Bacillus

Cell Transplant, 1994 Nov-Dec, 3(6), 515 - 27
A device to measure the oxygen uptake rate of attached cells: importance in bioartificial organ design; Foy BD et al.; Quantification of the dependence of cellular oxygen uptake rate (OUR) on oxygen partial pressure is useful for the design and testing of bioartificial devices which utilize cells . Thus far, this information has only been obtained from suspended cells and from cells attached to microcarriers . In this work, a device was developed to obtain the dependence of OUR on oxygen partial pressure for anchorage-dependent cells cultured in standard culture dishes . The device is placed and sealed on the top of the culture dish, and holds a Clark polarographic mini-electrode flush with the bottom surface of the device . It also houses a motor to spin a magnetic stir bar within the cell chamber to insure that the medium is well-mixed . Several characteristics of the device--such as oxygen leakage into the device chamber, electrode-lag time, and linearity of the electrode at low oxygen partial pressures--were quantified and their potential effect on the values of Vm (maximal OUR) and K0.5 (oxygen partial pressure at which OUR is half-maximal) were evaluated . Comparison of Vm and K0.5 values obtained with this device with previously published values for suspended rat hepatocytes, Bacillus cereus, and E . coli indicated that the technique provides values accurate within 30% as long as the cell under study has a K0.5 greater than approximately 1.0 mmHg . For hepatocytes cultured on 0.05 mm thickness collagen gel for 1 day (n = 4) and 3 days (n = 6), Vm was found to be 0.38 +/- 0.12 and 0.25 +/- 0.09 nmol O2/S/10(6) cells, respectively, and K0.5 was found to be 5.6 +/- 0.5 and 3.3 +/- 0.6 mmHg, respectively . This technique should aid in predicting bioreactor conditions such as flow rate, cell density, distance of cell from flow, and gas phase oxygen partial pressure which can lead to oxygen limitations . In addition, further studies of the effect of factors such as extracellular matrix composition, metabolic substrate, and drugs on the dependence of OUR on oxygen partial pressure for many anchorage-dependent cell types can be pursued with this technique.

Anal Biochem, 1994 Nov 1, 222(2), 461 - 4
A turbidometric assay for phospholipase C and sphingomyelinase; Torley L et al.; We describe a simple turbidometric assay for phosphatidylcholine-specific phospholipase C (PC-PLC) (EC 3.1.4.3), phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10), and sphingomyelinase (SMase) (EC 3.1.4.12), suitable for high-volume screening using unmodified substrates . Under the conditions described, 1 to 10 U/ml of PC-PLC (Bacillus cereus) induces a rapid and continuous increase in turbidity (0.4 to 0.6 AU at 410 nm) of phosphatidylcholine vesicles (1-10 mM) that highly correlates with hydrolysis . Turbidity increases with the formation of small homogenous particles, which is enzyme and substrate dependent . Analogously, PI-PLC (1-10 U/ml) causes a continuous increase in the turbidity of PI vesicles . SMase also causes a continuous increase in PC vesicle turbidity, but unlike like the glycerol phospholipases, SMase causes a discontinuous increase in vesicles of its proper substrate sphingomyelin (SM) . After 8-15% hydrolysis, SM vesicles are converted to large heterogeneous particles permitting detection of SMase activity by visual inspection . Thus, turbidity is a useful property to monitor SMases and C-type phospholipases that cleave vesicle-forming phospholipids . Furthermore, the assay is designed for the microtiter plate format, enabling the continuous and simultaneous monitoring of up to 96 wells.

J Dairy Res, 1994 Nov, 61(4), 529 - 35
Identification of Bacillus strains isolated from milk and cream with classical and nucleic acid hybridization methods; Tatzel R et al.; A total of 529 bacterial strains have been isolated from milk and cream sampled at different sites in a dairy production plant under conditions selective for aerobic sporeforming bacteria . Identification with classical methods based on morphological, physiological and biochemical criteria showed Bacillus licheniformis to be the most frequently occurring Bacillus sp . The investigation also revealed 62 unidentified strains . Classical identification methods were time consuming (3-7 d), lacked specificity and--because of their dependence on phenotypic gene expression--sometimes produced ambiguous results . Consequently, a colony hybridization method developed for the identification of B . licheniformis strains and using nonradioactive labelled 23S rRNA targeted oligonucleotide probes was also used . Identification of B . licheniformis with both classical and hybridization methods revealed diverging identification results for 70 strains.

