protein-encoding gene was cloned and sequenced . The inferred amino acid sequence of the putative toxic fragment has 37, 32, and 33% homology to the CryIIIA, CryIIIB, and CryIIID toxins, respectively . Interestingly, the 501 C-terminal amino acids show 41 to 48% amino acid identity with corresponding C-terminal amino acid sequences of other crystal proteins . Because of the toxicity of the fragment to the Colorado potato beetle and because of the distinct similarities of the toxic fragment with the other CryIII proteins, this gene was given a new subclass name (cryIIIC) within the CryIII class of coleopteran-active crystal proteins . CryIIIC represents the first example of a crystal protein with a silent activity towards coleopteran insect larvae . Natural CryIIIC crystals are not toxic . Toxicity is revealed only after an in vitro solubilization and activation step.

Urology, 1992 Aug, 40(2), 175 - 9
Clinical value of pathologic changes after intravesical BCG therapy of superficial bladder cancer; Bassi P et al.; Bladder pathologic features related to intravesical bacillus Calmette-Guerin (BCG) therapy in superficial bladder cancer (Ta, T1, Tis) were evaluated and related to clinical outcome . A total of 105 patients were treated with 75 mg Pasteur BCG weekly for six consecutive weeks . When tumor was not demonstrated a maintenance course was given . An additional six-week course was given when tumor recurrence or persistence, without progression, was observed after the induction course . An inflammatory change in the bladder was the most common pathologic finding . Granuloma was the only specific BCG-related feature and did not appear to be a prognostic factor because of low incidence (24%) and lack of correlation with clinical course . Dysplasia occurred more frequently (57%) in nonresponder patients and (26%) in responder patients, often heralding recurrence of tumor . All patients showing concurrent squamous and/or glandular metaplasia were unresponsive to BCG therapy . Histology and cytology did not correlate perfectly: cytology was ineffective in low-grade tumors and improved diagnostic accuracy, particularly when dysplasia was histologically evident.

Scand J Immunol, 1992 Aug, 36(2), 299 - 305
Hydrocortisone treatment of BCG-infected mice impairs the activation and enhancement of antimicrobial activity of peritoneal macrophages; Stokvis H et al.; The present study concerns the effect of hydrocortisone (HC) on the effector functions of Bacillus Calmette Guerin-purified protein derivative (BCG-PPD)-activated macrophages . Such activated macrophages release greater amounts of H2O2 and NO2-, inhibit the intracellular proliferation of T . gondii and kill L . monocytogenes more efficiently than resident macrophages . This activation was not fully expressed by macrophages from BCG-activated mice that had received a subcutaneous injection of HC 2 days before intraperitoneal injection of PPD, since the inhibition of the intracellular proliferation of T . gondii, the release of NO2- and the rate of intracellular killing of L . monocytogenes were lower than in macrophages from BCG-PPD-activated mice . However, treatment with HC did not impair the release of H2O2 by BCG-PPD-activated macrophages . The results show that the treatment of infected mice with HC inhibits their ability to develop adequate intracellular microbicidal mechanisms.

Rinsho Shinkeigaku, 1992 Aug, 32(8), 849 - 52
{MRI findings of brain-stem tuberculoma in a case of tuberculous meningitis}; Kubota A et al.; A 54-year-old woman developed headache and slight fever . When she consulted a physician, she could not move either of her eyes to the right . Cranial CT scan revealed no significant findings . Lumbar puncture was performed and CSF examination showed the cell count of 10,304/mm3, glucose level of 10 mg/dl, and total protein value of 270 mg/dl . Her symptoms and laboratory findings suggested meningitis and she was admitted to our hospital . Neurological examination revealed bilateral dilated pupils with sluggish light reflex, right gaze palsy, and hypesthesia of the left side of her face . A diagnosis of tuberculous meningitis was established by a positive test for acid-fast bacillus in CSF, and anti-tuberculous therapy was started at once . One month after the onset of symptoms, her main complaints were double vision and cerebellar ataxia . Both CT and MRI revealed a right brain-stem lesion . Pre-contrast CT could not clearly visualize the lesion but with contrast medium a homogeneously-enhanced circular lesion was shown . MRI on T2WI demonstrated the right brain-stem lesion to have a central bright core with hypointense periphery, which in turn was surrounded by hyperintensity . The lesion appeared isointense with cerebral white matter and the "central bright core" area was demonstrated to be slightly hypointense on T1WI . On post-contrast T1WI (with Gd-DTPA), the lesion showed strong homogeneous enhancement . The CT and MRI findings indicated a brain-stem tuberculoma, which was regarded as the cause of the ocular movement paralysis and cerebellar ataxia . As the clinical symptoms gradually resolved with anti-tuberculous treatment, the MRI appearance of the lesion also improved.(ABSTRACT TRUNCATED AT 250 WORDS)

Tuber Lung Dis, 1992 Aug, 73(4), 187 - 91
Humoral response to Mycobacterium tuberculosis in patients with human immunodeficiency virus infection; Barrera L et al.; The effect of the human immunodeficiency virus (HIV) on mycobacterial antibody production was investigated . Using an enzyme-linked immunosorbent assay (ELISA) for detecting IgG against Mycobacterium tuberculosis PPD, it was observed that individuals at risk of HIV infection show a pattern of humoral response to the tubercle bacillus similar to that previously found in the immunocompetent population not exposed to risk factors: 6 of 12 (50.0%) tuberculosis cases had elevated levels of antibodies to PPD and 27 of 30 (90.0%) asymptomatic individuals had antibody levels within the normal range . In an HIV-seropositive group without AIDS indicator diseases, 8 of 22 (36.4%) tuberculous patients had detectable mycobacterial antibodies whereas 156 of 164 (95.1%) non-tuberculous subjects did not . Among AIDS cases, only 1 of 20 (5.0%) patients with tuberculosis and none of 53 non-tuberculous subjects showed a positive result . The study evidenced an increasing humoral unresponsiveness to PPD in the progression of HIV infection to AIDS . Thus, a serodiagnostic method for detecting tuberculosis such as the ELISA here employed noticeably decreases its utility in the latency stage of the HIV infection, and it is practically useless in clinical AIDS.

Antibiot Khimioter, 1992 Aug, 37(8), 39 - 43
{Microbial ecology of laboratory animals as a model in evaluation of the immunomodulating and antimicrobial agents}; Shenderov BA et al.; Rats with altered microbial ecology and decreased colonization resistance due to neutropenia induced by cyclophosphamide were used as a model for estimating the effect of bacterial polysaccharides (lactulose and exopolysaccharide from Bacillus polymyxa) and fluoroquinolones (pefloxacin) . Monotherapy with pefloxacin had a favourable effect both on normalization on the intestinal microflora in the rats and their hemopoiesis (decreased neutropenia) . The highest correcting effect with respect to the lowered colonization resistance was observed when pefloxacin was used in combination with exopolysaccharide.

Chem Pharm Bull (Tokyo), 1992 Aug, 40(8), 2133 - 7
The study on phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis: synthesis of homogeneous substrates, substrate specificity and other properties; Kume T et al.; The properties of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were studied in detail . The enzyme was extremely thermostable in 0.1% bovine serum albumin and retained 73% of its activity at 100 degrees C for 10 min, while it was labile in the absence of albumin . The enzymatic activity was inhibited by HgCl2 or p-chloromercuriphenylsulfonic acid and restored by dithiothreitol . The kinetic parameters (Km and Vmax) of PI-PLC were determined for the mixed micelle of yeast phosphatidylinositol (PI)/Triton X-100 or sodium deoxycholate . Four PIs having different acyl chains: dilauroylphosphatidylinositol (DLPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI) and dioleoylphosphatidylinositol (DOPI) were synthesized from yeast PI through the processes of deacylation and reacylation, identified by infrared (IR) and Fourier transform nuclear magnetic resonance (FT-NMR) spectra, and subjected to the action of PI-PLC . All the synthetic PIs were hydrolyzed by this enzyme, with DLPI and DMPI being the best substrates . PI-PLC did not catalyze the hydrolysis of the phosphatidylnucleosides 5'-phosphatidylcytidine, 5'-phosphatidyluridine, 5'-phosphatidylthymidine, 5'-phosphatidyladenosine and 5'-phosphatidyl-2'-deoxyadenosine.

J Biochem (Tokyo), 1992 Aug, 112(2), 258 - 65
Leucine dehydrogenase from Bacillus stearothermophilus: identification of active-site lysine by modification with pyridoxal phosphate; Matsuyama T et al.; We have constructed an efficient expression plasmid for the leucine dehydrogenase gene previously cloned from Bacillus stearothermophilus . The recombinant enzyme was overproduced in Escherichia coli cells to a level of more than 30% of the total soluble protein upon induction with isopropyl beta-D-thiogalactopyranoside . The enzyme could be readily purified to homogeneity by heat treatment and a single step of ion-exchange chromatography . The purified enzyme was inactivated in a time-dependent manner upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride . The inactivation was completely prevented in the copresence of L-leucine and NAD+ . Concomitantly with the inactivation, several molecules of PLP were incorporated into each subunit of the hexameric enzyme . Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the modified protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled . Among these residues, Lys80 was predominantly labeled and, in the presence of L-leucine and NAD+, was specifically protected from the labeling . Furthermore, a linear relationship of about 1:1 was observed between the extent of inactivation and the amount of PLP incorporated into Lys80 . A slightly active mutant enzyme, in which Lys80 is replaced by Ala, was not inactivated at all by incubation with PLP, showing that the inactivation is correlated with the labeling of only Lys80 . Lys80is conserved in the corresponding regions of all the amino acid dehydrogenase sequences reported to date . These results suggest that Lys80 is located at the active site and plays an important role in the catalytic function of leucine dehydrogenase.

Biol Mass Spectrom, 1992 Aug, 21(8), 367 - 79
Isolation, mass spectrometric characterization, and protein phosphatase inhibition properties of cyclic peptide analogues of gramicidin-S from Bacillus brevis (Nagano strain); Thibault P et al.; Six substitutional variants of gramicidin-S, present in a commercial extract of Bacillus brevis, have been characterized using a combination of liquid chromatographic/mass spectrometric analysis of the crude extract, plus accurate mass measurements, tandem mass spectrometric investigations, and amino acid analyses, of isolated high-performance liquid chromatographic fractions . Two of these variants were shown previously (Nozaki and Muramatsu, J . Antibiotics 37, 689 (1984)) to involve replacement of one or both Val residues in gramicidin-S by aminobutyric acid, and these findings are confirmed here . One of the four unknown variants corresponds to substitution of one Val residue by Leu, while the other three all involve substitution of one of the Orn residues in gramicidin-S by Lys, or by citrulline (gamma-carbamoyl ornithine), or by gamma-formyl ornithine . The biological activities of the purified fractions were assessed with respect to their in vitro inhibition of protein phosphatases PP1 and PP2A . For both protein phosphatases inhibition levels fell in the micromolar range, about four orders of magnitude higher than IC50 values for other small cyclic peptides such as microcystins and nodularins.

Urol Clin North Am, 1992 Aug, 19(3), 581 - 9
Immunotherapeutic alternatives in superficial bladder cancer . Interferon, interleukin-2, and keyhole-limpet hemocyanin; Sargent ER et al.; Interleukin-2, IFNs, and TNF are biologic response modifiers that are part of an intricate network of interacting cytokines released during an immune response . Lack of sufficient endogenous cytokine activity secondary to the immunosuppressive effects of tumor growth may be overcome by direct, local application of biologic response modifiers or immunostimulants such as BCG or KLH . Bacillus Calmette-Guerin remains the most effective immunotherapeutic agent for superficial transitional-cell carcinoma . Although the mechanism of action is unknown, the weight of evidence suggests that local cytokine release is involved in the effector pathway . Recent data have shown that the local application of new single-agent immunotherapies can have an effect on superficial transitional-cell carcinoma and CIS similar to that of chemotherapeutic agents and nearly identical to that of BCG . But, unlike the situation with chemotherapy or BCG, these effects are attended by minimal or no toxicity . Chemotherapy and BCG failures have also shown responses to direct instillation of cytokines . Further understanding of the exact mechanism of action of these agents and of their interaction should lead to the optimal antitumor regimen with the least toxicity . Determining the degree of host immunoresponsiveness and which combination of cytokines or immunotherapeutic or chemotherapeutic agents is most effective for a specific tumor type is the challenge for the future.

Urol Clin North Am, 1992 Aug, 19(3), 549 - 56
Immunotherapy for superficial bladder cancer . A developmental and clinical overview; Morales A et al.; Superficial bladder cancer is one of the few solid human malignancies in which immunotherapy has been proved to be effective . Bacillus Calmette-Guerin was the vaccine which opened the door for this innovative approach . In an era of remarkable achievements in biotechnology, it is truly amazing that this throwback to the Stone Age of tumor immunology has not yet been replaced by a more (or equally) effective substitute . Potential candidates are already on the horizon and deserve a comprehensive evaluation . They must show not only that they are devoid of significant adverse effects but that they possess, beyond a doubt, superior antineoplastic activity . Even more remarkable is that one of the oldest vaccines still in use could emerge in a new role as an effective antineoplastic agent . Bacillus Calmette-Guerin has demonstrated an uncanny capacity for effectiveness as therapy for human diseases . Its protective effect against tuberculosis is well recognized, and its contribution to cancer therapy is widely known . A new and increasing repertoire has recently been presented: two separate groups of researchers have employed the vaccine successfully as a vehicle to express antigen-encoded genes from other pathogens . The exciting aspect of these recent studies resides in the demonstration that the altered vaccine is able to induce humoral and cell-mediated immune responses to the recombinant antigens, including the human immunodeficiency virus (HIV) . Thus, BCG once more attracts enormous interest from the scientific community for its versatility and potential as a therapeutic agent.

J Exp Med, 1992 Aug 1, 176(2), 581 - 6
Evidence for an alpha/beta T cell-independent mechanism of resistance to mycobacteria . Bacillus-Calmette-Guerin causes progressive infection in severe combined immunodeficient mice, but not in nude mice or in mice depleted of CD4+ and CD8+ T cells; Izzo AA et al.; Depleting thymectomized mice of CD4+ T cells, or CD4+ plus CD8+ T cells, rendered them incapable of resolving Bacillus-Calmette-Guerin (BCG) infection in their lives, spleens, kidneys, and lungs . However, it did not render them incapable of stabilizing infection in the latter three organs after an initial period of BCG growth . Athymic nude mice showed a similar capacity to control BCG growth in these organs after a certain stage of infection . In contrast, congenitally severe combined immunodeficient (SCID) mice appeared to offer no resistance to BCG infection, in that the organism grew progressively in all organs of these mice and was lethal for them beginning on day 55 of infection . The results suggest that, although CD4+ T cells are important for resolving BCG infection, an alpha/beta T cell-independent mechanism of resistance can be acquired at 2-3 wk of infection that is capable of inhibiting further BCG growth in all organs except the lungs . Because this mechanism is absent from SCID mice, it is likely that it depends on the functions of gamma/delta T cells, B cells, or both types of cells . In keeping with this possibility is the additional finding that SCID mice engrafted with lymph node cells depleted of CD4+ or CD8+ T cells were capable of expressing an appreciable level of resistance against BCG infection.

J Bacteriol, 1992 Aug, 174(15), 5051 - 6
Proteolytic processing of the mosquitocidal toxin from Bacillus sphaericus SSII-1; Thanabalu T et al.; The 97-kDa protein Mtx21, derived from the 100-kDa mosquitocidal protein (Mtx) from Bacillus sphaericus SSII-1 by the deletion of the putative signal sequence, was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and the fusion protein was purified by affinity chromatography . The fusion protein bound to glutathione agarose was cleaved with thrombin to release the Mtx21 protein . The 97-kDa Mtx21 protein was found to be toxic to Culex quinquefasciatus larvae with a 50% lethal concentration of 15 ng/ml . Treating Mtx21 with crude mosquito larval gut extracts gave rise to two major peptides of 70 and 27 kDa . Treating the 97-kDa Mtx21 protein with trysin also gave rise to a similar proteolytic cleavage pattern . N-terminal sequencing showed that the 27-kDa peptide was derived from the N-terminal region of the 97-kDa protein and that the 70-kDa protein was from the C-terminal region of the 97-kDa protein . The 27-kDa peptide has all the previously identified regions of homology with the catalytic peptides of the ADP-ribosyltransferase toxins, such as pertussis toxin S1 peptide, while the 70-kDa peptide has three internal regions of homology.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1992 Aug, 25(3), 138 - 48
Isolation and partial purification of cytochrome oxidases from Bacillus thuringiensis subsp . israelensis HD-567; Liu JK et al.; Analyses of the cell membrane fractions by spectral absorbance revealed the presence of cytochrome c and three cytochrome oxidases in Bacillus thuringiensis subsp . israelensis HD-567-cytochromes a+a3, d, and o . A modified procedure was used to purify the cytochrome c:o complex from this organism . The oxidase complex was first solubilized from a sonic-disrupted cell membrane fraction (R3 fraction) using deoxycholate and KCl . The resulting soluble fraction was further purified by Sephadex G-50 gel filtration and DEAE ionexchange chromatography . TMPD oxidase specific activity and cytochrome concentration were assayed to monitor the purification procedures . The F7 fraction (obtained after G-50 chromatography) contained cytochrome c (0.44 nmole/mg protein), cytochrome a+a3 (0.26 nmole/mg protein), and cytochrome o (0.38 nmole/mg protein), which had 6-fold, 3.4-fold and 18.9-fold increase, respectively, by comparison with the original R3 fraction . The TMPD oxidase specific activity of the F7 fraction also increased 4.8 fold . The F8 fraction (obtained after the final DEAE chromatography) contained a cytochrome c:o complex only (cytochrome c, 0.46 nmole/mg protein; cytochrome o, 0.16 nmole/mg protein), but no cytochrome a+a3 was found . Both the TMPD oxidase specific activity and the cytochrome o were attenuated greatly in comparison with the F7 fraction . SDS-PAGE analysis revealed that the F7 fraction contained numerous protein components, while the F8 fraction contained only a prominent major protein of 113.5 kD thought to be the cytochrome c:o complex, and a minor polypeptide (MW = 65.8 kD) . Although the final DEAE procedure removed many undesired polypeptides, TMPD oxidase activity and cytochrome o component were also lost greatly . Kinetic studies of this cytochrome c:o complex is in progress in our laboratory.

Wei Sheng Wu Xue Bao, 1992 Aug, 32(4), 305 - 7
{Transposition of Tn917 in Bacillus pumilus}; Geng Y et al.; Transposition Tn917 was introduced into Bacillus pumilus 289 by protoplast transformation with plasmid pTV32 . The temperature-sensitive replication property of pTV32 was maintained in B . pumilus . Tn917 was transposed efficiently in B . pumilus with 4.8 x 10(-4) transposition rate . The yield of auxotrophs was about 0.65% in all insertional mutants . It indicated a prospects for the use of Tn917 as a tool for insertional mutagenesis and genetic manipulation in B . pumilus.