Urologe A, 1994 Nov, 33(6), 540 - 6
{Cytokine therapy of superficial bladder carcinoma . Mechanisms of action and results of therapy}; Otto T et al.; The object of immunotherapy is the elimination of tumor cells mediated by modulation of the immune system . This can be achieved by different mechanisms, i.e . cell-mediated and humorally mediated immune reactions . Immunotherapy can be classified as passive, adoptive, active and non-conventional . Most clinical experience has been gathered with unspecific active immunotherapy . Superficial bladder carcinomas can be treated by intravesical application of Bacillus Calmette-Guerin (BCG) or of different cytokines, i.e., interferons or interleukin-2 . Since there has not so far been any standard immunotherapy for superficial bladder carcinoma, the efficacy of therapy with cytokines should be evaluated in clinical studies (phase II/III) only.

Biol Mass Spectrom, 1994 Nov, 23(11), 675 - 81
Rapid identification of specific mutations in the sequence of an enzyme variant produced by protein engineering using high-performance liquid chromatographic/fast atom bombardment mass spectrometric techniques; van Dongen WD et al.; Unknown, specific mutations in the sequence of an enzyme variant (a Bacillus subtilisin protease) produced by protein engineering were identified using High-performance Liquid Chromatographic/Fast Atom Bombardment Mass Spectrometric (HPLC/FAB MS) techniques . The variant and the highly homologous wild-type enzyme were treated with CNBr followed by tryptic digestion . The resulting peptides were analysed using HPLC/frit FAB MS . The peptides with molecular masses beyond the range of the HPLC/MS system under the chosen scanning conditions were collected using HPLC and subsequently analysed 'off-line' using static FAB MS . This procedure allowed the complete amino acid sequence determination of the variant protease using the known amino acid sequence of the wild-type enzyme as reference.

Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 358 - 63
Direct high-level secretion into the culture medium of tuna growth hormone in biologically active form by Bacillus brevis; Sagiya Y et al.; The characteristic features of the Bacillus brevis system developed by us are very high productivity of heterologous proteins and very low extracellular proteinase activity . However, the production level of eucaryotic proteins with this system was generally one or two orders of magnitude lower than that of bacterial proteins . Therefore, we have explored methods for increasing the production efficiency as to animal proteins . Signal peptide modification was found to be very effective for high-level secretion of tuna growth hormone (tGH) . Modification of the signal peptide with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a ten-fold increase in tGH production . Further elevation of the tGH yield to 240 mg/l was achieved by using a low proteinase mutant and a stable plasmid, and by culturing B . brevis under optimal conditions with the addition of some chemicals . Thus, biologically active tGH can be efficiently produced directly in the medium with this B . brevis system.

Lett Appl Microbiol, 1994 Nov, 19(5), 353 - 6
Comparison of the Tecra VIA kit, Oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin; Buchanan RL et al.; Two commercial serological kits (Oxoid BCET-RPLA and Tecra VIA) and a Chinese hamster ovary (CHO) cell cytotonicity assay for the detection of Bacillus cereus diarrhoeal enterotoxin were compared . Eleven B . cereus strains and one enterotoxigenic B . thuringiensis strain were evaluated . Both kits and the CHO cell assay yielded positive toxin responses for cell-free culture filtrates from eight out of 11 diarrhoeal enterotoxigenic strains . An emetic enterotoxin producing strain was negative with all three assays . Two B . cereus strains were negative using the BCET-RPLA kit, but positive with the Tecra VIA kit and CHO cell assay . The BCET-RPLA indicated significant levels of enterotoxin after samples were boiled, whereas the CHO cell and Tecra assays were negative . Overall, the cell culture assay was the most sensitive . However, the Tecra VIA kit provided similar results and was better suited for the rapid detection of B . cereus diarrhoeal enterotoxin.

Curr Microbiol, 1994 Nov, 29(5), 301 - 7
An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis; Porteous LA et al.; A rapid direct-extraction method was used to obtain DNA from environmental soil samples . Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells . The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonuclease PalI . The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi including Gossypium hirsucum (cotton), Pseudomonas, Bacillus, Streptomyces, and Colletotrichum . Up to 100 micrograms DNA/g (wet weight) of soil and 400 micrograms DNA/g of plant material were recovered . Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments . Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonuclease PalI, contained 12-20 DNA fragments, appearing as sample "fingerprints." Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.

Mikrobiologiia, 1994 Nov-Dec, 63(6), 986 - 92
{Biosynthesis of extracellular ribonuclease by Bacillus pumilus}; Znamenskaia LV et al.; Conditions for the biosynthesis of alkaline extracellular ribonuclease by Bacillus pumilus were studied and their comparison with that of the biosynthesis of ribonuclease by Bacillus intermedius was made . It was estimated that ribonuclease of Bacillus pumilus is synthesized post-exponentially, its synthesis is inhibited by inorganic phosphate and is stimulated by actinomycin D, as the synthesis of ribonuclease by Bacillus intermedius . At the same time essential differences in dynamic of enzyme formation, extent of phosphate inhibition and spores forming conditions of the comparing cultures were found.

Protein Eng, 1994 Nov, 7(11), 1379 - 86
Thermostabilization of the Bacillus circulans xylanase by the introduction of disulfide bonds; Wakarchuk WW et al.; The thermostability of the 20 396 Da Bacillus circulans xylanase was increased by the introduction of both intra- and intermolecular disulfide bridges by site-directed mutagenesis . Based on the 3-D structure of the enzyme, sites were chosen where favourable geometry for a bridge existed; in one case, to obtain favourable geometry additional mutations around the cysteine sites were designed by computer modelling . The disulfide bonds introduced into the xylanase were mostly buried and, in the absence of protein denaturants, relatively insensitive to reduction by dithiothreitol . The mutant proteins were examined for residual enzymatic activity after various thermal treatments, and were assayed for enzymatic activity at elevated temperatures to assess their productivity . We have examined one of these mutants by X-ray crystallography . All of the disulfide bond designs tested increased the thermostability of the B . circulans xylanase, but not all enhanced the activity of the enzyme at elevated temperatures.

J Biochem (Tokyo), 1994 Nov, 116(5), 955 - 9
Purification and properties of L-ornithine delta-aminotransferase from gramicidin S-producing Bacillus brevis; Takechi M et al.; In gramicidin S-producing Bacillus brevis, the addition of L-ornithine to the minimal medium with L-glutamate as the sole carbon and nitrogen source caused an 8-fold induction of L-ornithine delta-aminotransferase {EC 2.6.1.13} . The enzyme was purified to homogeneity . The native enzyme had a molecular weight of about 88,000 after gel filtration and consisted of two subunits with an identical in molecular weight of about 45,000 . The enzyme was specific for L-ornithine (Km = 1.05 mM) as an amino donor and for 2-oxoglutarate (Km = 6.25 mM) as an amino acceptor, and catalyzed the conversion of L-ornithine and 2-oxoglutarate, respectively, to glutamic-gamma-semialdehyde, which is spontaneously cyclized to delta 1-pyrroline-5-carboxylate and L-glutamate . The enzyme exhibits an absorption maximum at 425 nm at neutral pH, and 1 mol of pyridoxal phosphate is bound per subunit . The enzyme activity was irreversibly inhibited by gabaculine, and L-ornithine protected the enzyme from the inhibition . The N-terminal amino acid sequence revealed a noteworthy similarity between human and yeast L-ornithine delta-aminotransferases in residues 17-28 of the B . brevis enzyme.

J Mol Biol, 1994 Oct 28, 243(3), 530 - 2
Crystallization and preliminary X-ray diffraction studies of the lepidopteran-specific insecticidal crystal protein CrylA(a); Borisova S et al.; A trypsin-activated CrylA(a) protein from Bacillus thuringiensis has been purified and crystallized . Crystals belong to orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 53.3, b = 111.3 and c = 154.7 A . The crystals diffract to at least 2.2 angstrum resolution and are suitable for X-ray structural analysis . They contain a single molecule in the asymmetric unit.