Mol Microbiol, 1992 Aug, 6(15), 2095 - 8
The delta-endotoxin protein family displays a hydrophobic motif that might be implicated in toxicity; Pereyra-Alferez B et al.; A computer-based analysis of hydropathy and surface probability of representative members of each class of the Cry family of proteins was performed . A highly conserved hydrophobic motif within the previously described block, D2, is present not only in lepidopteran toxin genes but also in toxins active against diptera and coleoptera . An interesting feature of this hydrophobic motif is the presence of an aspartic residue (highly hydrophilic) in its middle part . Comparison with the amino acid sequence from diphtheria toxin showed that it also contains a hydrophobic motif similar to the one present in the Bacillus thuringiensis toxins . It also contains an aspartic residue in the middle part and some speculations are presented on the function of this specific region with regard to the toxic mechanism of action.

Eur J Biochem, 1992 Aug 1, 207(3), 1101 - 8
Acetylcholinesterases of the nematode Steinernema carpocapsae . Characterization of two types of amphiphilic forms differing in their mode of membrane association; Arpagaus M et al.; We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae . Two major AChEs are involved in acetylcholine hydrolysis . The first class of AChE is highly sensitive to eserine (IC50 = 0.05 microM) . The corresponding molecular forms are: an amphiphilic 14S form converted into a hydrophilic 14.5S form by mild proteolysis and two hydrophilic 12S and 7S forms . Reduction of the amphiphilic 14S form with 10 mM dithiothreitol produces hydrophilic 7S and 4S forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex . Sedimentation coefficients suggest that 4S, 7S, 12S forms correspond to hydrophilic monomer, dimer, tetramer and that the 14S form is also a tetramer linked to one structural element . The second class of AChE is less sensitive to eserine (IC50 = 0.1 mM) . Corresponding molecular forms are hydrophilic and amphiphilic 4S forms (monomers) and a major amphiphilic 7S form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C . This amphiphilic 7S form thus possesses a glycolipid anchor . It appears that Steinernema (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.

N Engl J Med, 1992 Jul 30, 327(5), 293 - 301
Identification of the uncultured bacillus of Whipple's disease; Relman DA et al.; BACKGROUND . Whipple's disease is a systemic disorder known for 85 years to be associated with an uncultured, and therefore unidentified, bacillus . METHODS . We used a molecular genetic approach to identify this organism . The bacterial 16S ribosomal RNA (rRNA) sequence was amplified directly from tissues of five unrelated patients with Whipple's disease by means of the polymerase chain reaction, first with broad-range primers and then with specific primers . We determined and analyzed the nucleotide sequence of the amplification products . RESULTS . A unique 1321-base bacterial 16S rRNA sequence was amplified from duodenal tissue of one patient . This sequence indicated the presence of a previously uncharacterized organism . We then detected this sequence in tissues from all 5 patients with Whipple's disease, but in none of those from 10 patients without the disorder . According to phylogenetic analysis, this bacterium is a gram-positive actinomycete that is not closely related to any known genus . CONCLUSIONS . We have identified the uncultured bacillus associated with Whipple's disease . The phylogenetic relations of this bacterium, its distinct morphologic characteristics, and the unusual features of the disease are sufficient grounds for naming this bacillus Tropheryma whippelii gen . nov . sp . nov . Our findings also provide a basis for a specific diagnostic test for this organism.

Orv Hetil, 1992 Jul 26, 133(30), 1877 - 81
{Clinical significance of serological markers of Helicobacter pylori}; Gerencser Z et al.; Helicobacter pylori (H . pylori) is a Gram-negative, curved bacillus known since 1983 . It is supposed to play role in the pathogenesis of certain gastroduodenic diseases, e.g . non-ulcer dyspepsia (NUD), chronic inflammation, ulcers, etc . Serum samples of 70 patients who were examined for stomach complaints with gastroscopy and those of 22 healthy persons were analysed . The purpose of the study was to evaluate anti-H . pylori IgG, IgM and IgA antibodies using ELISA method . Whether the antibodies can be detected or not, 8 possible variations exist, each of them denoting certain state of infection . These states are not always going parallel with the macroscopic pictures revealed by gastroscopy, but there are some obvious congruences . Results show that serologic examination cannot replace gastroscopy but on the other hand in follow-up tests and examinations as well as in understanding the aetiology of different gastroduodenal diseases it can play an important role.

Biochemistry, 1992 Jul 21, 31(28), 6603 - 7
Differential scanning calorimetric study of the thermal unfolding of beta-lactamase I from Bacillus cereus; Arriaga P et al.; The irreversible thermal unfolding of the class A beta-lactamase I from Bacillus cereus has been investigated at pH 7.0, using differential scanning calorimetry (DSC) and inactivation kinetic techniques . DSC transitions showed a single peak with a denaturation enthalpy of 646 kJ.mol-1 and were moderately scan rate dependent, suggesting that the process was partially kinetically controlled . The inactivation kinetics at constant temperature showed that the irreversible denaturation of the enzyme occurs as the sum of two exponential terms whose amplitudes are strongly temperature dependent within the transition range so that, at the lowest temperatures within this interval, irreversible inactivation would proceed mainly through the slow phase . The fraction of irreversibly denatured enzyme (D) as a function of temperature for a given scanning rate was calculated by numerical integration of the kinetic equation with temperature, using previously determined kinetic parameters . This D form was the most populated of the unfolded states only at temperatures well above the maximum in the calorimetric transition . Combination of the results of kinetic and DSC experiments has allowed us to separate the contribution of the final D state to the excess enthalpy change from the contribution arising from the reversibly denatured forms of the enzyme (I(i), i = 1,..., n), with the resulting conclusion that the scan rate dependence of the calorimetric traces was the result of two different dynamic effects, viz., the irreversible step and a slow relaxation process during formation of the reversibly denatured intermediate states . Finally, the problems of using results obtained at a single scan rate to validate the two-state kinetic model are commented on.

Biochim Biophys Acta, 1992 Jul 17, 1101(2), 236 - 9
The ATPases of Propionigenium modestum and Bacillus alcalophilus . Strategies for ATP synthesis under low energy conditions; Dimroth P; In Propionigenium modestum, ATP synthesis is coupled via delta mu Na+ to the decarboxylation of (S)-methylmalonyl-CoA . The low energy yield of this reaction implies that approx . 4 decarboxylation cycles are necessary to synthesize 1 molecule of ATP . Theoretical considerations in accord with experimental results suggest ATP synthesis in P . modestum at delta mu Na+ = -110 mV . Other anaerobic bacteria synthesize ATP at a delta mu H+ of similar size and alkaliphilic bacteria at pH 10.3 have a delta mu H+ of only -103 mV . In these cases, the H+(Na+) to ATP stoichiometry must be at least 4.

Biochem J, 1992 Jul 15, 285 ( Pt 2), 625 - 8
Increasing the thermostability of the neutral proteinase of Bacillus stearothermophilus by improvement of internal hydrogen-bonding; Eijsink VG et al.; In an attempt to increase the thermostability of the neutral proteinase of Bacillus stearothermophilus the buried Ala-170 was replaced by serine . Molecular-dynamics simulations showed that Ser-170 stabilizes the enzyme by formation of an internal hydrogen bond . In addition, the hydroxy group of Ser-170 could contribute to stability by filling an internal cavity . After the introduction of the mutation, using site-directed-mutagenesis techniques, an increase in stability of 0.7 +/- 0.1 degrees C was obtained.

Eur J Biochem, 1992 Jul 15, 207(2), 781 - 91
The structure of neutral protease from Bacillus cereus at 0.2-nm resolution; Stark W et al.; The crystal structure of the neutral protease from Bacillus cereus has been refined to an R factor of 17.5% at 0.2-nm resolution . The enzyme, an extracellular metalloendopeptidase, consists of two domains and binds one zinc and four calcium ions . The structure is very similar to that of thermolysin, with which the enzyme shares 73% amino-acid sequence identity . The active-site cleft between the two domains is wider in neutral protease than in thermolysin . This suggests the presence of a flexible hinge region between the two domains, which may assist enzyme action . The high-resolution analysis allows detailed examination of possible causes for the difference in thermostability between neutral protease and thermolysin.

FEMS Microbiol Lett, 1992 Jul 15, 73(3), 263 - 9
Isolation and sequence of a 2-kbp miniplasmid from Bacillus thuringiensis var . kurstaki HD-3a3b: relationship with miniplasmids of other B . thuringiensis strains; Marin R et al.; The miniplasmid profiles of 18 Bacillus thuringiensis strains belonging to 8 different serotypes were determined using an alkaline hydrolysis method for isolation of low molecular mass plasmids . Nearly all the strains contained covalently closed circular (CCC) DNA species ranging from 2 to 5 species per strain and from 1.5 to 10.5 kbp in size (values corresponding to CCC forms) . A 2-kbp plasmid from B . thuringiensis var . kurstaki HD-3a3b futura strain was used in Southern hybridization experiments to analyse relationships among the low molecular mass plasmids of different B . thuringiensis strains . This 2-kbp miniplasmid was present in most strains which show toxicity against lepidoptera . It was not present in those strains toxic against diptera (B . thuringiensis var . israelensis) or coleoptera (B . thuringiensis var . tenebrionis) . The 2-kbp miniplasmid from B . thuringiensis var . kurstaki HD-3a3b futura was cloned and fully sequenced . Sequence analysis of the 2058 bp of the miniplasmid revealed the presence of an ORF (630 bp, 210 amino acids in size) that is preceded by a consensus sequence of B . thuringiensis crystal protein gene transcription promoters . No significant homology was observed with known B . thuringiensis toxin nucleic acid sequences or with other known sequences.

Biochim Biophys Acta, 1992 Jul 13, 1122(1), 33 - 40
Chemical modification of cyclomaltodextrin glucanotransferase from Bacillus circulans var . alkalophilus; Mattsson P et al.; Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var . alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule . Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result . No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge . Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one . Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1 . Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min) . The inhibition was partially reversible . CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site . Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased . Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.

Biochem Pharmacol, 1992 Jul 7, 44(1), 192 - 5
Stimulation of macrophage tumouricidal activity by 5,6-dimethyl-xanthenone-4-acetic acid, a potent analogue of the antitumour agent flavone-8-acetic acid; Ching LM et al.; The new antitumour drug 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA; NSC 640488) was 14-fold more potent than the investigational chemotherapeutic drug flavone-8-acetic acid (NSC 347512) in stimulating tumouricidal activity in cultures of resident murine peritoneal macrophages . The tumouricidal activity of thioglycollate-elicited and Bacillus Calmette-Guerin-primed macrophages was also significantly enhanced by 5,6-MeXAA . Stimulation of macrophage tumouricidal activity by 5,6-MeXAA was not affected by inhibitors of superoxide and nitric oxide production, but was reduced by cyclosporin A, an inhibitor of protein secretion . Inhibitors of neutral proteases had no effect . Cortisone, dexamethasone, indomethacin, dibutyryl cAMP, prostaglandin E2 and prostacyclin, but not prostaglandin F2 alpha, inhibited stimulation, suggesting the involvement of tumour necrosis factor-alpha (TNF) . However, antibodies to TNF did not inhibit stimulation . The results suggest that 5,6-MeXAA acts on macrophages in a manner similar to that of endotoxin, utilizing a pathway which includes arachidonic acid metabolism and requiring cell-cell contact with target cells for a tumouricidal effect.

Biochemistry, 1992 Jul 7, 31(26), 6011 - 8
Extensive comparison of the substrate preferences of two subtilisins as determined with peptide substrates which are based on the principle of intramolecular quenching; Gron H et al.; Subtilisins are serine endopeptidases with an extended binding cleft comprising at least eight binding subsites . Interestingly, subsites distant from the scissile bond play a dominant role in determining the specificity of the enzymes . The development of internally quenched fluorogenic substrates, which allow polypeptides of more than 11 amino acids to be inserted between the donor and the acceptor, has rendered it possible to perform a highly systematic mapping of the individual subsites of the active sites of subtilisin BPN' from Bacillus amyloliquefaciens and Savinase from Bacillus lentus . For each enzyme, the eight positions S5-S'3 were characterized by determination of kcat/KM values for the hydrolysis of substrates in which the amino acids were systematically varied . The results emphasize that in both subtilisin BPN' and Savinase interactions between substrate and S4 and S1 are very important . However, it is apparent that interactions between other subsites and the substrate exert a significant influence on the substrate preference . The results are rationalized on the basis of the structural data available for the two enzymes.

J Sykepleien . 1992 Jul 6;80(12):19.
{Nursing under a different sky . Swaziland}; Berg-Moe G; PIP: Swaziland is a kingdom with 800,000 inhabitants bordering on Mozambique and South Africa with about 50% of the population under 15 years of age . The experience of a nurse in a small clinic in the course of several years is recounted . Swaziland ranks 3rd in the world in alcohol abuse which often leads to wounds requiring suturing . Penicillin is given prophylactically with a paracetamol preparation for analgesia . As a rule, every injured person will get a .5 ml tetanus injection for prophylaxis . The most serious conditions of polyclinic patients are hepatitis, bilharzia, diarrhea, pellagra, pneumonia, and malnutrition . A great number of patients have sexually transmitted diseases, and the rate of AIDS infection is not known . According to 1 study 60-80% of the population in reproductive age will die of AIDS in the course of a 5-year period . The majority of people are impervious to counseling about their sexual behavior in spite of educational programs on the radio, in schools, and in work places . Condoms are not popular, since they are not considered manly . Pregnant women receive iron and multivitamin tablets in the course of pregnancy . Many pregnant women are anemic, and 70% give birth at home, the rest in a hospital or clinic . During delivery they get no analgesia, and there are few complications . The average weight of the newborn is 3.5 kg, although none of the women are under 150 cm . A little after birth all children are vaccinated with bacillus Calmette-Guerin (BCG) and polio, later with diphtheria-pertussis-tetanus (DPT) and measles .

Public Health Rep, 1992 Jul-Aug, 107(4), 477 - 80
An outbreak of Bacillus cereus food poisoning--are caterers supervised sufficiently; Slaten DD et al.; Bacillus cereus is an uncommonly reported cause of foodborne illness in the United States . In May 1989, an outbreak of B . cereus gastroenteritis occurred among 140 guests who had attended a catered wedding reception in Napa, CA . Investigation established Cornish game hens served at the event as the vehicle for disease transmission (OR = 29, P = 0.0001) . Although the spores of B . cereus are ubiquitous, large numbers of toxin-producing organisms (more than 10(5) per gram of food) are required for illness to occur . In the Napa outbreak, bacterial multiplication was facilitated at several points during the preparation and transportation of the food . While a licensed restaurant kitchen was used, the facilities were clearly inadequate for the event . At present, the California Health and Safety Code does not address the scope of catering operations . As caterers increase in number, there will be a growing need for governmental oversight to ensure that food production on a large scale is conducted safely.

Biochem J, 1992 Jul 1, 285 ( Pt 1), 25 - 33
Plasma-membrane-intercalated heparan sulphate proteoglycans in an osteogenic cell line (UMR 106-01 BSP); McQuillan DJ et al.; The heparan sulphate (HS) proteoglycans associated with the cell layer of a rat osteosarcoma cell line {UMR 106-01 (BSP)} were compared with similar cell-associated proteoglycans from other cells, and their interaction with the plasma membrane was studied . HS proteoglycans were metabolically labelled by incubation of cell cultures with {3H}glucosamine or {3H}leucine and {35S}sulphate . HS proteoglycan core protein preparation generated by heparitinase digestion of the major species from UMR 106-01 (BSP) cells co-migrated on PAGE with identical preparations from ovarian granulosa cells and parathyroid cells (at approximately 70 kDa) . The hydrophobic nature of the major HS proteoglycans from these diverse cell lines, based on elution position from octyl-Sepharose, were also comparable . Linkages of the HS proteoglycan to the cell membrane were investigated by labelling plasma-membrane preparations with a lipid soluble photoactivatable reagent, 3-(trifluoromethyl)-3- (m-{125I}iodophenyl)diazirine (TID), which selectively labels plasma-membrane-spanning peptide domains . Purified HS proteoglycan from UMR 106-01 (BSP) cells was shown to be accessible to the {125I}TID, and the core protein portion of the molecule was labelled, confirming its close association with the plasma membrane . Approx . 36% of 35S-labelled HS proteoglycans were released from the cell surface by phospholipase C (Bacillus thuringiensis), which specifically cleaves phosphatidylinositol-linked proteins . In the presence of insulin, the metabolism of the phospholipase C-sensitive population was unaltered; however, release of the phospholipase C-insensitive population into the medium was increased . These data indicate that a subpopulation of HS proteoglycans are covalently bound to the plasma membrane by a glycosylphosphatidylinositol structure, with the remainder representing those species directly inserted into the plasma membrane via a hydrophobic peptide domain . These observations are similar to those reported for ovarian granulosa cells {Yanagishita & McQuillan (1989) J . Biol . Chem . 264 17551-17558}, and thus may represent a general phenomenon for many cell types.

Appl Environ Microbiol, 1992 Jul, 58(7), 2211 - 8
Biosorption of dichlorodiphenyltrichloroethane and hexachlorobenzene in groundwater and its implications for facilitated transport; Lindqvist R et al.; The potential for enhanced mobility of hydrophobic pollutants by cotransport with bacteria in saturated soils was evaluated from measurements of biosorption of 14C-labeled hexachlorobenzene and dichlorodiphenyltrichloroethane (DDT) to five strains of soil and sewage bacteria . The sorption process could be described by a linear partition equation and appeared to be reversible, but desorption kinetics were slow and/or partly irreversible . The DDT partition coefficients varied with equilibration time, possibly reflecting DDT-induced changes in the physiology of the bacteria . The partition coefficients, normalized to the masses of the bacteria, ranged from 250 to 14,000 for live cells, but the largest coefficients were associated with autoclaved cells of a Pseudomonas sp . The sorptive capacity of the bacterial biomass was greater for DDT than for hexachlorobenzene but was not correlated to overall bacterial hydrophobicity, measured by hydrophobic interaction chromatography . In a column study, 1.2 x 10(9) cells of a Bacillus sp . strain per ml enhanced DDT transport about 8-fold, whereas an advective-dispersive-sorptive equilibrium model for two mobile phases, water and free-living bacteria, suggested a 14-fold enhancement, based on the DDT partition coefficient . The disagreement was in part due to a retarded nonequilibrium movement of the bacteria . Model calculations based on literature data covering a wide range of organisms and compounds suggested that 10(6) cells ml-1 would increase the mobility of very hydrophobic compounds (log octanol-water partition coefficient {K(ow) of greater than or equal to 6), whereas higher densities of bacteria (10(8) cells ml-1) would have a significant impact on compounds with a log K(ow) of greater than or equal to 4.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Microbiol, 1992 Jul, 30(7), 1882 - 4
Bilophila wadsworthia isolates from clinical specimens; Baron EJ et al.; Bilophila wadsworthia is an anaerobic, gram-negative, asaccharolytic, bile-resistant, catalase-positive bacillus that is usually urease positive and was originally recognized in specimens of peritoneal fluid and tissue from patients with appendicitis . Additional isolations from clinical specimens, including a scrotal abscess, mandibular osteomyelitis, axillary hidradenitis suppurativa, pleural fluid, joint fluid, and blood, are described here.

Clin Exp Immunol, 1992 Jul, 89(1), 110 - 4
Cellular immune response to the cell walls of Mycobacterium leprae in leprosy patients and healthy subjects exposed to leprosy; Roche PW et al.; Cell walls of M . leprae consist of complex arrangements of carbohydrate, lipid, peptidoglycan and protein molecules . Recently, extractable proteins of a wide range of molecular weights were identified as components of the cell wall . We have examined the cellular immune responses of Nepali leprosy patients to a cell wall preparation of M . leprae enriched for these proteins . Strong lymphocyte proliferative responses to the antigens were present in half of the paucibacillary leprosy patients and in the majority of healthy control subjects with occupational exposure to leprosy . Patients with multibacillary disease responded poorly and patients with tuberculosis had intermediate responses . Proliferative responses to the cell wall protein fraction were strongly correlated to the proliferative responses to sonicates of the whole leprosy bacillus . Immunization of mice with cell wall proteins resulted in inhibition of growth of M . leprae following foot-pad inoculation with viable organisms . Therefore cell-mediated immune responses to the extractable proteins of the cell wall may play a role in protective immunity against M . leprae infection.

J Med Microbiol, 1992 Jul, 37(1), 77 - 80
Cytotoxicity of Bacillus piliformis; Riley LK et al.; Seven isolates of B . piliformis, the agent of Tyzzer's disease, obtained from various host species, were examined for cytotoxic activity by incubating culture filtrates on BRL 3A rat-hepatocyte and 3T3 mouse-fibroblast cell lines . One isolate exhibited cytopathic effects on BRL 3A cells, but not on 3T3 cells . Three other isolates were strongly cytotoxic for 3T3 cells but only slightly so for BRL 3A cells . The remaining three isolates showed no cytotoxicity for either cell line . The cytotoxic products were greater than 100 kDa in mol . wt, thermolabile, and partly destroyed by trypsin treatment . The data show that some B . piliformis isolates produce cytotoxic proteins, which may contribute to the pathogenesis of Tyzzer's disease.

J Bacteriol, 1992 Jul, 174(13), 4463 - 74
Heat killing of bacterial spores analyzed by differential scanning calorimetry; Belliveau BH et al.; Thermograms of the exosporium-lacking dormant spores of Bacillus megaterium ATCC 33729, obtained by differential scanning calorimetry, showed three major irreversible endothermic transitions with peaks at 56, 100, and 114 degrees C and a major irreversible exothermic transition with a peak at 119 degrees C . The 114 degrees C transition was identified with coat proteins, and the 56 degrees C transition was identified with heat inactivation . Thermograms of the germinated spores and vegetative cells were much alike, including an endothermic transition attributable to DNA . The ascending part of the main endothermic 100 degrees C transition in the dormant-spore thermograms corresponded to a first-order reaction and was correlated with spore death; i.e., greater than 99.9% of the spores were killed when the transition peak was reached . The maximum death rate of the dormant spores during calorimetry, calculated from separately measured D and z values, occurred at temperatures above the 73 degrees C onset of thermal denaturation and was equivalent to the maximum inactivation rate calculated for the critical target . Most of the spore killing occurred before the release of most of the dipicolinic acid and other intraprotoplast materials . The exothermic 119 degrees C transition was a consequence of the endothermic 100 degrees C transition and probably represented the aggregation of intraprotoplast spore components . Taken together with prior evidence, the results suggest that a crucial protein is the rate-limiting primary target in the heat killing of dormant bacterial spores.

J Surg Oncol, 1992 Jul, 50(3), 144 - 8
Adjuvant immunotherapy in melanoma: a new approach; Elias EG et al.; Patients with metastatic cutaneous melanoma to two or more regional lymph nodes have an extremely poor prognosis despite radical lymphadenectomy . In an attempt to improve the survival and to determine the safety of a new method of tumor specific adjuvant immunotherapy in such a high risk group of patients, nine patients were studied . Three to four weeks after regional lymphadenectomy, each of them received a single intradermal injection of Bacillus Calmette-Guerin . Three weeks later, they were immunized by allogenic melanoma cells obtained from live donors with distant metastases . Each patient received three vaccinations, each from a different donor (except in one), to avoid development of HLA response, but maintaining exposure to melanoma antigens . No cultured melanoma cells were used . Each vaccine consisted of mitomycin-C treated tumor cells mixed with purified protein derivative (PPD) of tuberculin given intradermally once per month for 3 months . The patients were then observed with no further treatment . Utilizing the leukocyte migration inhibition test, there was some in vitro evidence of tumor specific cell mediated response which seemed to disappear 1-2 months postimmunization . At 5 years, five of the nine patients (55%) were alive free of disease . No autoimmune diseases were detected in any of the immunized patients . A major hindering factor for such an approach was the limited availability of the allogenic melanoma cells.

Am J Surg Pathol, 1992 Jul, 16(7), 650 - 7
Immunocytochemical identification of Rochalimaea henselae in bacillary (epithelioid) angiomatosis, parenchymal bacillary peliosis, and persistent fever with bacteremia; Reed JA et al.; We report the immunocytochemical identification of Rochalimaea henselae, a newly recognized fastidious, Gram-negative, Warthin-Starry-positive organism, as the common pathogen in bacillary angiomatosis (BA), bacillary peliosis (BP) of the liver and spleen, and persistent fever with bacteremia in immunocompromised patients . Immunogenic proteins of the R . henselae strain isolated from the blood of a febrile immunocompromised patient with BP of the liver were used to produce primary immune serum in rabbits . Using immunocytochemical procedures, the polyclonal antiserum reacted strongly not only with the immunizing strain of the bacteria, but also with other blood isolates of R . henselae (five cases) from both immunocompromised and immunocompetent patients and with the organisms present in the tissue lesions of cutaneous BA (five cases) and BP of the liver (two cases) and spleen (one case) . The blood isolates and BA and BP tissue samples were obtained from widely separated geographic areas . The antiserum was weakly cross-reactive with cultures of Rochalimaea quintana, an organism closely related to R . henselae, but this reactivity was eliminated by specific adsorption . The antiserum did not cross-react with the Warthin-Starry-positive organisms associated with cat scratch disease (Afipia felis), syphilis (Treponema pallidum), Lyme disease (Borrelia burgdorferi) or chronic active gastritis (Helicobacter pylori) . Likewise, the antiserum did not identify organisms in eight cases of Kaposi's sarcoma, a disorder of immunocompromised patients that is clinically similar to BA . Further studies are needed to determine the prevalence of this newly recognized organism as well as its possible involvement in other angioproliferative diseases.

Protein Eng, 1992 Jul, 5(5), 421 - 6
The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease; Eijsink VG et al.; Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B . thermoproteolyticus (85% sequence identity) . Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior . The mutations had small effects on the thermostability, even after drastic changes, such as Leu284----Trp and Met168----Trp . The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4 degrees C . These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude . Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core.

Protein Eng, 1992 Jul, 5(5), 413 - 20
Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket; Teplyakov AV et al.; Serine endoproteases such as trypsins and subtilisins are known to have an extended substrate binding region that interacts with residues P6 to P3' of a substrate . In order to investigate the structural and functional effects of replacing residues at the S4 substrate binding pocket, the serine protease from the alkalophilic Bacillus strain PB92, which shows homology with the subtilisins, was mutated at positions 102 and 126-128 . Substitution of Val102 by Trp results in a 12-fold increase in activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpNA) . An X-ray structure analysis of the V102W mutant shows that the Trp side chain occupies a hydrophobic pocket at the surface of the molecule leaving a narrow crevice for the P4 residue of a substrate . Better binding of sAAPFpNA by the mutant compared with the wild type protein as indicated by the kinetic data might be due to the hydrophobic interaction of Ala P4 of the substrate with the introduced Trp102 side chain . The observed difference in binding of sAAPFpNA by protease PB92 and thermitase, both of which possess a Trp at position 102, is probably related to the amino acid substitutions at positions 105 and 126 (in the protease PB92 numbering) . Kinetic data for the variants obtained by random mutation of residues Ser126, Pro127 and Ser128 reveal that the activity towards sAAPFpNA increases when a hydrophobic residue is introduced at position 126.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Eng, 1992 Jul, 5(5), 405 - 11
Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus; van der Laan JM et al.; The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 A resolution . The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg . The model of the PB92 protease has been refined to an R-factor of 14.0% and contains 1882 protein atoms, two calcium ions and 188 water molecules . The overall folding of the polypeptide chain closely resembles that of the subtilisins . Furthermore, almost all of the secondary structure elements found in subtilisin Carlsberg are also present in the PB92 protease . The major differences between the two structures are located around the deletion regions (residues 37 and 158-161 in subtilisin Carlsberg) and in two loops which are known to be the most variable parts of subtilisin structures . Flexibility of one of these loops (residues 126-130 in the PB92 protease) is believed to account for the induced-fit mechanism of substrate binding.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1515 - 26
Binding of purified Bacillus sphaericus binary toxin and its deletion derivatives to Culex quinquefasciatus gut: elucidation of functional binding domains; Oei C et al.; Highly larvicidal strains of Bacillus sphaericus produce a binary toxin composed of 51 and 42 kDa proteins which binds to sharply delineated regions of the gastric caecum and posterior midgut of susceptible larvae of the mosquito Culex quinquefasciatus . To investigate the role of the individual subunits and the organization of functional binding regions within the toxin, plasmids were constructed for the expression in Escherichia coli of the toxin proteins and their NH2- and COOH-terminal deletion derivatives as fusions with glutathione S-transferase (GST) . Toxin proteins were purified by affinity chromatography followed by cleavage from the GST carrier with thrombin . The LC50 values for the purified toxin proteins and their deletion derivatives were determined . The binding patterns of fluorescently labelled toxin suggested that the 51 kDa protein is the primary binding component of the toxin and mediates the regional binding and internalization of the 42 kDa protein . Examination of the toxin deletion derivatives revealed that the NH2-terminal region of the 51 kDa protein was required for binding to the larval gut, whilst the COOH-terminal region was responsible for interacting with the 42 kDa protein . Toxicity was strongly correlated with the subsequent internalization of the toxin, probably by endocytosis.

Nippon Hinyokika Gakkai Zasshi, 1992 Jul, 83(7), 1052 - 61
{Experimental and clinical study of intravesical BCG therapy . The role in the prevention of recurrence of superficial bladder tumor}; Hirano A; The effects of Tokyo strain bacillus Calmette-Guerin (BCG), which is available in Japan for treatment, were studied in an experimental murine bladder tumor (MBT-2) model prior to clinical study for treatment of superficial bladder tumor . The results were as follows: Tokyo-strain BCG is more effective on the local injection around the tumor than on systemic administration . BCG therapy is more effective at earlier time of small tumor burden . BCG also has a prophylactic effect against the tumor growth . Clinical trial of intravesical instillation of BCG was performed on patients with superficial bladder tumor for prophylaxis of tumor recurrence after transurethral resection of the tumor . In patients of 145 primary cases, the tumor recurrence rate after BCG therapy was estimated, comparing with that of historical control in our department . The historical control groups are consisted of 50 patients who were treated by some intravesical chemotherapy after TUR and 38 patients, who were treated by TUR alone . The tumor recurrence rate in BCG group was significantly lower than that in both control groups . No relationship between PPD responsiveness and the tumor recurrence rate could be detected . On the other hand, in the patients of 36 recurrent cases, evaluation was performed by the tumor recurrence rate comparing with those during the two years prior to BCG therapy . The results demonstrated a statistically significant decrease in recurrent tumor following BCG therapy . Although most of the adverse effects in this study such as bladder irritability, flu-like syndrome and macroscopic hematuria were minimal and tolerable . There were no significant side effects or serious complications attributable to BCG therapy in this series . These results indicate that intravesical Tokyo strain BCG instillation provide prophylactic effects against recurrence of superficial bladder cancer.

Microbiologica, 1992 Jul, 15(3), 291 - 5
Molecular cloning of a specific DNA probe for the identification of Bacillus licheniformis; Bollet C et al.; Bacillus licheniformis has been found to be one of the dominant nosocomial species of Bacillus: laboratories dealing with nosocomial infections must be able to identify Bacillus up to the species level . To date, no DNA probes have been isolated for B . licheniformis although there is a clear need for a direct detection by polymerase chain reaction . The isolation of a B . licheniformis-specific DNA probe, as described in this paper, represents the first step toward accomplishing this goal.

J Dairy Sci, 1992 Jul, 75(7), 1870 - 6
Depletion of intramuscularly injected ceftiofur from the milk of dairy cattle; Jaglan PS et al.; Ceftiofur sodium, a new broad-spectrum cephalosporin, has been approved in the US, Canada, and several other countries throughout the world to treat bovine respiratory disease in cattle and dairy cows . In Experiment 1, 6 lactating cows were intramuscularly treated with 2.29 mg of {14C}ceftiofur/kg of BW daily for 5 d . In Experiment 2, 30 additional cows at three locations were similarly treated with 2.2 mg of ceftiofur (unlabeled)/kg of BW . Milk was collected every 12 and 24 h after each dose and every 12 h up to 5 d after the last dose . The majority of milk samples, both during treatment (12 and 24 h after each dose) and after the last dose (up to 5 d following ceftiofur treatment), were negative by screening procedures based on microbial inhibition (Delvotest-P, Bacillus stearothermophilus disk assay, and cylinder plate assays) . The receptor-binding Charm Test II assay, which has a limit of detection of .005 ppm of ceftiofur, gave positive tests for milk samples up to 48 h following treatment . When the Charm Test II assay is used with .008 IU/ml of penicillin as a positive control, 44% of the samples from individual cows were negative at 12 h posttreatment . Ninety percent of the samples from individual cows were negative at 24 h after the last treatment . The use of ceftiofur in dairy cattle in accordance with the label directions does not result in total residues in milk higher than the FDA-calculated safe concentration of 1-ppm ceftiofur equivalents . The milk from individual cows did not test positive by the commercial screening assays examined in this study, except for the Charm Test II . The Charm Test II was 90% negative using the Charm Sciences criteria at 24 h after the last treatment.

CLAO J, 1992 Jul, 18(3), 197 - 201
Early diagnosis of infectious keratitis with in vivo real time confocal microscopy; Chew SJ et al.; The tandem scanning confocal microscope (TSM) was adapted for in vivo examination of the cornea in rabbits with experimental bacterial and fungal keratitis . Compared to slit lamp biomicroscopy, the TSM provides superior lateral and axial resolution and serial optical sectioning capability, which may be useful for identification of corneal pathogens in the early stages of infection . We used the TSM to examine normal rabbit eyes infected with bacteria (Bacillus cereus) and a filamentous fungus (Aspergillus) . We also examined a human cornea removed by penetrating keratoplasty after a clinical diagnosis of amoebic keratitis . In the early stages of bacterial infection, slit lamp examination revealed a nonspecific minimal stromal haze and limbal injection indistinguishable from sterile ulcers and epithelial defects . With the TSM, bacteria were visible as highly refractile bodies in the epithelium and superficial stroma . Branching fungal hyphae were also easily identified by the TSM, as were Acanthamoeba cysts and parasites in the subepithelial stroma . Our results indicate that this technique may provide a new modality for quickly and accurately identifying the agent of corneal infection, thereby facilitating prompt and appropriate treatment.

Mol Gen Genet, 1992 Jul, 234(1), 129 - 37
Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase; Kuroda A et al.; A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli . Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27,513 . The gene was designated as cwlM, for cell wall lysis . The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region . Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity . The gene product purified from E . coli cells harboring a cwlM-bearing plasmid exhibited a M(r) value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) . The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B . licheniformis and B . subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference . CWLM prepared from B . subtilis cells harboring a plasmid containing cwlM had a similar M(r) value to that from E . coli . Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.

Mikrobiologiia, 1992 Jul-Aug, 61(4), 672 - 7
{Comparative electron microscopic study of Bacillus thuringiensis cells grown in a chemostat}; Blokhina TP et al.; It has been shown that the culture of Bacillus thuringiensis subsp . gall . 69-6 dissociates into R- and S-variants in chemostat . Although under some conditions the sporogenesis and the synthesis of S-variant toxin began two hours earlier than these processes in R-variant cells which were observed, respectively, after 10 or 12 hours from the beginning of the experiment, the intensities of sporogenesis and toxin production as well as the exit of spores and toxin excretion from cells were similar after 24 hours . The resistance to the bacteriophage present in chemostat was the advantage of S-variant cells . The data obtained by electron microscopy indicate that the phagoresistance is caused by the structural organization of the S-variant cell wall . Its peptidoglycan component is thin and is distinguished by crumb structure . By means of negative contrast microscopy it was found that the surface T-layer of R-variant cell wall was characterized by the tetragonal packaging of protein subunits indicating the regular orientation of phagoreceptors in it . The redistribution of protein subunits in the T-layer of S-variant cell wall prevented from the adsorbance of bacteriophages on the cell surface . The adsorbance of phages on the surface of R-variant cells was observed rather often . It led to the degradation of peptidoglycan, the formation of protoplasts and lysis.

Mikrobiologiia, 1992 Jul-Aug, 61(4), 577 - 84
{Influence of carbon sources on the biological activity and morphology of Bacillus thuringiensis parasporal crystals}; Iudina TG et al.; The influence of the carbon source on biological activity and morphological properties of the parasporal crystals of Bacillus thuringiensis subsp . kurstaki cultivated on three different media has been studied . Biological activity of delta-endotoxins contained in crystals was tested according to their impact on insects and microorganisms . Variations in carbon sources composition resulted in changes of biological activity and morphology of crystals, caused by alterations in synthesis rate of various delta-endotoxins contained in the crystals . Variations in the content of a certain carbon source (e.g . starch) in the medium produced no effect on the crystals properties, and the content variations of the delta-endotoxins content was proportional to the producer biomass . Control of the antibacterial effect of delta-endotoxins allows a valid evaluation of their biological activity under various growth conditions as well as comparison of the biological potential of entomopathogenic chemicals for plant protection containing delta-endotoxins with different specificity of insecticidal activity.