J Biol Chem, 1994 Oct 28, 269(43), 26836 - 41
Common pathways of cytochrome P450 gene regulation by peroxisome proliferators and barbiturates in Bacillus megaterium ATCC14581; English N et al.; Bacillus megaterium contains a barbiturate-inducible cytochrome P450BM-3, which catalyzes the hydroxylation of fatty acids . We report the intriguing finding that peroxisome proliferators, a major class of epigenetic carcinogen, are also extremely potent inducers of this enzyme being up to 50-fold more potent than one of the most effective barbiturates, secobarbital . Similar to barbiturates, the mechanism of induction appears to involve the direct binding of the peroxisome proliferator to the transcriptional repressor (Bm3R1), resulting in its dissociation from its DNA operator . These observations provide evidence that peroxisome proliferators can interact with a transcription factor to modulate gene expression . The data also demonstrate that the effects of these compounds are highly conserved through evolution and that there are important common denominators in the regulation of gene expression by peroxisome proliferators and the barbiturates . Evidence is presented to indicate that this may involve effects on unsaturated fatty acid homeostasis.

Proc Natl Acad Sci U S A, 1994 Oct 25, 91(22), 10417 - 21
Native-like in vivo folding of a circularly permuted jellyroll protein shown by crystal structure analysis; Hahn M et al.; A jellyroll beta-sandwich protein, the Bacillus beta-glucanase H(A16-M), is used to probe the role of N-terminal peptide regions in protein folding in vivo . A gene encoding H(A16-M) is rearranged to place residues 1-58 of the protein behind a signal peptide and residues 59-214 . The rearranged gene is expressed in Escherichia coli . The resultant circularly permuted protein, cpA16M-59, is secreted into the periplasm, correctly processed, and folded into a stable and active enzyme . Crystal structure analysis at 2.0-A resolution, R = 15.3%, shows cpA16M-59 to have a three-dimensional structure nearly identical with that of the parent beta-glucanase . An analogous experiment based on the wild-type Bacillus macerans beta-glucanase, giving rise to the circularly permuted variant cpMAC-57, yields the same results . Folding of these proteins, therefore, is not a vectorial process depending on the conformation adopted by their native N-terminal oligopeptides after ribosomal synthesis and translocation through the cytoplasmic membrane.

Biochemistry, 1994 Oct 25, 33(42), 12644 - 8
Structural and functional roles of the cysteine residues of Bacillus stearothermophilus farnesyl diphosphate synthase; Koyama T et al.; p-(Chloromercuri)benzoic acid inhibited the catalytic activity of Bacillus stearothermophilus farnesyl diphosphate synthase (FPP synthase), which contains only two cysteine residues at positions 73 and 289 . In order to explore the role of the cysteine residues, either or both of them were replaced with phenylalanine or serine . Five mutant enzymes, C73F, C73S, C289F, C289S, and C73S-C289S, were overproduced in Escherichia coli and purified to homogeneity . All of them were active as farnesyl diphosphate synthase, showing specific activities comparable to that of the wild-type enzyme . These results indicate that neither of the cysteines is essential for catalytic function . The C73F mutant, however, was very sensitive to heat treatment, while C73S was as highly stable as the wild type . The Km value of C289F for isopentenyl diphosphate is 10 times that of the wild type . The wild-type enzyme was converted into an oxidized form which was separable from the native enzyme on ion-exchange chromatography, and this conversion was accelerated by cupric ions . Similar conversion has previously been reported by several researchers, who found the occurrence of two forms of pig liver FPP synthase and who attributed this phenomenon to the oxidoreduction of sulfhydryl and disulfide groups . However, even the C73S-C289S mutant, which has no cysteine residues, was also converted into an oxidized form as in the case of the wild-type enzyme . These results provide evidence that residues other than cysteine are involved in the conversion of this enzyme into the oxidized form.

FEBS Lett, 1994 Oct 24, 353(3), 259 - 63
A comparison and analysis of the toxicity and receptor binding properties of Bacillus thuringiensis CryIC delta-endotoxin on Spodoptera littoralis and Bombyx mori; Sanchis V et al.; The binding of L-{35S}methionine in vivo labelled CryIC toxin to its receptor in brush border membrane vesicle (BBMV's) prepared from Spodoptera littoralis and Bombyx mori was studied . Both insect species were highly susceptible to the CryIC toxin in bioassays, B . mori being 7-fold more sensitive to CryIC than S . littoralis (LC50's of 10 ng/cm2 and 70 ng/cm2, respectively) . Competition and direct binding experiments revealed saturable high-affinity binding sites on BBMV's from both insects which had similar binding characteristics for the CryIC toxin (Kd = 10 nM, Bmax = 8 to 9 pmol/mg BBMV's and IC50 = 37 nM for both inspect species) . Thus a specific receptor for the CryIC toxin is present in both insect species and the 7-fold greater potency of CryIC towards B . mori is not due to qualitative or quantitative differences in binding affinity or receptor site concentration . Dissociation experiments also indicated that the binding of {35S}CryIC to B . mori BBMV's is partially reversible.