J Parenter Sci Technol, 1992 Jul-Aug, 46(4), 117 - 23
Temperature profiles and sterilization within a dead-ended tube; Young JH et al.; Use of steam-in-place (SIP) sterilization has increased as the complexity of biotechnology processing equipment has increased . Extensive biological testing is required prior to use of this equipment as no quantitative guidelines exist for the design of SIP sterilizable equipment . Dead-ended geometries present the most difficult challenge to SIP sterilization, but data are not available as to the effects of tube orientation, length and diameter on time required for sterilization . This study examines the effects on sterilization of location within a dead-ended tube and orientation of the tube with respect to the gravitational vector . Temperature profiles and biological kill of Bacillus stearothermophilus were determined for four tube orientations . Kill kinetics were characterized by time to start of kill and cycle log reduction (CLR) times . Both values increased with increasing distance up the tube and orientation of the tube in a more horizontal position . CLR values were as much as ten times greater than those resulting from saturated steam . Projected sterilization times were determined and found to be very dependent on tube orientation . Recommendations are given for sterilization and validation testing of dead-ended geometries.

Int J Food Microbiol, 1992 Jul, 16(3), 209 - 14
Heat-resistant fungi in the soil; Jesenska Z et al.; The occurrence of heat-resistant fungi has been demonstrated in samples of soil from the Slovak Republic . The heat-resistant species isolated were Byssochlamys nivea, Dichotomomyces cejpii, Eupenicillium baarnense, Neosartorya fischeri, Talaromyces avellaneus, Tal . bacillisporus, Tal . emersonii, Tal . flavus, Tal . trachyspermus, Tal . wortmanii, Botryotrichum piluliferum, Gilmaniella humicola and Nodulisporium sp . Some of them were isolated for the first time from Czechoslovakian soil . For the various soil samples examined, the occurrence of heat-resistant fungi varied qualitatively and quantitatively . Further research is needed to identify conditions affecting the occurrence of heat-resistant fungi in soil.

Genetika, 1992 Jul, 28(7), 82 - 8
{Inversion polymorphism in the malaria mosquito Anopheles messeae . XI . The group effect of larval infection with Bacillus thuringiensis subsp . israeliensis bacteria}; Gordeev MI et al.; Genetic consequences of the group and individual infections of Anopheles messeae larvae with bacteria Bacillus thuringiensis subsp . israelensis (Bti) were examined . The group effect of infections was manifested by increase in mortality and the speed-up of selection of chromosomal inversions resistant to Bti . Connection between the group and individual selections during the process of populations' adaptation is under discussion.

Environ Health Perspect, 1992 Jul, 97, 167 - 70
Effect of 60Co gamma-irradiation on the nonspecific cytotoxicity of alveolar macrophages in vitro; Gong Y et al.; This paper reports on the effect of radiation on the nonspecific cytotoxicity of rat alveolar macrophages (AM) . AM (effector cells) of bacille Calmette-Guerin-activated Wistar rats were irradiated with 60Co gamma rays in vitro to give doses of 0, 100, 300, and 500 Gy . Three hours after irradiation, the AM were cultured with human lung adenocarcinoma AGZY83-a and HeLa target cells in 125I-deoxyuridine-containing media for 6 hr and the cytotoxicity indexes determined . The results indicated that the cytotoxicity indexes of AM against human lung adenocarcinoma cells and HeLa cells were 94.3 +/- 0.3% and 81.3 +/- 1.9%, respectively . The cytotoxicity indexes using an effector/target cell ratio (E/T) of 10 seemed to be greater than with ratios of 20 and 30 . The cytotoxicity indexes of AM (7 rats), irradiated to give doses of 0, 100, 300, and 500 Gy, against adenocarcinoma cells at an E/T ratio of 10 were 87.9 +/- 8.4%, 65.4 +/- 14.1%, 47.5 +/- 17.5%, and 36.7 +/- 9.7%, respectively . The significance of the nonspecific cytotoxicity of AM in the immunological elimination of tumors and the inhibitory effect of radiation on AM cytotoxicity are discussed.

Can J Microbiol, 1992 Jul, 38(7), 694 - 5
Serotyping of Bacillus thuringiensis environmental isolates by extracellular heat-stable somatic antigens; Ohba M et al.; A total of 525 Bacillus thuringiensis environmental isolates, belonging to the five flagellar (H) serovars (alesti, sotto, kenyae, aizawai, and morrisoni), were serotyped by extracellular heat-stable somatic antigens (HSSAs) . The isolates belonging to a given H serovar were assigned to a single HSSA serogroup at a high frequency, 87-100% . This indicates that the extent of HSSA variation within a single H serovar is small in the field populations of these B . thuringiensis serovars.

Biochimie, 1992 Jul-Aug, 74(7-8), 695 - 704
Cloning and sequencing of the Bacillus megaterium spoIIA operon; Tao YP et al.; The spoIIA operon of Bacillus megaterium has been cloned and the nucleotide sequence determined . The spoIIA sequence contains three open reading frames coding for putative proteins of 116 aa, 147 aa, and 253 aa; the first and the third genes are preceded by a ribosomal binding site . The genes are in the same order as those of B subtilis and B licheniformis . The deduced amino acid sequences of these three open reading frames show 78-92% homology with SpoIIAA, SpoIIAB and SpoIIAC of B subtilis and B licheniformis . Northern hybridization revealed that B megaterium also has two spoIIA transcripts, 1.77 kb and 2.92 kb, attaining maximum expression at t1 and t3, respectively . In addition, homology to a possible penicillin binding protein gene upstream and the first part of a spoVA operon downstream has been identified on the 3.34-kb fragment . The spoIIA and the downstream spoVA promoter regions are highly conserved among these three species . Sequence analysis of the spoVA promoter revealed a region upstream to the -35 that is highly conserved across Bacillus species.

Int J Syst Bacteriol, 1992 Jul, 42(3), 439 - 45
Bacillus methanolicus sp . nov., a new species of thermotolerant, methanol-utilizing, endospore-forming bacteria; Arfman N et al.; The generic position of 14 strains of gram-positive bacteria able to use methanol as a growth substrate was determined . All are obligately aerobic, thermotolerant organisms that are able to grow at temperatures of 35 to 60 degrees C . Nine of the strains produce oval spores at a subterminal-to-central position in slightly swollen rod-shaped cells . DNA-DNA hybridization studies, 5S rRNA sequence analysis, and physiological characteristics revealed that all 14 strains cluster as a well-defined group and form a distinct new genospecies . Analysis of the 16S and 5S rRNA sequences indicated that this new species is distinct from Bacillus brevis but closely related to B . firmus and B . azotoformans . The name proposed for this new species is B . methanolicus . The type strain, PB1, has been deposited in the National Collection of Industrial and Marine Bacteria as NCIMB 13113.

Anticancer Res, 1992 Jul-Aug, 12(4), 1079 - 81
Hyperthermic potentiation of bleomycin cytotoxicity in the presence of Bacillus thuringiensis subsp . israelenis delta-endotoxin; Yokoyama Y et al.; We previously reported that a 25-kDa Bacillus thuringiensis subsp . israelensis (BTI) delta-endotoxin potentiated the antitumor activity of bleomycin (BLM) in both in vitro and in vivo systems . We report here a characteristic hyperthermic synergy toward the cytocidal activity of BLM in the presence of BTI endotoxin in cultured L1210 cells . Cytocidal gain, i.e., the ratio of the IC50 of BLM in the absence versus the presence of BTI delta-endotoxin, reached ca . 20 at 40 degrees C, suggesting that a more effective heat-BLM combination treatment may be achieved in the presence of BTI delta-endotoxin.

Histopathology, 1992 Jul, 21(1), 1 - 12
Whipple's disease: a histological, immunocytochemical and electronmicroscopic study of the immune response in the small intestinal mucosa; Ectors N et al.; Whipple's disease is a multisystem disorder with protean manifestations and with poorly understood aetiopathogenesis . It is unclear how the immune system reacts, whether it functions normally or not, whether it protects the organism or is defeated in one way or another by the 'Whipple bacillus' . The purpose of our study was to assess humoral and cellular immunity at the level of the intestinal mucosa . This histochemical, immunocytochemical and electronmicroscopic study, based on 16 cases, has shown that the changes in components of the mucosal immune system in Whipple's disease are quite different from normal . The phagocytic capacity of the macrophages, assessed microscopically, is abnormal, the number of intra-epithelial lymphocytes is increased, the CD 4/CD 8 cell ratio is decreased and the IgM positive cells in the lamina propria outnumber the IgA positive cells . These changes may be inter-dependent.

Mol Microbiol, 1992 Jul, 6(13), 1801 - 7
Phylogenetic relationships among the agent of bacillary angiomatosis, Bartonella bacilliformis, and other alpha-proteobacteria; Relman DA et al.; Bacillary angiomatosis (BA) and chronic bartonellosis are bacterial infections of humans which result in an unusual vascular proliferative tissue response . In order to determine their phylogenetic relationships, we have determined greater than 95% of the 16S rRNA sequences for these two organisms by amplification directly from infected BA tissue and from a Bartonella bacilliformis lyophilized culture . The BA agent and B . bacilliformis are closely related alpha-proteobacteria (98.5%), although the BA agent is more closely related to Rochalimaea quintana (99.1%) . Contrary to previous belief, the BA agent is distinct from, and less closely related to, the cat scratch bacillus (Afipia felis) (90.7%) . We propose a novel secondary structure in a hypervariable region of the 16S rRNA which is useful for alignment of primary sequences and which may be useful for design of nucleic acid probes.

FEMS Microbiol Lett, 1992 Jul 1, 73(1-2), 63 - 8
Comparison of Bacillus thuringiensis subsp . israelensis CryIVA and CryIVB cloned toxins reveals synergism in vivo; Angsuthanasombat C et al.; When the gene for the mosquitocidal protein CryIVA was expressed in two strains of Bacillus thuringiensis (Bt) cured of their resident delta-endotoxin genes, the protein accumulated as large inclusions . The inclusions produced in the Bt subsp . kurstaki recipient strain were twice as soluble at alkaline pH as the inclusions produced in Bt subsp . israelensis . Solubilized protoxins were activated by treatment with mosquito gut extracts or trypsin for varying lengths of time and tested for in vitro cytotoxicity on cell lines of three genera of mosquito . CryIVA treated with any of the mosquito gut extracts for 6 h showed significant toxicity against Anopheles gambiae cells and slight activity on Culex quinquefasciatus cells . For CryIVB, the only significant cytotoxicity observed was against Aedes aegypti cells after treatment with Aedes gut extract . In in vivo bioassays, both CryIVA, purified from either of the Bt recipient strains, and CryIVB inclusions were similarly toxic to A . aegypti and A . gambiae larvae but CryIVA was 25-fold more toxic to C . quinquefasciatus . Synergism in vivo between the two toxins was revealed when results from assaying single toxins and mixtures were compared . Mixtures of CryIVA and CryIVB proved to be 5-fold more toxic to Culex than either toxin used singly and showed a reduced but similar synergism when tested against Aedes and Anopheles larvae . The synergism was not duplicated in vitro using cell lines from these three insects.

J Invertebr Pathol, 1992 Jul, 60(1), 5 - 9
Genomic variations in mosquitocidal strains of Bacillus sphaericus detected by M13 DNA fingerprinting; Abadjieva A et al.; The genomic variation of Bacillus sphaericus reference and local strains belonging to different serotypes was examined by DNA fingerprinting . A phage M13 DNA probe detected a number of variable fragments in the restriction digests of total strain DNAs . The patterns of band distribution showed a certain homology among mosquitocidal strains, expressed by similarity index D and might be a reliable criterion for assessing the level of genomic similarity between closely related strains . An important advantage of DNA fingerprinting is the differentiation of one bacterial strain from another, both expressing common phenotype and possessing highly similar genomic portions . The strain variation revealed by the M13 probe will be useful for characterization of individual strains within a serotype . It could help as well to solve some uncertain cases based on the results obtained by other methods of identification.

J Invertebr Pathol, 1992 Jul, 60(1), 10 - 4
Characterization and toxicity to mosquito larvae of four Bacillus sphaericus strains isolated from Brazilian soils; Schenkel RG et al.; Four Bacillus sphaericus strains, S1, S2, S5, and L2, isolated from Brazilian soils, were found to be toxic to larvae of the mosquitoes Culex pipiens and Anopheles stephensi at a level similar to that of strain 2362 which is now used operationally . Like strain 2362, the four strains belonged to the serotype H5 and produced major proteins of apparent molecular weights of 125, 110, 56, and 43 kDa . These latter two proteins were immunologically related to toxins of the same molecular weight as B . sphaericus 2362 . Although the four Brazilian strains were very similar to strain 2362, gas chromatography analysis of the fatty acids revealed that these strains were different from strain 2362 and from each other, except for a possible similarity between strains S1 and S5.

Mol Microbiol, 1992 Jul, 6(14), 1959 - 67
IS231D, E and F, three new insertion sequences in Bacillus thuringiensis: extension of the IS231 family; Rezsohazy R et al.; IS231 constitutes a family of insertion sequences widespread among Bacillus thuringiensis subspecies . Three new IS231 variants have been isolated from B . thuringiensis subspecies finitimus (IS231 D and E) and israelensis (IS231F) . Like the previously described IS231A, B and C, these 1.7 kb elements display single open reading frames encoding 477/478-amino-acid proteins which share between 72% and 88% identity with those of the other members of the family . Sequence comparisons also reveal that all the iso-IS231 terminal inverted repeats are strongly conserved 20 bp sequences . A region susceptible to forming a stable hairpin structure is found just upstream of the open reading frame . Nucleotide substitutions occurring on one strand of the hairpin stems are compensated for by complementary changes at facing positions, giving credence to the hypothesis that this secondary structure plays a role in the regulation of transposition . Examination of IS231 D, E and F flanking sequences reveals that IS231F is bordered by a 12 bp direct repeat . No direct repeats were found flanking IS231D or IS231E.

Comp Biochem Physiol B, 1992 Jul, 102(3), 605 - 10
Isolation and partial characterization of binding proteins for immobilized delta endotoxin from solubilized brush border membrane vesicles of the silkworm, Bombyx mori, and the common cutworm, Spodoptera litura; Indrasith LS et al.; 1 . Brush border membrane vesicles (BBMV) were prepared from the Bombyx mori, and Spodoptera litura, midguts . BBMV was solubilized . 2 . Activated delta endotoxin from Bacillus thuringiensis were immobilized . 3 . Solubilized BBMV proteins were applied to the toxin column and the proteins bound were analyzed by SDS-PAGE . 4 . In the case of B . mori M(r) = 220,000, 150,000 and 130,000 and in the case of S . litura 160,000 bands were detected . 5 . The bindings were inhibited by N-acetyl galactosamine and mannose . 6 . The binding proteins applied onto a Con A column and eluted by 0.1 M methyl-alpha-glucose, suggesting that hybrid type sugar sidechain may involve in the interaction.

Mol Gen Mikrobiol Virusol, 1992 Jul-Aug, (7-8), 20 - 3
{Preparation and regeneration of protoplasts from Bacillus megaterium cells dried by sublimation}; Zerov IuP et al.; Bacterial protoplasts are widely used in genetical research, for instance, in protoplasts fusion experiments and the transfer of heterologous DNA into bacterial cells . The usage of a new fresh grown culture of bacteria in every experiment restricts the reproducibility of the results preventing the technique becoming widespread . The use of antioxidants as components of stabilizing medium for sublimation drying of Bacillus megaterium cells supported cellular viability in bacterial culture . It also made possible preservation of such cellular fundamental properties as the ability to form protoplasts and regenerate the cell wall . Efficiencies of protoplasts formation and generation are similar for lyophilized and fresh grown cells . Cellular properties are conserved for 6 months of storage at least . Experiments with a lot of lyophilized biomass samples are highly reproducible . The potential of the technique was demonstrated in obtaining the hybrid Bacillus megaterium colonies by fusion of protoplasts derived from lyophilized genetically marked strains stored for up to 6 months.

Cancer Res, 1992 Jun 15, 52(12), 3460 - 6
Expression of class II major histocompatibility complex molecules correlates with human colon tumor vaccine efficacy; Ransom JH et al.; Vaccination of colon cancer patients with X-irradiated autologous tumor cells and Bacillus Calmette-Guerin results in a significant reduction in tumor recurrence . A study was undertaken to determine whether the expression of tumor-associated antigens, expression of major histocompatibility complex molecules, or the cellular composition of the vaccine cells correlates with vaccine efficacy . A significant increase in the percentage of histocompatibility leukocyte antigen (HLA) class II molecule-expressing tumor cells was the only marker with a positive correlation . Because HLA class II molecule expression is not a prognostic marker in control patients, it was hypothesized that HLA class II molecules are involved in the induction of tumor immunity in patients treated with the autologous colon tumor vaccine . Enhancement of HLA class II molecule-expressing cells could be induced in X-irradiated colon tumor cells injected into the skin of mice when the cells were mixed with gamma-interferon . Therefore, addition of gamma-interferon to the colon tumor vaccine, resulting in increased numbers of HLA class II molecule-expressing cells, could potentiate the generation of tumor immunity.

Biochim Biophys Acta, 1992 Jun 19, 1100(3), 332 - 8
The relation between the soluble factor associated with H(+)-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli; Cunningham IJ et al.; Although in mitochondria, Escherichia coli and Rhodobacter capsulatus the H(+)-transhydrogenases are intrinsic membrane proteins, in Rhodospirillum rubrum a water-soluble component (Ths) and a membrane-bound component are together required for activity . Ths was selectively removed from chromatophore membranes of Rhs . rubrum and was purified to homogeneity by precipitation with (NH4)2SO4 and ion-exchange, affinity dye and gel exclusion chromatography . The latter indicated an Mr of approx . 74,000 under non-denaturing conditions but analysis of the pure protein by SDS-PAGE revealed a single polypeptide, Mr 43,000 . Antibodies against this polypeptide inhibited transhydrogenase activity of chromatophores and decreased the capacity of Ths to restore activity to depleted membranes . They reacted with a polypeptide of Mr 43,000 in crude cell extract, chromatophore membranes and chromatophore washings but not with transhydrogenase polypeptides from the membranes of E . coli, Rb . capsulatus or animal mitochondria . The N-terminal amino acid sequence of the 43,000 polypeptide was strongly homologous with the reported N-terminal regions of mitochondrial transhydrogenase and the alpha subunit of the E . coli protein . The break between the alpha and beta polypeptides of E . coli transhydrogenase is such that both components are membrane-associated . In contrast, these results suggest that in the Rhs . rubrum enzyme Ths has been formed by a break closer to the N-terminus, thus avoiding the putative trans-membrane helical segments and yielding a relatively hydrophilic subunit, which is water-soluble . There is a predicted similarity between Ths and the reported sequence of alanine dehydrogenase from Bacillus but Ths did not have any alanine dehydrogenase activity.