Med Clin (Barc), 1994 Oct 22, 103(13), 490 - 3
{How many cases of tuberculosis are not reported?}; Garcia Rodriguez JF et al.; BACKGROUND: The aim of this study was to evaluate the degree of registry of tuberculosis and the factors associated to the same . METHODS: A retrospective study of the cases of respiratory tuberculosis diagnosed in the hospital A . Marcide-Novoa Santos (El Ferrol . La Coruna . Spain) from 1990 to 1992 was carried out . Identification was obtained from the registries of microbiology and pathology and the clinical history files . Registered cases were obtained from the nominal notifications to the Epidemiology Department of the local health service department . Sex, age, place of residence, previous history of tuberculosis, HIV, diagnostic method, localization of the tuberculosis, registration and reporting physician were evaluated for each patient . RESULTS: Three hundred ninety-three cases were identified of which 78 (19.8%) had been registered . Age and pulmonary localization were the variable influencing the degree of registration . Reporting was greater in the age group from 0 to 10 years (p < 0.05) . Pulmonary tuberculosis was the most reported type although only 22.4% of the cases were declared . Bacilloscopy was positive in sputum in 190 patients and declared in 46 (24.2%) . The degree of registration increased significantly over three years (p < 0.000001) . Sex, previous history of tuberculosis, infection by the HIV and diagnostic method did not influence the degree of registration . CONCLUSIONS: Seventy-five percent of the cases with positive bacilloscopy in sputum were not declared . The degree of declaration has improved over time, however, remains deficient being 2.7 fold lower than the total number of cases diagnosed in 1992.

Ugeskr Laeger, 1994 Oct 17, 156(42), 6175 - 80
{Cat-scratch disease and bacillary angiomatosis . An old and a new infectious disease with common etiology?}; Engbaek K et al.; A review of cat-scratch disease (CSD) and bacillary angiomatosis (BA) is presented on the basis of published articles . Two newly identified bacteria--Rochalimaea henselae and Afipia felis--have been isolated from patients with CSD . Preliminary investigations seem to indicate that A . felis is an uncommon cause of the disease . CSD may appear as a local suppurative lymphadenopathy or a systemic infection . BA is caused by Rochalimaea species and may appear as cutaneous, mucous or visceral angiomas or bacteremia . It may be a special manifestation of CSD in immunocompromised patients . A description is given of the various pathological pictures and differential diagnosis, and an evaluation is made of the different diagnostic methods, namely visualisation of bacteria in the lesions with Warthin-Starry's silver impregnation, isolation of bacteria, demonstration of bacteria with gene technique and detection of antibodies . The treatment of the disease is discussed.

Biochem J, 1994 Oct 15, 303 ( Pt 2), 555 - 8
Effects of site-specific mutagenesis of tyrosine 105 in a class A beta-lactamase; Escobar WA et al.; Tyr-105 is a conserved residue in the Class A beta-lactamases and is in close proximity to the active-site . Tyr-105 in beta-lactamase from Bacillus licheniformis was converted into Phe by site-directed mutagenesis . This mutation caused no significant effect on the structure of the enzyme and had only small effects on the catalytic properties . In particular, in comparison to the wild-type, kcat . for benzylpenicillin was increased slightly, whereas it was decreased slightly for several other substrates . For each substrate examined, Km increased 3-4-fold in the mutant compared with the wild-type enzyme . Examination of the effect of pH on the catalytic reaction revealed only small perturbations in the pK values for the acidic and basic limbs of the kcat./Km pH profiles due to the mutation . Overall effects of the Y105F substitution on the catalytic efficiency for different penicillin and cephalosporin substrates ranged from 14% to 56% compared with the wild-type activity . We conclude that Tyr-105 is not an essential residue for beta-lactamase catalysis, but does contribute to substrate binding.