J Immunol, 1992 Jun 15, 148(12), 3820 - 9
IL-4 plays a dominant role in the differential development of Tho into Th1 and Th2 cells; Abehsira-Amar O et al.; We have analyzed the evolution of the pattern of lymphokine secretion by Th cell lines specific for either the synthetic terpolymer Glu60Ala30Tyr10 (GAT) or killed bacillus Calmette Guerin . When cultured in the presence of exogenous rIL-2 as a growth factor, GAT-specific Th cell lines secreted mainly IL-4, whereas bacillus Calmette Guerin-specific lines produced predominantly IL-2 . However, culturing in the presence of rIL-4 or of anti-IL-4 mAb and rIL-2 led to the establishment of Th2-like and Th1-like lines, respectively, regardless of their Ag specificity . Inasmuch as we show that the proliferative response of mature Th1 and Th2 cells was identical in the presence of IL-4, these results indicate that IL-4 influences the development of Th cell subsets . To understand the mode of IL-4 action, we isolated immature GAT-specific Tho clones able to secrete IL-2 and IL-4 . Two types of Tho cells were isolated: ThoA cells that secreted IL-2 and IL-4, but not IFN-gamma, and ThoB cells that secreted IL-2, IL-4, and IFN-gamma . We show for the first time that such cells are indeed Th precursors able to differentiate into Th1 or Th2 cells . We demonstrate that IL-4 positively and negatively controls the differentiation of Tho cells into Th2 and Th1 cells, respectively . When cultured in rIL-4, Tho cells stop secreting IL-2 and IFN-gamma, but maintain IL-4 secretion . Moreover, endogenous IL-4 produced by Tho cells has similar effects: when cultured in rIL-2 alone, Tho cells either keep their immature phenotype or become Th2 cells, but do not become Th1 cells . In contrast, neutralization of secreted IL-4 completely prevents the differentiation of Tho into Th2 cells, but permits the development of Th1 cells . The presence of exogenous IFN-gamma does not affect the development of Tho into Th1 and Th2 cells, because it does not modify either mode of IL-4 action . However, it influences the ratio between the two types of Tho cells: when IL-4 is neutralized, added IFN-gamma can induce IFN-gamma secretion by ThoA cells and thereby facilitate their passage into ThoB cells . Taken together, our results demonstrate that IL-4, in addition to mediating T cell growth, is a principal factor that controls the differential development of Tho cells into Th1 and Th2 cells.

J Clin Microbiol, 1992 Jun, 30(6), 1469 - 73
Tracking laboratory contamination by using a Bacillus cereus pseudoepidemic as an example; Morrell RM Jr et al.; From 1 March to 31 May 1990, Bacillus cereus was recovered from 24 of 5,534 (0.49%) blood cultures and 22 of 1,088 (2.02%) other body fluid cultures . The rarity of this organism as a pathogen and comparison with previous baseline rates led to the conclusion that it was a pseudoepidemic involving some form of culture contamination . Generalized precautions taken without specific knowledge of the contaminant source reduced the recovery rate of the organism . Recovery rates for the organism returned to normal baseline prevalence after environmental cultures and epidemiological analysis led to the sterilization of a contaminated water bath used for boiling thioglycollate media . The problems encountered in this investigation are examined, and a systematic approach to clinical laboratory epidemiology is outlined.

Biochemistry, 1992 Jun 9, 31(22), 5183 - 93
Phospholipids chiral at phosphorus . Stereochemical mechanism for the formation of inositol 1-phosphate catalyzed by phosphatidylinositol-specific phospholipase C; Bruzik KS et al.; The phosphatidylinositol-specific phospholipase C (PI-PLC) from mammalian sources catalyzes the simultaneous formation of both inositol 1,2-cyclic phosphate (IcP) and inositol 1-phosphate (IP) . It has not been established whether the two products are formed in sequential or parallel reactions, even though the latter has been favored in previous reports . This problem was investigated by using a stereochemical approach . Diastereomers of 1,2-dipalmitoyl-sn-glycero-3-(1D- {16O,17O}phosphoinositol) ({16O,17O}DPPI) and 1,2-dipalmitoyl-sn-glycero-3-(1D-thiophosphoinositol) (DPPsI) were synthesized, the latter with known configuration . Desulfurization of the DPPsI isomers of known configurations in H2(18)O gave {16O,18O}DPPI with known configurations, which allowed assignment of the configurations of {16O,17O}DPPI on the basis of 31P NMR analyses of silylated {16O,18O}DPPI and {16O,17O}DPPI (the inositol moiety was fully protected in this operation) . (Rp)- and (Sp)-{16O,17O}DPPI were then converted into trans- and cis-{16O,17O}IcP, respectively, by PI-PLC from Bacillus cereus, which had been shown to proceed with inversion of configuration at phosphorus {Lin, G., Bennett, F . C., & Tsai, M.-D . (1990) Biochemistry 29, 2747-2757} . 31P NMR analysis was again used to differentiate the silylated products of the two isomers of IcP, which then permitted assignments of IcP with unknown configuration derived from transesterification of (Rp)- and (Sp)-{16O,17O}DPPI by bovine brain PI-PLC-beta 1 . The results indicated inversion of configuration, in agreement with the steric course of the same reaction catalyzed by PI-PLCs from B . cereus and guinea pig uterus reported previously . For the steric course of the formation of inositol 1-phosphate catalyzed by PI-PLC, (Rp)- and (Sp)-{16O,17O}DPPI were hydrolyzed in H2(18)O to afford 1-{16O,17O,18O}IP, which was then converted to IcP chemically and analyzed by 31P NMR . The results indicated that both B . cereus PI-PLC and the PI-PLC-beta 1 from bovine brain catalyze conversion of DPPI to IP with overall retention of configuration at phosphorus . These results suggest that both bacterial and mammalian PI-PLCs catalyze the formation of IcP and IP by a sequential mechanism . However, the conversion of IcP to IP was detectable by 31P NMR only for the bacterial enzyme . Thus an alternative mechanism in which IcP and IP are formed by totally independent pathways, with formation of IP involving a covalent enzyme-phosphoinositol intermediate, cannot be ruled out for the mammalian enzyme . It was also found that both PI-PLCs displayed lack of stereo-specifically toward the 1,2-diacylglycerol moiety, which suggests that the hydrophobic part of phosphatidylinositol is not recognized by PI-PLC.

J Appl Bacteriol, 1992 Jun, 72(6), 493 - 9
Myceloid cell formation in Arthrobacter globiformis during osmotic stress; Deutch CE et al.; Arthrobacter globiformis was grown in a semi-defined liquid medium containing added solutes to determine the effects of osmotic stress on its reproduction and cell morphology . There was a progressive reduction in the specific growth rate during exponential phase as the concentration of NaCl was increased, although the final yields of the cultures during stationary phase were not affected . Clusters of branching myceloid cells rather than the typical bacillary forms predominated during exponential phase . These myceloids did not undergo complete septation and persisted into stationary phase . Similar responses were observed with potassium sulphate as the exogenous solute but less dramatic morphological effects were found with added polyethylene glycol or sucrose . The myceloids formed in response to osmotic stress could not be disrupted mechanically but were more sensitive than normal cells to lysozyme, particularly during stationary phase . Addition of osmoprotective compounds such as proline, glutamate, glycine betaine, or trehalose to the growth medium did not significantly relieve the effects of osmotic stress on growth rate or morphology . A . simplex also formed myceloid cells during osmotic stress but A . crystallopoietes did not . These results indicate that arthrobacters exhibit characteristic responses to osmotic stress and suggest these bacteria may contain novel osmoprotective compounds.

J Vet Med Sci, 1992 Jun, 54(3), 443 - 6
Production of the vacuolation factor of Bacillus cereus isolated from vomiting-type food poisoning; Shinagawa K et al.; Vacuole response in HEp-2 cells was induced with culture supernatants of Bacillus cereus strains isolated from outbreaks of vomiting- and diarrheal-type food poisoning grown in rice flour and laboratory media . High vacuole response was obtained with culture supernatants of B . cereus strains isolated from vomiting-type food poisoning grown in cooked rice suspension or on a cooked rice plate, whereas no response was obtained with those of the same strains grown in brain heart infusion and trypto-soya broth media . The vacuole activity appeared only after spore formation of B . cereus . The activity was stable to proteolytic enzymes, heating, and exposing to pH 2.0 and 11.0 . Of 124 strains isolated from B . cereus food poisoning that were tested, the vacuole activity was observed by 68 of 110 (61.8%) of the strains isolated from the vomiting-type food poisoning but not by all strains (14 strains) from diarrheal-type ones . Moreover, the vacuole response in the HEp-2 cells was found to be induced by 56 of 76 (73.7%) of the serotype H-1 strains isolated from vomiting-type food poisoning.

J Clin Microbiol, 1992 Jun, 30(6), 1617 - 9
Stability of the recombinant hepatitis B core antigen; Nath N et al.; The recombinant gene for hepatitis B core antigen (HBcAg) was cloned and expressed, and the protein was purified from Escherichia coli cultures . Purified HBcAg was tested for the effects of various physical and chemical agents on its immunoreactivity by a paramagnetic particle-based enzyme immunoassay . Recombinant HBcAg retained its immunoreactivity when heated at 70 degrees C for 60 min but was inactivated at 85 degrees C in 10 min . It was stable between pHs 5 and 10.5 but not at pHs 2 and 13.5 . Treatment with sodium dodecyl sulfate (SDS), ethanol, and methanol caused a significant loss in HBcAg reactivity . The proteolytic enzymes papain and bacterial protease (type VIII from Bacillus licheniformis) degraded HBcAg significantly, but trypsin and chymotrypsin did not . The effect of combined SDS and 2-mercaptoethanol on recombinant HBcAg was an immediate loss in immunoreactivity, followed by rapid recovery to about 50% of the initial level . This level was maintained for 24 to 48 h and was followed by an almost total loss of HBcAg in about 120 h.

J Am Vet Med Assoc, 1992 Jun 1, 200(11), 1678 - 81
Immunotherapy of periocular squamous cell carcinoma with metastasis in a pony; McCalla TL et al.; A 5-year-old Pony of America mare was referred for evaluation of inflamed upper and lower right eyelids . Squamous cell carcinoma of the eyelids and ulcerative keratitis secondary to self-trauma were diagnosed . Initial treatment of the eyelid neoplasia with 2 applications of cryotherapy failed to resolve the lesions, and immunotherapy with bacillus of Calmette-Guerin (BCG) was instituted . Multiple injections of BCG over a 17-week period resulted in progressive shrinkage of the tumor mass, but regional metastasis to the ipsilateral submandibular lymph node occurred . Six months later, ocular neoplastic lesions were not evident, and the lymph node had regressed in size . Eighteen months after the diagnosis of metastatic disease, signs of recurrence were not noticed in either the primary or secondary tumor sites . Squamous cell carcinoma of the equine eyelid historically carries a poor prognosis for resolution . Immunotherapy for equine ocular squamous cell carcinoma should be considered as a treatment alternative to cryosurgery, radiotherapy, hyperthermy, and CO2 laser ablation, especially in cases involving the eyelid.

Br J Oral Maxillofac Surg, 1992 Jun, 30(3), 165 - 7
Gram negative aerobic bacillus pneumonia in oral and maxillofacial surgery--a case for comment; Mitchell DA et al.; A case illustrating the potentially fatal complication of endogenous Gram Negative Aerobic Bacillus (GNAB) septicaemia secondary to nosocomial pneumonia is presented along with current theories as to its aetiology . The technique of selective decontamination of the digestive tract is designed and advocated to prevent such occurrences; oral and maxillofacial surgeons should be aware of this approach . It may be, however, that by using much simpler manoeuvres such as changes in policy regarding gastric stress ulcer prophylaxis, the already small risk of such an occurrence will be further reduced . Awareness of this condition will allow a higher index of suspicion when presented with catastrophic septic complications on the ITU and aid in more rational planning of antimicrobial therapy.

Appl Environ Microbiol, 1992 Jun, 58(6), 1823 - 31
Biodegradation of bisphenol A and other bisphenols by a gram-negative aerobic bacterium; Lobos JH et al.; A novel bacterium designated strain MV1 was isolated from a sludge enrichment taken from the wastewater treatment plant at a plastics manufacturing facility and shown to degrade 2,2-bis(4-hydroxyphenyl)propane (4,4'-isopropylidenediphenol or bisphenol A) . Strain MV1 is a gram-negative, aerobic bacillus that grows on bisphenol A as a sole source of carbon and energy . Total carbon analysis for bisphenol A degradation demonstrated that 60% of the carbon was mineralized to CO2, 20% was associated with the bacterial cells, and 20% was converted to soluble organic compounds . Metabolic intermediates detected in the culture medium during growth on bisphenol A were identified as 4-hydroxybenzoic acid, 4-hydroxyacetophenone, 2,2-bis(4-hydroxyphenyl)-1-propanol, and 2,3-bis(4-hydroxyphenyl)-1,2-propanediol . Most of the bisphenol A degraded by strain MV1 is cleaved in some way to form 4-hydroxybenzoic acid and 4-hydroxyacetophenone, which are subsequently mineralized or assimilated into cell carbon . In addition, about 20% of the bisphenol A is hydroxylated to form 2,2-bis(4-hydroxyphenyl)-1-propanol, which is slowly biotransformed to 2,3-bis(4-hydroxyphenyl)-1,2-propanediol . Cells that were grown on bisphenol A degraded a variety of bisphenol alkanes, hydroxylated benzoic acids, and hydroxylated acetophenones during resting-cell assays . Transmission electron microscopy of cells grown on bisphenol A revealed lipid storage granules and intracytoplasmic membranes.

Br J Dermatol, 1992 Jun, 126(6), 535 - 41
The clinical spectrum of bacillary angiomatosis; Webster GF et al.; Bacillary angiomatosis is a recently recognized bacterial infectious disease that is seen mainly in patients with the acquired immunodeficiency syndrome . Including this publication, 45 patients have been described in the medical literature . In this report we describe examples of the clinical presentations of bacillary angiomatosis and review therapeutic strategies.

Eur J Immunol, 1992 Jun, 22(6), 1667 - 9
High frequency of cord blood lymphocytes against mycobacterial 65-kDa heat-shock protein; Fischer HP et al.; A high frequency of nonadherent mononuclear cells in human cord blood proliferates in response to mycobacterial 65-kDa heat-shock protein . The frequency range in cord blood is not different from that in peripheral blood of Bacillus Calmette-Guerin vaccinated adults . In comparison we found 10 to 100 times lower frequencies to purified protein derivative in nonadherent cord blood mononuclear cells than in adult peripheral blood mononuclear cells . These findings may provide experimental support for Cohen's theory of the immunological homonculus.

J Bacteriol, 1992 Jun, 174(11), 3750 - 6
Physical maps of the genomes of three Bacillus cereus strains; Carlson CR et al.; NotI restriction maps of the chromosomes from Bacillus cereus ATCC 10876, ATCC 11778, and the B . cereus type strain ATCC 14579 have been established and compared with the previously established map of B . cereus ATCC 10987 . Between 10 and 14 NotI fragments were observed, ranging from 15 to 1,300 kb, in digests of DNA from the various strains . The sizes of the genomes varied between 5.4 and 6.3 Mb . The maps were constructed by hybridization of 42 random probes, prepared from B . cereus ATCC 10987 libraries, to fragments from partial and complete NotI digests, separated by pulsed-field gel electrophoresis . Nine probes were specific for ATCC 10987 only . Probes for five B . subtilis and five B . cereus genes were also used . The NotI restriction fragment patterns of the four strains were strikingly different.

J Exp Med, 1992 Jun 1, 175(6), 1717 - 28
Novel responses of human skin to intradermal recombinant granulocyte/macrophage-colony-stimulating factor: Langerhans cell recruitment, keratinocyte growth, and enhanced wound healing; Kaplan G et al.; Recombinant granulocyte/macrophage-colony-stimulating factor (rGM-CSF), prepared from Chinese hamster ovary (CHO) cells and Escherichia coli, was administered to 35 patients with the borderline and polar lepromatous forms of leprosy by the intradermal and subcutaneous routes at doses of 7.5-45.0 micrograms/d for 10 d . With each of these doses and routes, increases in the number of circulating eosinophils were noted . After the intradermal injection, the local skin sites demonstrated zones of roughening and micronodularity that appeared within 24-48 h and persisted for more than 6 d . Reinjection of sites led to enhanced areas of epidermal reaction . GM-CSF prepared from CHO cells was a more potent inducer of this effect . GM-CSF given by the subcutaneous route, at higher doses, failed to initiate these changes . At the microscopic level, the epidermis became thickened (+75%) with increased numbers and layers of enlarged keratinocytes . These contained increased numbers of ribosomes and prominent nucleoli, and were imbedded in a looser meshwork of the zona Pellucida . The modified keratinocytes remained MHC class II antigen negative throughout the course of the response . A major change in the dermis was the progressive accumulation of CD1+, Birbeck granule-positive cells . These Langerhans were recognizable at 48 h after intradermal injection and reached maximum numbers by 4 d . During this period the number of epidermal Langerhans cells remained relatively constant . No increment in dermal Langerhans cells occurred when GLM-CSF was injected by the subcutaneous route . No appreciable increase in the numbers of T cells and monocytes was noted, and granulocytes and eosinophils were largely present within the dermal microvasculature . 4-mm punch biopsies taken from injected sites and adjacent controls were compared in terms of the rapidity of wound healing . 22 of 26 sites demonstrated more rapid filling and hemostasis, whereas four were equivalent to controls . We conclude that rGM-CSF, when introduced into the skin, leads to enhanced keratinocyte growth, the selective recruitment of Langerhans cells into the dermis, and enhanced wound healing of the prepared site . There was no evidence of an enhanced cell-mediated response to Mycobacterium leprae, and bacillary numbers remained unchanged.

J Infect Dis, 1992 Jun, 165(6), 1138 - 41
Interactions between live Bartonella bacilliformis and endothelial cells; Garcia FU et al.; Bartonella bacilliformis, a gram-negative, flagellated, motile bacterium, is the etiologic agent of verruca peruana . It is found within the verruca, where it can form large cytoplasmic (Rocha-Lima) inclusions in endothelial cells . Previously, an activity has been described in homogenates of B . bacilliformis that in vitro increases the proliferation of endothelial cells and their production of tissue-type plasminogen activator (t-PA) and in vivo is angiogenic . The aim of the present study was to determine if live B . bacilliformis similarly stimulated endothelial cells and produced the Rocha-Lima inclusion . By measuring proliferation of cells and the production of t-PA in vitro, it was found that the live bacteria increased both parameters in a fashion similar to the homogenates of B . bacilliformis . Interaction between the bacteria and endothelial cells appeared to be necessary for proliferation . On electron microscopy, bacteria penetrated the endothelial cell within 1 h, forming a small membrane-bound inclusion . By 12 h, a large membrane-bound inclusion, similar to the Rocha-Lima inclusion, containing numerous bacteria was present . These data provide further evidence that B . bacilliformis has an angiogenic activity and that the bacteria are at least in part responsible for the vascular proliferation of the verruca.