Biochem J, 1994 Oct 15, 303 ( Pt 2), 423 - 8
The role of tryptophan 97 of cytochrome P450 BM3 from Bacillus megaterium in catalytic function . Evidence against the 'covalent switching' hypothesis of P-450 electron transfer; Munro AW et al.; The 'Covalent Switching' hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems {Baldwin, Morris and Richards (1991) Proc . R . Soc . London B 245, 43-51} . We have investigated the effect of alteration of the conserved tryptophan (Trp-97) in cytochrome P-450 BM3 (P-450 102) from Bacillus megaterium . Replacement of Trp-97 with Ala, Phe or Tyr results in a decrease in the natural haem content and alters the resting spin state of the remaining haem in the purified mutant enzymes . However, kinetic analyses indicate that the mutant enzymes retain high levels of catalytic activity . C.d . and e.p.r . spectroscopy also reveal little alteration in secondary structure or change in the pattern of haem ligation . These findings cast doubt on the covalent switching mechanism of intermolecular electron flow in the P-450s, but indicate that this residue plays a role in the association of the haem prosthetic group.

Structure, 1994 Oct 15, 2(10), 945 - 51
Stability and function: two constraints in the evolution of barstar and other proteins; Schreiber G et al.; BACKGROUND: Barstar is the intracellular inhibitor of barnase, an extracellular RNAse of Bacillus amyloliquefaciens . The dissociation constant of the barnase-barstar complex is 10(-14) M with an association rate constant between barnase and barstar of 3.7 x 10(8) s-1 M-1 . The rapid association arises in part from the clustering of four acidic residues (Asp35, Asp39, Glu76 and Glu80) on the barnase-binding surface of barstar . The negatively charged barnase-binding surface of barstar effectively 'steers' the inhibitor towards the positively charged active site of barnase . RESULTS: Mutating any one of the four acidic side chains of barstar to an alanine results in an approximately two-fold decrease in the association rate constant, while the dissociation rate constant increases from five orders of magnitude for Asp39-->Ala, to no significant change for Glu80-->Ala . The stability of barstar is increased by all four mutations, the increase ranging from 0.3 kcal mol-1 for Asp35-->Ala or Asp39-->Ala, to 2.1 kcal mol-1 for Glu80-->Ala . CONCLUSIONS: The evolutionary pressure on barstar for rapid binding of barnase is so strong that glutamate is preferred over alanine at position 80, even though it does not directly interact with barnase in the complex and significantly destabilizes the inhibitor structure . This, and other examples from the literature, suggest that proteins evolve primarily to optimize their function in vivo, with relatively little evolutionary pressure to increase stability above a certain threshold, thus allowing greater latitude in the evolution of enzyme activity.

J Immunol, 1994 Oct 15, 153(8), 3684 - 90
Synergistic cooperation between phorbol ester and IFN-gamma for induction of nitric oxide synthesis in murine peritoneal macrophages; Jun CD et al.; The role of protein kinase C (PKC) in the induction of nitric oxide (NO) synthesis in murine peritoneal macrophages was examined . Phorbol ester, a PKC activator, had no effect on NO synthesis by itself, whereas IFN-gamma alone had modest activity . When phorbol ester was used in combination with IFN-gamma, there was a marked cooperative induction of NO synthesis in a dose-dependent manner . This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) mRNA, as determined by Northern blotting . The optimal effect of phorbol ester was shown at 6 h after treatment with IFN-gamma . Phorbol ester also induced the release of NO to the incubation medium by bacillus Calmette-Guerin-infected peritoneal macrophages . Prolonged incubation of cells with phorbol ester, which down-regulates PKC activity, abolished the synergistic cooperative effect on NO production with IFN-gamma . In addition, such PKC inhibitors as staurosporin or polymyxin B reduced NO production induced by IFN-gamma plus phorbol ester . When the cells were treated with both actinomycin D and phorbol ester after IFN-gamma stimulation, more NO was produced and more iNOS mRNA was expressed than in the cells treated with actinomycin D alone . On the basis of these observations, we conclude that PKC might not be directly involved in the expression of NO synthase, but, instead, might be involved in the stabilization of the iNOS mRNA already expressed by the treatment of IFN-gamma.