FEBS Lett, 1992 Jun 1, 303(2-3), 193 - 6
Delivery of an accessory signal for cell activation by exogenous phosphatidylinositol-specific phospholipase C; Jamshedur Rahman SM et al.; Digestion of phosphatidylinositol (PI) or glycosylphosphatidylinositol (GPI) anchors of membrane proteins on the external cell surface with exogenous PI-specific phospholipase C (PIPLC) from Bacillus thuringiensis was shown to transmit a signal into the thymocyte to modulate the TCR/CD3 complex-induced signal delivery for cell activation . This was demonstrated for very early protein tyrosine phosphorylation, early c-fos transcription and late DNA synthesis . For this effect preincubation of the cells with PIPLC was required, but there was no evidence of involvement of any soluble products released from the cell surface by PIPLC in the signaling, suggesting a crucial role of the membrane-bound counterpart (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate . A possible role for this accessory signal in the microorganism-linked control of the (diacylglycerol or diradylglycerol) of the PI/GPI hydrolysate . A possible role for this accessory signal in the microorganism-linked control of the T cell receptor function is discussed.

Curr Opin Oncol, 1992 Jun, 4(3), 435 - 41
Bladder cancer; Ozen H; In the search for sensitive and specific tumor markers for bladder carcinoma, expression of various oncogenes and gene products (such as c-erb B-2, p53) and epidermal growth factor receptor merits particular attention . Although the results are not yet conclusive, important predictive markers are about to emerge from ongoing studies in this field . Bacillus Calmette-Guerrin treatment in superficial bladder cancer is probably the most successful immunotherapy in humans . But there is still a large knowledge deficit in the issues of optimal dose schedule and mechanisms of action . Although promising results of neoadjuvant chemotherapy in patients with invasive bladder cancers are reported, we must be cautious about changing our conventional approach until the results of large scale, controlled, randomized studies evaluating the survival are published.

Appl Biochem Biotechnol, 1992 Jun, 33(3), 193 - 203
Purification and general biochemical properties of thermostable pullulanase from Bacillus stearothermophilus G-82; Kambourova MS et al.; Thermostable extracellular pullulanase, produced by Bacillus stearothermophilus G-82 was purified to homogeneity from supernatants of continuous culture by ultrafiltration, ammonium sulphate precipitation, chromatography on Sephadex G-100, and DEAE cellulose . A mol wt of 53,000 was determined by gel filtration and 56,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) . The isoelectric point (pI) was 4.2 . The pullulanase contained predominantly acidic amino acids . The enzyme was optimally active at a temperature of 60 degrees C and pH 7.0 . It preserved 100% of its activity after 10 min treatment at 60 degrees C . The thermostability was considerably increased in the presence of pullulan . Ca2+ did not increase activity or thermostability . Enzyme activity was fully inhibited by N-bromosuccinimide and partially by phenylmethylsulfonyl fluoride . Bacillus stearothermophilus G-82 pullulanase was able to hydrolyze alpha 1-6 as well as alpha 1-4 glucosidic bonds in pullulan, amylopectin, amylose, glycogen, and dextrin . The enzyme showed highest affinity to pullulan (Km = 0.14).

J Am Mosq Control Assoc, 1992 Jun, 8(2), 184 - 6
An episode of resistance to permethrin in larvae of Simulium squamosum (Diptera: Simuliidae) from Cameroon, after 3 1/2 years of control; Hougard JM et al.; Because of the biting nuisance from females of Simulium squamosum, a 30 km section of the Sanaga River (Cameroon) was treated since 1987 with permethrin for the control of larval populations . In 1990, resistance to permethrin occurred in a small proportion of the larvae, with a resulting 2-4x increase of the LC95 for dead larvae (moribund larvae considered as live) . In 1991, after a 6-month interruption of the treatments, susceptibility to permethrin returned to the initial level, and was similar to the susceptibility of S . squamosum larvae from a non-treated section of the Sanaga . In the context of a small-scale control program, resistance to permethrin can be reversible, and it can be avoided by rotation with other types of insecticides such as Bacillus thuringiensis serovar, israelensis.

J Am Mosq Control Assoc, 1992 Jun, 8(2), 149 - 55
Reduction of mortality rates of Bacillus thuringiensis var . israelensis aqueous suspensions due to freezing and thawing; Tousignant ME et al.; When studying the behavior (carry, dispersion, persistence) of Bacillus thuringiensis var . israelensis (B.t.i.) formulations used in the treatment of rivers or streams for black fly control, a large number of samples containing small quantities of B.t.i . are required for proper analysis . Freezing is a useful procedure to prevent enzymatic alteration or bacterial growth in samples before bioassays are to be performed . Using Aedes atropalpus neonate larvae, we studied the effect of freezing and thawing of B.t.i . aqueous suspensions by looking at mortality response parameters such as the slope and the LC50 of the probit regression . Initial concentration values of 1, 5, 10 and 20 mg/liter at the moment of freezing of the B.t.i . suspensions did not significantly affect toxicity . The number of freeze-thaw cycles greatly increased the LC50 values without much change to the slope of the log-probit regressions . We derived an equation that allowed us to compensate for the loss of toxicity of a given B.t.i . sample, knowing the number of freeze-thaw cycles.

J Am Mosq Control Assoc, 1992 Jun, 8(2), 143 - 8
Fate of Bacillus sphaericus and Bacillus thuringiensis serovar israelensis in the aquatic environment; Yousten AA et al.; Bacillus sphaericus spores were suspended in bottles of filtered (0.45 microns) freshwater and seawater under various conditions of temperature, pH and salinity . Heat resistant culturable counts (spores) slowly decreased with time . Spores suspended in dialysis bags submerged in a freshwater pond or in flowing seawater underwent a more rapid drop in heat resistant spore counts than did spores held in bottles . Thus, laboratory studies may overestimate spore longevity in the environment . Spore settling rate was related to the nature of particulate material in the water column . Paraspores (or perhaps spores and toxin) of B . thuringiensis serovar israelensis (B.t.i.) had a greater tendency to adhere to and settle with suspended sediment and fine particulates than did paraspores of B . sphaericus . These observations may at least partially explain the greater persistence of B . sphaericus larvicidal activity in field tests than that of B.t.i..

J Am Mosq Control Assoc, 1992 Jun, 8(2), 126 - 30
Efficacy of Bacillus thuringiensis var . israelensis against larvae of the southern buffalo gnat, Cnephia pecuarum (Diptera: Simuliidae), and the influence of water temperature; Atwood DW et al.; Susceptibility of southern buffalo gnat larvae to Bacillus thuringiensis var . israelensis (B.t.i.) was studied during 1986-88 . Tests were conducted using Vectobac 12AS in a mini-gutter aquatic bioassay system . Mini-gutter tests using a 4.5 ppm B.t.i . concentration and a 10 min exposure period confirmed a positive correlation between water temperature and B.t.i . effectiveness . Significantly lower larval mortality occurred as water temperature decreased below 9 degrees C, indicating that proper timing of B.t.i . application is essential to maximize larval control . Even with this temperature limitation, larval control using B.t.i . should provide an economically effective means of preventing outbreaks of the southern buffalo gnat.

Antibiot Khimioter, 1992 Jun, 37(6), 5 - 7
{Selection and physiological study of culture of Bacillus circulans-- producer of butirosin}; Vikhanskii IuD et al.; For isolating a highly active variant of the butirosin-producing culture, a strain forming trace amounts of the antibiotic substance was used . Exposure to nitrosomethylbiuret and nitrosoguanidine and the use of selective media containing streptomycin and butirosin resulted in a 30-fold increase in the strain productivity . Thin layer chromatography of the produced antibiotic substance in the solvent system developed by the authors, mass spectrometry and assay of the antimicrobial spectrum in regard to ++gram-positive and ++gram-negative bacteria by using the known aminoglycosidine-inactivating enzymes revealed that the substance was identical to butirosin . Along with the major product, the fermentation broth contained up to 5 per cent of ribostamycin.

Immunology, 1992 Jun, 76(2), 217 - 24
Dendritic cells efficiently immunoselect mycobacterial-reactive T cells in human blood, including clonable antigen-reactive precursors; Pancholi P et al.; Given the persistence of tuberculosis throughout the world, the delineation of mechanisms that lead to protective immunity to Mycobacterium tuberculosis is important . We have evaluated the presenting function of human dendritic cells for mycobacterial antigens, since these antigen-presenting cells (APC) are particularly effective in initiating antigen-specific T-cell responses . Dendritic cells from blood prove to be active APC for mycobacteria-specific proliferative responses by CD4+ T cells from bacillus Calmette-Guerin (BCG)-vaccinated individuals . In the first 24-48 hr of the response, dendritic cells that have been pulsed with mycobacterial antigens, including live BCG, effectively bind T cells forming discrete cell clusters . The clusters represent about 1% of the applied T cells . Clusters are highly enriched in mycobacterial reactivity while the non-clusters are depleted . Clustered T cells can be used as a starting point to expand antigen-specific cell lines . Mitogen and allogeneic feeder cells were used as APC to expand the mycobacterial-reactive lines, because the antigen-specific T cells had been preselected by virtue of their binding to antigen-pulsed dendritic cells . We discuss the advantages of obtaining antigen-reactive T cells by using dendritic cells as immunoadsorbents . These lines should help delineate the range of mycobacterial antigens and T-cell responses that participate in host responses to mycobacteria.

Biosci Biotechnol Biochem, 1992 Jun, 56(6), 890 - 5
Accumulation of alkaliphilic Bacillus penicillinase cleaved within the signal sequence in cytoplasm of Escherichia coli; Aono R; Alkaliphilic Bacillus penicillinase produced by Escherichia coli is distributed in several subcellular compartments according to cultivation conditions . The penicillinase that accumulated in particular subcellular fractions of E . coli grown under different conditions was purified and characterized . Periplasmic or extracellular penicillinase (24 kDa) was mature protein, indicating that the putative precursor (27 kDa) was processed at the correct amino acid residue, probably by signal peptidase I . Cytoplasmic penicillinase contained two unusual proteins (25 kDa) that are produced by proteolytic cleavage of the precursor within its signal sequence.

J Am Mosq Control Assoc, 1992 Jun, 8(2), 166 - 72
Toxicity and residual action of the photoactivated compound, cyano-alpha-terthienyl, and its efficacy for reducing pre-imaginal populations of mosquitoes; Dosdall LM et al.; The photoactivated compound, cyano-alpha-terthienyl (cyano-alpha-T), was highly toxic to pre-imagines of the mosquitoes Culex restuans, Cx . tarsalis and Culiseta inornata when synergized with piperonyl butoxide (PBO) . Lethal concentrations for 50% mortality, determined during an outdoor trial using caged fourth-instar Culex spp . larvae, were 19.4, 15.4 and 12.9 g/ha at 24, 48 and 72 h after treatment, respectively . No residual activity of cyano-alpha-T was observed beyond 24 h following treatment . In artificial pool tests, greatest population reductions were achieved using dosages of 20 and 40 g/ha; statistically significant reductions were not observed following applications of 5 g/ha . Cyano-alpha-T plus PBO was more effective for reducing mosquito populations than alpha-terthienyl (alpha-T) plus PBO at comparable dosages, although it exhibited slightly lower insecticidal activity at a dosage of 20 g/ha than a formulation of Bacillus thuringiensis var . israelensis (Vectobac 12 AS, 0.12 ml/m2) . Greatest effectiveness of cyano-alpha-T plus PBO was observed in pools with low organic content relative to pools high in organic content.

J Gen Microbiol, 1992 Jun, 138 ( Pt 6), 1159 - 66
Differentiation of mosquito-pathogenic strains of Bacillus sphaericus from non-toxic varieties by ribosomal RNA gene restriction patterns; Aquino de Muro M et al.; DNA from 17 strains of Bacillus sphaericus, including representatives of all the established DNA homology groups, was cleaved with EcoRI or HindIII and fragments were separated by agarose gel electrophoresis . Southern blots of this DNA were hybridized to a radioactively labelled DNA probe prepared from the cloned 16S rrnB ribosomal RNA operon of Escherichia coli . Banding patterns of the chromosomal DNA digests and the autoradiograms were specific to DNA homology groups I (B . sphaericus sensu stricto), IIA (mosquito-pathogenic strains), IIB (B . fusiformis) and V, but groups III and IV were not clearly distinguished . This suggests that the mosquito-pathogenic strains represent a separate subspecies.

FEMS Microbiol Lett, 1992 Jun 1, 72(2), 127 - 32
Streptomyces lividans possesses a GroEL-like chaperonin; Marco S et al.; Streptomyces lividans grown at 45 degrees C produces a GroEL-like chaperonin . This protein is specifically synthesized in bacterial cell cultures upon heat shock induction . It has a similar size (62 kDa) to the GroEL-like proteins from Escherichia coli and Bacillus subtilus and shows immunological cross-reaction with serum raised against GroEL from E . coli . The S . lividans 62-kDa protein assembles into oligomers around 20S that show a morphology consistent with a barrel showing six-fold and seven-fold symmetries as previously described in E . coli and B . subtilis.

Mol Microbiol, 1992 Jun, 6(11), 1533 - 7
Involvement of a possible chaperonin in the efficient expression of a cloned CryIIA delta-endotoxin gene in Bacillus thuringiensis; Crickmore N et al.; The Bacillus thuringiensis cryIIA delta-endotoxin gene is found as the third-gene in a three-gene operon, with a sporulation-dependent promoter lying upstream of the first gene, orf1 . We show here that the polypeptide product of the middle gene (orf2) is required for efficient expression of the toxin gene . In the absence of a functional ORF2 polypeptide the toxin does not form the crystalline inclusions characteristic of other known Bacillus thuringiensis toxins . We discuss the importance of this finding with respect to the possible role of chaperonins in the crystallization of these proteins.

Wei Sheng Wu Xue Bao, 1992 Jun, 32(3), 167 - 75
{Modification and expression of insecticidal protein structural gene of Bacillus thuringiensis var . aizawai 7-29}; Guo S et al.; The regulative region (181bp) and the fifth toxic active domain (217bp) were removed from the insecticidal protein gene of Bacillus thuringiensis var . aizawai 7-29 . After the synthesis of the adaptor (15bp) that contains initiation codon (ATG) and the PCR synthesis of the fifth toxic active domain (229bp) that contains stop codon (TAA), were inserted into on 5' truncated and 3' truncated of the coding fod N-terminal peptid's DNA fragment, that to become a modified structural gene . The modified structural gene can be play initiatic translation-function and stop translation-function during translation of insecticidal protein . The insecticidal protein was determined by western blotting, showed the expression of modified structural gene in Escherichia coli JM 103 . The bioassay of insecticidal proteins showed the 3' truncated and 5' truncated of insecticidal gene was higher toxic active than the 3' truncated of insecticidal gene in Escherichia coli JM 103.

Rinsho Byori, 1992 Jun, 40(6), 670 - 2
{Activities and isozymes of adenosine deaminase and lactate dehydrogenase in tuberculous pleural effusion with special reference to the presence of mycobacterium tuberculosis}; Kurata N et al.; We have previously shown that tuberculous pleurisy possesses a high level of adenosine deaminase (ADA) which is predominantly composed of ADA2 . In this paper, we report the cases of tuberculous pleural effusion which contained mainly ADA1 . In these cases, mycobacterium tuberculosis was positive by smear examination and/or culture and granulocytes were found to be major components . Analysis of lactate dehydrogenase (LDH) revealed that its activity was high and LDH5 occupied about 50% of total activity . In the tubercle bacillus negative cases, lymphocytes were the main components and the levels of LDH containing mostly LDH3 were low . It was assumed that the difference in LDH activity and isozyme pattern is due to the differential presence of leukocytes in pleurisy i.e., granulocytes and lymphocytes in tubercle bacillus positive and negative pleurisy, respectively . In conclusion, tuberculous pleural effusions can be divided into two groups on the basis of ADA and LDH activities and isozymes which may reflect the presence of mycobacterium tuberculosis.

J Am Mosq Control Assoc, 1992 Jun, 8(2), 156 - 8
Efficacy of a flowable concentrate formulation of Bacillus thuringiensis (H-14) against larval mosquitoes in southern Iran; Zaim M et al.; A flowable concentrate formulation of Bacillus thuringiensis (H-14) {Bactimos FC (1000 ITU/mg)} was evaluated for the control of mosquito larvae in simulated ponds and natural breeding sites in Kazeroun (Fars Province), southern Iran . A comparison was made with Abate emulsifiable concentrate . Bactimos FC caused 93-96% anopheline and 97% culicine larval mortality 24 h posttreatment in simulated ponds and natural breeding sites, when used at the rate of 0.2 cc/m2 . Abate (0.015 cc/m2) resulted in significantly higher anopheline (98.1%) and culicine (100%) mortality at 24 h posttreatment . There was a relatively sharp decline in larval mortality 48 h posttreatment when Bactimos FC was applied . Five-day applications were suggested to prevent pupal production.

J Mol Biol, 1992 May 20, 225(2), 543 - 9
Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogue; Hansen S et al.; The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution . The final R-factor is 19.7% . We have also studied the binding of two other inhibitors, iodide and iodate, to PLC . X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives . A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding . The results have so far been ambiguous but are discussed briefly . Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site . PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy . The ninth blocks the entrance to the active site cleft and is of higher occupancy . Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.

Eur J Biochem, 1992 May 15, 206(1), 103 - 7
Substrate preferences of glutamic-acid-specific endopeptidases assessed by synthetic peptide substrates based on intramolecular fluorescence quenching; Breddam K et al.; The substrate preferences of the easily available Glu/Asp-specific enzymes from Staphyllococcus aureus (V8), Bacillus licheniformis and Streptomyces griseus have been extensively investigated using a series of synthetic peptide substrates, containing an N-terminal anthraniloyl group and a 3-nitrotyrosine close to the C-terminus, allowing the fluorimetric monitoring of substrate hydrolysis by the decrease in intramolecular quenching . All three enzymes hydrolysed Glu-Xaa peptide bonds approximately 1000-fold faster than Asp-Xaa bonds and they are consequently more appropriately termed Glu-specific enzymes . The difference in kcat/Km for the hydrolysis of substrates with Glu and Asp is primarily due to a difference in kcat . The enzymes appear to hydrolyse all types of Glu-Xaa bonds, although those with Xaa as Asp and, in particular, Xaa as Pro, are hydrolysed with very low rates . The influence of the nature of the amino acid residues at the substrate positions P2, P3, P4, P'1 and P'2 has been determined and it is shown that the enzyme from S . griseus exhibits the most narrow substrate preference . The results are useful in connection with fragmentation of proteins for sequencing purposes as well as for cleavage of fusion proteins.

J Biol Chem, 1992 May 15, 267(14), 9580 - 8
Features of apparent nonchemiosmotic energization of oxidative phosphorylation by alkaliphilic Bacillus firmus OF4; Guffanti AA et al.; Oxidative phosphorylation by extremely alkaliphilic Bacillus species violates two major predictions of the chemiosmotic hypothesis: the magnitude of the chemiosmotic driving force, the delta p (electrochemical proton gradient), is too low to account for the phosphorylation potentials observed during growth at pH 10.5 without using a much higher H+/ATP stoichiometry than used during growth at pH 7.5, and artificially imposed diffusion potentials fail to energize ATP synthesis above about pH 9.5 (Guffanti, A . A., and Krulwich, T . A . (1989) Annu . Rev . Microbiol . 43, 435-463) . To further examine the latter observation, large valinomycin-mediated potassium diffusion potentials were imposed across starved cells of Bacillus firmus OF4 at various pH values from pH 7.5 to 10.5 . As the external pH increased above pH 8, there was a sharp decrease in the rate of ATP synthesis in response to an imposed diffusion potential . The rate of ATP synthesis fell to zero by pH 9.2 and 9.4, respectively, in the presence and absence of a small inwardly directed Na+ gradient . Electrogenic Na+/H+ antiport and Na+/alpha-aminoisobutyric acid symport proceeded at substantial rates throughout . When synthesis was energized by an electron donor, cells under comparable conditions synthesized ATP at rapid rates up to pH 10.5 . The proton transfers that occur during respiration-dependent oxidative phosphorylation at pH 10.5 may depend upon specific complexes . Cells grown at pH 7.5, which have one-third the levels of the caa3-type terminal oxidase, and slightly lower levels of certain other respiratory chain complexes than pH 10.5-grown cells, support only low rates of ATP synthesis at pH 10.5, although energy-dependent symport and antiport rates are comparable with those in pH 10.5-grown cells . A model is presented for oxidative phosphorylation by the alkaliphilic Bacillus that involves a nonchemiosmotic direct intramembrane transfer of protons from specific respiratory chain complexes to the F0 sector of the ATPase, whereas remaining respiratory chain complexes extrude protons into the bulk to generate the bulk potential required both for ATP synthesis and other bioenergetic work . A pK-regulated gate or a delocalized proton pathway that fails to work above pH 9.5 are suggested as possible features that account for the loss of efficacy of a bulk-imposed diffusion potential in energizing ATP synthesis above pH 9.4.

Appl Environ Microbiol, 1992 May, 58(5), 1650 - 5
Cloning and expression of the cryIVD gene of Bacillus thuringiensis subsp . israelensis in the cyanobacterium Agmenellum quadruplicatum PR-6 and its resulting larvicidal activity; Murphy RC et al.; A mosquitocidal cyanobacterium has been developed by introducing the mosquito-toxic cryIVD gene from Bacillus thuringiensis subsp . israelensis into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6 (Synechococcus sp . strain PCC 7002) . The cryIVD gene was introduced into the cyanobacterium on a derivative of the PR-6 expression vector pAQE19 delta Sal in which the cryIVD gene was translationally fused to the initial coding sequence of the highly expressed PR-6 cpcB gene . Coomassie blue staining and immunoblot analysis of gel-fractionated cell extract polypeptides indicate that the cpcB-cryIVD gene fusion is expressed at high levels in the cyanobacterial cells, with little or no apparent degradation of the cryIVD gene product . Larvicidal assays revealed that freshly hatched Culex pipiens mosquito larvae readily ingested the transformed cyanobacteria and that the cells proved to be toxic to the larvae.

J Clin Microbiol, 1992 May, 30(5), 1327 - 30
Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids; Floyd MM et al.; Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Guerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M . tuberculosis and M . bovis by elution and relative retention times is possible . Mycolic acid patterns of BCG eluted from the column 0.5 min before M . tuberculosis or M . bovis, resulting in relative retention times for two peaks not seen in the pattern of M . tuberculosis or M . bovis . Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains . These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M . tuberculosis and M . bovis.

Biochim Biophys Acta, 1992 May 7, 1131(1), 111 - 3
Nucleotide and derived amino acid sequence of the subtilisin from the antarctic psychrotroph Bacillus TA39; Narinx E et al.; The nucleotide sequence of the subtilisin-encoding gene from the antarctic psychrotroph Bacillus TA39 was determined . The primary structure of the subtilisin precursor is composed of 420 amino acids giving rise to a mature enzyme of 309 amino acids . Asp-145, His-185 and Ser-361 are the proposed catalytic residues of the active site.

J Gen Microbiol, 1992 May, 138 ( Pt 5), 987 - 96
Development of a synthetic medium for continuous anaerobic growth and ethanol production with a lactate dehydrogenase mutant of Bacillus stearothermophilus; San Martin R et al.; A synthetic medium was developed by the pulse and medium-shift technique for the continuous cultivation of Bacillus stearothermophilus strain LLD-15 (NCIMB 12428) under anaerobic conditions . This mutant strain lacks L-lactate dehydrogenase activity, and is a promising candidate for the production of ethanol from pentoses and hexoses, using a high-temperature two-stage process . The final medium contained four amino acids and five vitamins, and growth characteristics in this medium compared well with those in complex medium containing yeast extract and tryptone . At 70 degrees C, the medium was capable of supporting good anaerobic and aerobic growth at 10 g input sucrose l-1 . High ethanol production indicated that pyruvate metabolism probably occurred via the combined activity of the pyruvate-formate-lyase pathway and pyruvate dehydrogenase.

J Biochem (Tokyo), 1992 May, 111(5), 584 - 8
The effect of amino acid deletion in subtilisin E, based on structural comparison with a microbial alkaline elastase, on its substrate specificity and catalysis; Takagi H et al.; Subtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering . In this study, we analyzed the substrate specificity of B . subtilis subtilisin E based on the structure of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity . Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft . To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis . When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase . The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes.

Genetika, 1992 May, 28(5), 66 - 72
{Isolation and analysis of protease-deficient mutants of Bacillus amyloliquefaciens}; Plotnikova TG et al.; Four types of protease negative mutants of Bacillus amyloliquefaciens A50 were selected after four stages of step-by-step UV light mutagenesis . EDTA, and PMSF were used as inhibitors of protease activity to characterize the protease negative mutants with regard to the protease type . The electrophoretic patterns of proteases from culture medium of B . amyloliquefaciens protease deficient mutants were studied . The proinsulin stability in the culture medium of different mutants was analysed . Protease deficient B . amyloliquefaciens strain A50-32 may be used as a recipient for cloning and expression of a foreign proteins genes.

Naturwissenschaften, 1992 May, 79(5), 202 - 12
{Molecular biology and regulatory mechanisms of antibiotic production in Bacillus}; Marahiel MA; Several species of the genus Bacillus produce linear and cyclic peptide antibiotics nonribosomally through multienzyme complexes by the so-called thiotemplate mechanism . Molecular genetic studies have shown that some peptide antibiotic biosynthesis genes are organized in operons and that they are expressed postexponentially under conditions that also activate the process of endospore formation in Bacillus . Furthermore, DNA-sequence analysis of some multifunctional peptide synthetase genes revealed that they contain a highly conserved and repeated domain structure . The same domain was also found to be conserved within a superfamily of peptide synthetases and adenylate-forming enzymes of diverse origins . Based on sequence homology and functional similarity I conclude that those enzymes bearing domain(s) represent a family of superenzymes which may have a common evolutionary origin.

Immunology, 1992 May, 76(1), 129 - 32
Influence of H-2 genes on growth of Mycobacterium tuberculosis in the lungs of chronically infected mice; Brett S et al.; Mice infected by intraperitoneal injection of Mycobacterium tuberculosis were studied over a 23-week period . They showed progressive infection in the lung (with increasing microbial count and granuloma size) whereas viable bacillary counts remained largely stationary in the spleen and in the liver . The influence of H-2 genes on the progression of the lung infection was studied in four congenic strains of animals with B10 and three congenic strains of animals with BALB backgrounds . H-2k mice had significantly higher bacterial counts in the lung than H-2b mice on both B10 and BALB backgrounds, BALB . K (H-2k) mice were also more susceptible than BALB/c (H-2d) mice . Results with recombinant strains showed that bacillary counts and granulomatous infiltration were lower in the B10 (KbAbE-Db) compared with B10.A(3R) (KbAbEbDd) strain and in B10.A(4R) (KkAkE-Db) compared with B10.BR (KkAkEkDk) mice . This resistance to the late expansion of tuberculous infection in the lungs may be associated with the lack of an expressed I-E molecular or with the expression of the Db molecule.

Krankenpfl J, 1992 May, 30(5), 204 - 6
{In Africa as a nurse}; Haag M; PIP: The author relates her experience in Benin during a 3 and 1/2 year tenure as a nurse under the aegis of the German Development Agency . In Malanville, she was responsible for starting the operating room, caring for hygiene, sterility, and the related training of domestic staff . A septic and aseptic operating room was set up along with a storage room for instruments, a sterilization room, and a changing room . For the operating and surgical station, the following personnel were available: 2 nurses with 3 years of training, 1 nurse with 2 years of training, and 3 orderlies without training . A nurse with 3 years of training was assigned to the author to carry on the project after her departure . The standard of operating care was very low . It took a month to teach the staff what was not sterile . There was a even problem with putting on sterile gloves which required an exercise in patience . There were an average of 5 relatives per patient taking care of the patient and cooking . The undernutrition center for infants had 6 beds with 2 German nurses who administered Bacillus Calmette-Guerin (BCG), diphtheria, polio, and tetanus vaccinations . Their activity was strengthened by nutrition counselling and plans for underweight and malnourished children . Abrupt weaning that resulted in harmful diarrhea and vomiting was prevalent . Clinical signs of marasmus and kwashiorkor were frequent . In the middle of 1990, AIDS educators informed students of the public school as well as registered prostitutes about condom use . In the hospital, there were about 900 births per year, and women were asked to follow recommendations for prenatal care, especially to achieve anemia prevention by getting iron tablets . They were urged to deliver in the clinic, not at home assisted by untrained midwives . Oxytocin and syntometrin were available as was a hand-driven, vacuum evacuation pump . This experience made a lasting impression on the author who has resolved to go to another developing country to train traditional birth attendants in midwifery .

J Invertebr Pathol, 1992 May, 59(3), 286 - 9
A novel bioassay system for evaluating the toxicity of Bacillus thuringiensis israelensis against mosquito larvae; Misch DW et al.; A bioassay system employing acutely toxic concentrations of a spore-crystal mixture of Bacillus thuringiensis subspecies israelensis against fourth instar larvae of Aedes aegypti (L.) is described . Individual larvae are separately exposed to toxin in glass-lined miniature wells or scintillation vials . This method is free from the deaths due to predation among larvae . Such larval deaths are commonly encountered in bioassay groups of 25 larvae as currently specified in the World Health Organization guidelines . Our method offers shortened testing time, increased accuracy, and improved statistical precision.

Clin Infect Dis, 1992 May, 14(5), 1045 - 9
Capnocytophaga bacteremia in a patient with Hodgkin's disease following bone marrow transplantation: case report and review; Bilgrami S et al.; Capnocytophaga is a gram-negative, capnophilic, facultatively anaerobic bacillus that normally inhabits the oral cavity . We report the case of a patient who developed capnocytophaga bacteremia following autologous bone marrow transplantation for Hodgkin's disease, and we review other reported cases of capnocytophaga bacteremia in immunocompromised patients . In our case infection followed pretransplantation conditioning and was associated with severe oral mucositis and neutropenia . Antibiotic therapy resulted in clinical resolution of infection . Capnocytophaga bacteremia should be included in the differential diagnosis of febrile neutropenia in immunocompromised patients (e.g., those undergoing bone marrow transplantation) especially in the presence of mucositis and gingival bleeding.

Biochem J, 1992 May 1, 283 ( Pt 3), 665 - 71
Expression in Escherichia coli of a sub-gene encoding the lipoyl and peripheral subunit-binding domains of the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus; Hipps DS et al.; A sub-gene encoding the N-terminal 170 residues of the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was over-expressed in Escherichia coli . The expressed polypeptide consists of the lipoyl domain, inter-domain linker and peripheral subunit-binding domain; these were found to have folded into their native functional conformations as judged by reductive acetylation of the lipoyl domain, limited proteolysis of the linker region and ability to bind the dihydrolipoamide dehydrogenase dimer . The di-domain was largely (80%) unlipoylated; a small proportion (4%) was correctly modified with lipoic acid and the remainder (16%) was aberrantly modified with octanoic acid . A polyclonal antiserum was raised that recognized both the di-domain and the individual component domains . The 400 MHz 1H-n.m.r . spectrum of the di-domain showed resonances corresponding to those seen in spectra of the lipoyl domain, plus others characteristic of amino acid residues in the flexible linker region . Further, as yet unidentified, resonances are likely to be derived from the peripheral subunit-binding domain . The existence and independent folding of the peripheral subunit-binding domain is thus confirmed and its purification in large-scale amounts for detailed structural analysis is now possible.

Mol Microbiol, 1992 May, 6(9), 1211 - 7
Identification and characterization of a novel Bacillus thuringiensis delta-endotoxin entomocidal to coleopteran and lepidopteran larvae; Tailor R et al.; A new class of Bacillus thuringiensis delta-endotoxins, or insecticidal control proteins (ICPs), is defined by an apparently cryptic protein with a unique primary structure and novel entomocidal specificity for certain coleopteran and lepidopteran species . The discovery of a new group of ICPs will extend the use of this natural insecticide in integrated pest-management systems.

J Gen Virol, 1992 May, 73 ( Pt 5), 1297 - 301
Characterization of the discontinuities in rice tungro bacilliform virus DNA; Bao Y et al.; The dsDNA of rice tungro bacilliform virus (RTBV) has two discontinuities, one on each strand, each in a specific position as found in other pararetroviruses . The 5' end of discontinuity 1 was mapped to nucleotide 1 of the published RTBV DNA sequence which suggests that tRNAiMet serves as a primer for negative strand DNA synthesis . This 5' terminus contains up to two ribonucleotides and the 3' terminus overlaps it by five to 25 nucleotides . The discontinuity 2 (D2) did not map to a purine-rich region as has been found in other similar viruses . Both the 5' and 3' termini of D2 were heterogeneous in position giving structures varying from a gap of 10 nucleotides to an overlap of 103 nucleotides.

J Clin Microbiol, 1992 May, 30(5), 1357 - 60
Pseudoepidemic of Nocardia asteroides associated with a mycobacterial culture system; Patterson JE et al.; Nocardia isolations increased from 0.7 to 11.7/1,000 acid-fast bacillus and mycological cultures (P less than 0.000001) . Only three isolations from one patient represented infection . Pseudoepidemic strain identity was confirmed by DNA fingerprinting; the isolate causing infection was distinct . The end of the pseudoepidemic was associated with changing the needle sterilizer and prolonging needle sterilization time on the BACTEC 460 machine . To our knowledge, this is the first reported Nocardia asteroides pseudoepidemic.

J Clin Microbiol, 1992 May, 30(5), 1338 - 40
Assay of urease-inhibiting activity in serum from children infected with Helicobacter pylori; Thomas JE et al.; In order to provide a basis for obtaining further information concerning the host response to Helicobacter pylori urease, four assay methods for detecting urease-inhibiting activity in serum were examined . A quantitative assay, established in a COBAS BIO centrifugal fast analyzer and based on detection of the consumption of NADH by glutamate dehydrogenase stimulated by ammonia production, was considered most suitable for large-scale serological work . Serum samples from 63 children (aged 5 to 16 years), 28 of whom had seropositive H . pylori gastritis, were assayed . One of the serum samples in this latter group showed significant inhibitory activity . This serum sample was one of 13 in the seropositive group known to bind to urease antigen . It showed no inhibitory activity against Bacillus pasteurii or jack bean urease . Protein A binding and heat treatment indicated that the inhibitory activity was immunoglobulin G mediated . The patient from whom this sample was collected showed no distinctive features in his illness . The COBAS BIO analyzer-based urease inhibition assay provides a new tool for studying one aspect of the host response to H . pylori infection.

Am J Clin Pathol, 1992 May, 97(5), 713 - 8
Bacillary epithelioid angiomatosis involving the liver, spleen, and skin in an AIDS patient with concurrent Kaposi's sarcoma; Steeper TA et al.; The simultaneous findings of bacillary epithelioid angiomatosis and Kaposi's sarcoma of the skin with visceral hepatosplenic bacillary epithelioid angiomatosis is reported in a patient with acquired immune deficiency syndrome . Liver biopsy showed periportal peliotic spindle cell foci that initially were misinterpreted as Kaposi's sarcoma . After antibiotic therapy induced rapid clinical improvement, repeated liver biopsy showed resolution of the previously noted lesions . Although the violaceous skin lesions all appeared similar clinically, some resolved completely and some progressed . One of the latter was biopsied and had the histologic features of Kaposi's sarcoma . The differential diagnosis is discussed.

J Urol, 1992 May, 147(5), 1439 - 43
Sequential immunocytological evaluation of murine transitional cell carcinoma during intralesional bacillus Calmette-Guerin and interleukin-2 immunotherapy; Sosnowski JT et al.; The antitumor effect of intralesionally administered recombinant interleukin-2 was highly effective (90% complete response) in murine bladder cancer . We postulated that interleukin-2 may be integral to the Bacillus Calmette-Guerin-induced antitumor response in human bladder cancer . Flow cytometric evaluation of the tumor infiltrates was compared before and after intralesional treatment of an established, untreated murine bladder tumor model with recombinant interleukin-2, Bacillus Calmette-Guerin or saline . Large increases in the number of tumor infiltrating immune cells occurred between the day of randomization and the second day (one day after the first treatment) in all three groups . However, since tumor volume was reduced by treatment, the ratios of the immune cells to tumor volume was increased . The ratios of T(helper), T(cytotoxic)/suppressor cells, macrophages, and natural killer cells to tumor volume were 1.5 to 3.4 times higher in the interleukin-2 and Bacillus Calmette-Guerin groups in comparison to the saline group . The ratio of T(helper)/T(cytotoxic)/suppressor cells however, remained approximately the same despite treatment . Over the next 22 days all subpopulations of tumor infiltrating immune cells decreased in number and frequency to less than measurable levels . The similar modulation of infiltrating immune cell subpopulations by Bacillus Calmette-Guerin and interleukin-2 may indicate that the production of interleukin-2 is part of the tumor modulating mechanism of Bacillus Calmette-Guerin.

J Urol, 1992 May, 147(5), 1256 - 8
Dose-response of bacillus Calmette-Guerin in the treatment of superficial bladder cancer; Morales A et al.; Although bacillus Calmette-Guerin (BCG) has been recognized as an effective therapy for superficial bladder cancer, dose-response studies are not available . Such studies are important because the administration of the vaccine is not devoid of significant side effects when the standard dose of 120 mg . is used . It has been speculated that smaller doses may be equally effective but carry fewer side effects . A controlled study comparing 2 doses of BCG was conducted as an initial step to explore this possibility . A total of 97 patients with a diagnosis of superficial (stages TIS, Ta and T1) bladder cancer was assigned to receive either 60 or 120 mg . BCG intravesically weekly for 6 weeks . The higher dose resulted in a better response for stages TIS, Ta (for prophylaxis of recurrence) and T1 . However, the differences were not statistically significant . When stage TIS and Ta tumors coexisted, a significantly better response was recorded for the high dose . The overall success for the 2 treatments was 67% and 37% for the high and low doses, respectively . These differences reached statistical significance (p less than 0.02) . Side effects were significantly less in number and severity in patients receiving the smaller dose of the vaccine.

J Bacteriol, 1992 May, 174(9), 3087 - 91
Purification and characterization of a Bacillus polymyxa beta-glucosidase expressed in Escherichia coli; Painbeni E et al.; The beta-glucosidase encoded by the bglA gene from Bacillus polymyxa was overproduced in Escherichia coli by using a plasmid in which bglA is under control of the lacI promoter . Induction with isopropyl-beta-D-thiogalactopyranoside allowed an increase in the specific activity of the enzyme of about 100 times the basal level of gene expression . The enzyme was purified by a two-step procedure involving salting out with ammonium sulfate and ion-exchange chromatography with DEAE-cellulose . Fractions of beta-glucosidase activity recovered by this procedure, after electrophoresis in an acrylamide gel and staining with silver nitrate, yielded a single band of protein . This band was shown by a zymogram to correspond to beta-glucosidase activity . The purified protein showed an apparent molecular mass of 50 kDa and an isoelectric point of 4.6, values in agreement with those expected from the nucleotide sequence of the gene . Km values of the enzyme, with either cellobiose or p-nitrophenyl-beta-D-glucoside as the substrate, were determined . It was shown that the enzyme is competitively inhibited by glucose . The effects of different metallic ions and other agents were studied . Hg2+ was strongly inhibitory, while none of the other cations tested had any significant effect . Ethanol did not show the stimulating effect observed with other beta-glucosidases . The mechanism of enzyme action was investigated . High-pressure liquid chromatography analysis with cellobiose as the substrate confirmed previous data revealing the formation of two products, glucose and another, unidentified, compound . Results presented here indicate that this compound is cellotriose formed by transglycosylation.

Cancer Res, 1992 May 1, 52(9), 2440 - 6
Locoregional administration of etoposide, but not of interleukin 2, facilitates active specific immunization in guinea pigs with advanced carcinoma; Claessen AM et al.; Using a guinea pig line 10 hepatocellular carcinoma model for advanced metastatic disease, we studied the therapeutic effect of local cytotoxic drug treatment at the tumor site as compared to, and in combination with, active specific immunization . In addition, locoregional treatment with interleukin 2 (IL-2) was studied . Intratumoral administration of the cytotoxic drug etoposide (VP-16), but not of IL-2, when started in a late stage of tumor growth and continued for 3 wk, caused full regression of all intradermally implanted tumors and cured a small number of animals (14%) . When the primary tumor was removed at the onset of treatment, administration of VP-16 and, to a lesser degree, IL-2 at the former tumor site led to improvement of cure rates (up to 30%) . Complete cure always coincided with the induction of antitumor immunity . Since both VP-16 and IL-2, when locally administered, strongly augment T-cell-mediated immune responses, the observed therapeutic effect was partially attributed to potentiation of a T-cell-mediated antitumor response . Active specific immunization (ASI) using viable irradiated tumor cells admixed with Bacillus Calmette-Guerin also aims at induction of specific antitumor immunity . In late-stage disease, ASI alone induced cure rates of 39% . Combination of ASI with local cytotoxic drug treatment, but not with locoregional administration of IL-2 at the former primary tumor site, led to very high cure rates (up to 78%) . Cured animals were always resistant to a second challenge with line 10 tumor cells . Routinely, one systemic injection with cyclophosphamide was given at the start of all treatment protocols . Omission of CY strongly reduced the cure rates obtained with ASI and locoregional VP-16 treatment . The high cure rates likely relate to the fact that locally administered cytotoxic drugs are capable of reversing immune tolerance, besides exerting direct antitumor action within tumor-draining lymphoid tissues . The present results therefore support our view that local cytotoxic drug treatment should be further explored for its incorporation in antitumor therapies such as ASI, aiming at maximal clinical benefit and minimal toxicities.

Arch Biochem Biophys, 1992 May 1, 294(2), 654 - 61
P450BM-3: reduction by NADPH and sodium dithionite; Peterson JA et al.; Microsomal P450s catalyze the monooxygenation of a large variety of hydrophobic compounds, including drugs, steroids, carcinogens, and fatty acids . The interaction of microsomal P450s with their electron transfer partner, NADPH-P450 reductase, during the transfer of electrons from NADPH to P450, for oxygen activation, may be important in regulating this enzyme system . Highly purified Bacillus megaterium P450BM-3 is catalytically self-sufficient and contains both the reductase and P450 domains on a single polypeptide chain of approximately 120,000 Da . The two domains of P450BM-3 appear to be analogous in their function and homologous in their sequence to the microsomal P450 system components . FAD, FMN, and heme residues are present in equimolar amounts in purified P450BM-3 and, therefore, this protein could potentially accept five electron equivalents per mole of enzyme during a reductive titration . The titration of P450BM-3 with sodium dithionite under a carbon monoxide atmosphere was complete with the addition of the expected five electron equivalents . The intermediate spectra indicate that the heme iron is reduced first, followed by the flavin residues . Titration of the protein with the physiological reductant, NADPH, also required approximately five electron equivalents when the reaction was performed under an atmosphere of carbon monoxide . Under an atmosphere of argon and in the absence of carbon monoxide, one of the flavin groups was reduced prior to the reduction of the heme group . The titration behavior of P450BM-3 with NADPH was surprising because no spectral changes characteristic of flavin semiquinone intermediates were observed . The results of the titration with NADPH can only be explained if (a) there was "rapid" intermolecular electron transfer between P450BM-3 molecules, (b) there is no kinetic barrier to the reduction of P450 by the one-electron-reduced form of the reductase, and (c) the "air-stable semiquinone" form of the reductase does not accumulate in this complex multidomain enzyme.

Cutis, 1992 May, 49(5), 318 - 20
Multiple scattered granulomatous skin lesions in cat scratch disease; Calzavara-Pinton PG et al.; We report a patient with cat scratch disease who presented with multiple scattered nodular lesions on the legs . Examination of skin biopsy specimens revealed a granulomatous pattern . In our opinion, this is a previously undescribed secondary cutaneous reaction of cat scratch disease . The pathogenesis of this reaction is unclear but some data suggest that the eruption might be caused by a hematogenous spread of cat scratch disease bacteria to the skin . Pathogenetic relationships with so-called bacillary angiomatosis, recently described in patients with acquired immunodeficiency syndrome, are reviewed here.

Plasmid, 1992 May, 27(3), 237 - 41
New plasmid vector and host system for genetic engineering in Bacillus sphaericus; Qiao M et al.; A new plasmid, pNQ116, was constructed in Bacillus sphaericus by cloning a promoter fragment from B . sphaericus Ts-1 into pNQ112 . The plasmid (CmrKmr, 5.23 kb) contains a restriction endonuclease polylinker used for cloning foreign genes, and its cat-86 gene is expressed at high levels from the Ts-1 promoter . This plasmid vector has been transformed into B . sphaericus AS 1.270, AS 1.465, AS 1.469, and 2362, at frequencies of 10(2)-10(3) transformants per microgram of DNA, and is maintained stably under nonselective conditions in these host strains . The presence of pNQ116 in B . sphaericus 2362 does ot interfere with the mosquito larvicidal activity of the organism.

Med Parazitol (Mosk), 1992 May-Jun, (3), 33 - 5
{A trial of the possible joint use of mermithids and bacterial preparations for the control of mosquito larvae}; Vladimirova VV et al.; The action of bacterial insecticides based on Bacillus thuringiensis H-14 and B . sphaericus in a dose of 0.005-2 mg/l on the mermithids Romanomermis iyengari, R . culicivorax and R . jingdeensis was studied . It was shown that though bacterial agents suppressed mermithid preparasite larvae density about 30-40%, the survivors might invade 100% of mosquito larvae . The viability and fertility of adult mermithids did not change under the influence of bactoculicide in doses 5-50 times higher than those used against mosquitoes in practice . In combined field trials of sphaerolarvicide (1.5 kg/ha) and R . iyengari mermithids the infectivity of Anopheles sacharovi larvae reached 96% . Mermithids in combination with bactoculicide and sphaerolarvicide might be useful in integrated control systems because these bacterial agents are harmless for mermithids of all stages in doses useful against mosquito larvae.

Rev Med Interne, 1992 May-Jun, 13(3), 200 - 4
{Inflammatory neuropathies}; Said G; Inflammatory neuropathy is the term used for all neuropathies associated with an inflammatory infiltrate of the nerves and/or nerve roots . Broadly speaking, there are two types of inflammatory neuropathies: those caused by an identified infectious agent, and those of uncertain origin for which an autoimmune process is usually blamed . Among the neuropathies of infective origin, leprosy is the most important owing to its frequency and to the physiopathological and therapeutic problems it still poses to clinicians and researchers, since the form and severity of nerve lesions depend on cellular immunity to the bacillus' antigen rather than on the bacillus itself . Retroviral infections, caused by the virus of AIDS more than by the virus of tropical spastic paraplegia, are responsible for numerous neuropathies the mechanisms of which are discussed here . The principal inflammatory neuropathies of uncertain origin are polyradiculitis and its different forms, and the heterogeneous group of neuropathies associated with Sjogren's syndrome.

J Biomol NMR, 1992 May, 2(3), 227 - 33
Positive theta-angles in proteins by nuclear magnetic resonance spectroscopy; Ludvigsen S et al.; Non-glycine residues with positive theta-angles have been identified in four proteins, barley serine proteinase inhibitor CI-2, bacterial ribonuclease (barnase) of Bacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy . By accurate measurements of the coupling constant (3)JHNHalpha and integration of the nuclear Overhauser HN-Halpha cross peak, positive theta-angles could be determined reliably to 60 degrees +/- 30 degrees, in full agreement with the crystal structures for lysozyme, barnase and serine proteinase inhibitor CI-2 . The work emphasizes that positive theta-angles can also occur in non-glycine residues and in the four proteins, positive theta-angles have been observed for the residue types aspartic acid, asparagine, arginine, serine, glutamine, histidine, tyrosine, tryptophan and phenylalanine . The measured (3)JHNHalpha coupling constants and the intensity of the intraresidue HN-Halpha NOEs agree well with the solution structures of three of the proteins, using the existing parametrization of the Karplus curve (Pardi, A., Billeter, M . and Wuthrich, K . (1984) J . Mol . Biol., 180, 741-751; Ludvigsen, S . Andersen, K.V . and Poulsen, F.M . (1991) J Mol . Biol., 217, 731-736).

J Invertebr Pathol, 1992 May, 59(3), 295 - 302
The parasporal inclusion of Bacillus thuringiensis subsp . shandongiensis: characterization and screening for insecticidal activity; Pietrantonio PV et al.; The parasporal body of Bacillus thuringiensis subsp . shandongiensis was characterized in terms of its structure, protein composition, and toxicological properties against several types of insects . The crystals of B . thuringiensis shandongiensis appear to consist of a major protein of 144 kDa present in an spherical inclusion, as determined by transmission electron microscopy, titration curve analysis, and SDS-PAGE of the solubilized crystals . A second protein of ca . 60 kDa is present in trace amounts and appears to be associated with a small bar-shaped inclusion . The 144-kDa protein has been characterized by isoelectric point determination, N-terminal amino acid sequence analysis, amino acid analysis, and immunological cross reactivity . Its N-terminal amino acid sequence differed from that of other B . thuringiensis crystal proteins . The 144-kDa protein was not immunologically related to the crystal proteins of two toxic serovars (B . thuringiensis israelensis and B . thuringiensis kurstaki HD-1) and one nontoxic serovar (B . thuringiensis indiana), as shown in immunoblots probed with antiserum raised against the 144-kDa B . thuringiensis shandongiensis protein, the B . thuringiensis israelensis crystal proteins, and the trypsin resistant fragment of B . thuringiensis kurstaki P1 proteins . In contrast to most B . thuringiensis serovars, B . thuringiensis shandongiensis crystals did not dissolve at pH 12 . Solubilization was achieved in sodium bicarbonate at pH 8.3 and in the presence of 25 mM dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1992 Apr 30, 184(2), 612 - 7
Prediction and Fourier transform infrared spectroscopy estimation of the secondary structure of a Bacillus licheniformis endo-beta-1,3-1,4-D-glucanase; Querol E et al.; The secondary structure of a recombinant Bacillus licheniformis endo-beta-1,3-1,4-D-glucanase (EC.3.2.1.73) has been estimated by Fourier Transform Infrared Spectroscopy and also predicted by the algorithm of Chou and Fasman . From the curve fitting of the deconvolved IR spectrum, the most probable distribution of the secondary structural classes appears to be about 40% beta-sheet, 25% reverse turn, 24% non-ordered and 11% alpha-helix . From theoretical prediction of secondary structure the protein would present 37% beta-sheet, 31% reverse turn, 22% non-ordered and 10% alpha-helix.

Clin Chim Acta, 1992 Apr 30, 207(1-2), 1 - 9
Assay of gamma-glutamyltransferase with amino acid dehydrogenases from Bacillus stearothermophilus as auxiliary enzymes; Kondo H et al.; A spectrophotometric assay for serum gamma-glutamyltransferase (gamma-GT, EC 2.3.2.2) based on the increasing absorbance at 340 nm due to NADH formation is described . Using gamma-glutamyl dipeptides as the donor substrate, amino acids formed by the gamma-glutamyl transfer from the donor substrate to the acceptor substrate glycylglycine are stoichiometrically converted by the corresponding amino acid dehydrogenases from Bacillus stearothermophilus to produce their keto acids and NADH . gamma-Glutamylalanine was found to be the most specific and sensitive donor substrate . The method can be mechanized, is free of interference and correlates well with conventional methods.

Biochem Biophys Res Commun, 1992 Apr 30, 184(2), 692 - 9
Characterization of the pH-mediated solubility of Bacillus thuringiensis var . san diego native delta-endotoxin crystals; Koller CN et al.; Native crystals of Bacillus thuringiensis var . san diego, a coleopteran-specific delta-endotoxin, were metabolically labelled with {35S}methionine . Specific activity was 82,000 CPM/micrograms (2.44 Ci/mmol) . Using a universal buffer formulated with the same ionic strength at every pH, we determined that native crystals dissolve above pH 10 and below pH 4 . At the acidic pH, the rate of solubilization was substantially slower than at the alkaline pH . Recrystallization rates for the toxin were similar regardless of solubilization conditions . The banding patterns in denatured polyacrylamide gel electrophoresis were unaffected by solubilization conditions . Toxicity was higher for soluble toxin compared to crystal toxin, but virtually identical for the acidic and alkaline produced solutions . Acid solubilization is significant because of the acidic midgut of susceptible Coleoptera.

Biochim Biophys Acta, 1992 Apr 23, 1125(2), 166 - 70
Synthetic phospholipids as substrates for phospholipase C from Bacillus cereus; Ries U et al.; The substrate requirement of phospholipids for hydrolysis with phospholipase C from Bacillus cereus was studied with synthetic lipids well-defined in structure and configuration . For optimal activity, the glycerol molecule must contain three substituents: phosphocholine in sn-3-, an ester bond in sn-2- and an ether- or ester bond in sn-1-position . The length of the ester or ether chains is of minor importance . Any deviation from these structural requirements results in a large decrease in the hydrolysis rate . These essential structural and configurational elements for optimal activity for the B . cereus enzyme are perfectly combined in the platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine . This molecule is one of the best substrates for hydrolysis with the bacterial phospholipase C.

Proc R Soc Lond B Biol Sci, 1992 Apr 22, 248(1321), 1 - 7
A broad-spectrum cytolytic toxin from Bacillus thuringiensis var . kyushuensis; Knowles BH et al.; Bacillus thuringiensis (Bt) var . kyushuensis synthesizes a mosquitocidal crystalline inclusion containing several proteins ranging from 140 to 14 kDa . We have identified a 25 kDa protein protoxin in this inclusion which is not cytolytic, but when activated proteolytically to 23-22 kDa products is cytolytic to mosquito, lepidopteran and mammalian cells, can release entrapped glucose from liposomes and forms cation-selective channels in a planar lipid bilayer . This broad-spectrum cytolytic toxin is related antigenically to the 23 kDa toxin from Bt var . darmstadiensis strain 73-E10-2, but not to the 25 kDa CytA toxin of Bt var . israelensis . The cytolytic activity of these Bt var . kyushuensis toxins, like that of the latter two toxins, can be neutralized by incubation with liposomes containing phospholipids.

Biochem J, 1992 Apr 15, 283 ( Pt 2), 327 - 31
The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus . Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli; Ploux O et al.; The 8-amino-7-oxopelargonate synthase {6-carboxyhexanoyl-CoA:L-alanine carboxyhexanoyltransferase (decarboxylating); EC 2.3.1.47} from Bacillus sphaericus involved in biotin biosynthesis was purified from an Escherichia coli overproducing strain . The purification afforded an electrophoretically homogeneous enzyme with a specific activity of 0.67 unit/mg . The purified enzyme is a monomer of 41 kDa . N-Terminal sequencing of the first 14 amino acid residues showed complete agreement with the predicted sequence from the bioF gene . The pure enzyme showed the characteristic absorption band (425 nm) of pyridoxal 5'-phosphate-dependent enzymes . Furthermore, the holoenzyme was resolved during an affinity step yielding the inactive apoenzyme, which recovered activity and the 425 nm-absorption band on dialysis against pyridoxal 5'-phosphate . Km values for L-alanine and pimeloyl-CoA were respectively 3 mM and 1 microM.

Biochemistry, 1992 Apr 14, 31(14), 3555 - 9
Translocation mediated by domain II of Pseudomonas exotoxin A: transport of barnase into the cytosol; Prior TI et al.; Pseudomonas exotoxin A (PE) is a protein toxin composed of three structural domains . Functional analysis of PE has revealed that domain I is the cell-binding domain and that domain III functions in ADP ribosylation . Domain II was originally designated as the translocation domain, mediating the transfer of domain III to the cytosol, because mutations in this domain result in toxin molecules with normal cell-binding and ADP-ribosylation activities but which are not cytotoxic . However, the results do not rule out the possibility that regions of PE outside of domain II also participate in the translocation process . To investigate this problem, we have now constructed a toxin in which domain III of PE is replaced with barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens . This chimeric toxin, termed PE1-412-Bar, is cytotoxic to a murine fibroblast cell line and to a murine hybridoma resistant to the ADP-ribosylation activity of PE . A mutant form of PE1-412-Bar with an inactivating mutation in domain II at position 276 was significantly less toxic . Because the cytotoxic effect of PE1-412-Bar was due to the ribonuclease-activity of barnase molecules which had been translocated to the cytosol, we conclude that domain II of PE is not only essential but also probably sufficient to carry out the translocation process.

Proteins, 1992 Apr, 13(2), 158 - 61
Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus; Huang K et al.; Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed . Two mutants of the enzyme have been prepared . In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R) . In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y) . In addition, an inadvertent C97G mutant was present . Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature . Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer . The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant . These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively) . The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.

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