Biochem Biophys Res Commun, 1994 Oct 14, 204(1), 428 - 35
Two o-type oxidases in Methylobacillus flagellatum KT; Muntyan MS et al.; Two oxidases of the o-type in membranes of the methanol-grown obligate methylotroph Methylobacillus flagellatum KT were distinguished . For this purpose the kinetic analysis of the laser flash-induced optical absorbance changes of CO-oxidase complexes under reducing conditions was used . The ratio of these oxidases in membranes greatly depended on the phases of bacterial growth . One of the oxidases appeared to belong to the Escherichia coli o-type oxidase family being more sensitive to KCN (Ki = 1 microM) . It showed monophasic CO recombination kinetics with tau 25-30 ms and was expressed in the early exponential phase of growth . The other oxidase seemed to be similar to the Bacillus sp . FTU o-type oxidase being less sensitive to KCN (Ki = 6 microM), having three-phasic CO reassociation kinetics with tau 35-70 microseconds, 0.25-0.5 ms and 2-4 ms and dominating in the stationary growth phase . Pyridine haemochrome spectra showed haems A and D to be absent from the bacterial membranes.

J Med Chem, 1994 Oct 14, 37(21), 3668 - 70
Synthesis of N-acetylglucosamine-modified ara-C and its effect on ovarian cancer cells; Fujimoto H et al.; 1-beta-D-Arabinofuranosylcytosine (ara-C) was modified by reaction of tetra-N-acetylchitotetraose ((GlcNAc)4) using the transglycosylation activity of thermostable chitinase (EC 3.2.1.14) from Bacillus licheniformis X-7u . The structure of the modified ara-C was determined to be either beta 1-3'- or beta 1-5'-linked GlcNAc-ara-C or (GlcNAc)2-ara-C . The total yield of these glycosylated ara-Cs was about 10% . GlcNAc-ara-C and (GlcNAc)2-ara-C depressed the growth of G-401 cancer cells, while 5-O-beta-D-galactopyranosyl-beta-D-arabinofuranosylcytosine (Gal-ara-C) had no effect on G-401 cells.

Biochemistry, 1994 Oct 11, 33(40), 12056 - 62
Evidence for conformational dynamics and molecular aggregation in cytochrome P450 102 (BM-3); Black SD et al.; The native molecular weight of affinity-purified cytochrome P450 102 from barbiturate-induced Bacillus megaterium has been studied by sedimentation methods and HPLC size-exclusion chromatography . Sedimentation velocity experiments yielded an s020,w = 9.244 S for the holocytochrome, but the diffusion coefficient was unexpectedly large and varied widely with centrifugal field, ionic strength, and protein concentration . Addition of 50 mM DL-dithiothreitol (DTT) caused a small decrease in the value of s020,w, but D20 still did not behave as expected . The sedimentation coefficients were consistent with a molecular weight of about 200,000, and the diffusion coefficients indicated molecular aggregation . Sedimentation equilibrium analyses suggested that the native enzyme was a mixture of monomer, dimer, trimer, and tetramer . However, after incubation of P450 102 with DTT, sedimentation equilibrium demonstrated that the enzyme was dimeric (molecular weight 236,000) . HPLC size-exclusion chromatography of the cytochrome showed the presence of four peaks, which corresponded to 1.45-mer, 2.06-mer, 3.02-mer, and a higher molecular weight fraction; aggregated forms accounted for about 52% of the P450 102 . Incubation of the enzyme with DTT caused a shift toward the 1.45-mer, but dimer, trimer, and the high molecular weight peak still persisted; the shift was not attributable to disulfide bond reduction . The 1.45-mer was determined to be a monomeric species of significantly asymmetric geometry . Together, the results indicated that cytochrome P450 exists with monomer, dimer, trimer, etc . in equilibrium, contrary to the expectation that this soluble P450 would be monomeric.(ABSTRACT TRUNCATED AT 250 WORDS)

Virology, 1994 Oct, 204(1), 91 - 100
Rice tungro bacilliform virus: transcription and translation in protoplasts; Chen G et al.; Protoplasts from cell suspension cultures of Oryza sativa (monocot) and Orychophragmus violaceus (dicot) support transcription from the rice tungro bacilliform virus (RTBV) promoter and translation of the resulting mRNA despite the presence of a long leader sequence with strong secondary structure and 12 short open reading frames . Transcriptional elements located both upstream and downstream of the transcription initiation site are defined by deletion analysis and the functional TATA motif is determined . Expression of an open reading frame downstream of the entire leader is more efficient than that downstream of truncated derivatives . For optimal expression sequences in the 5' and 3' parts of the leader are required.

J Urol, 1994 Oct, 152(4), 1275 - 80
Bacillus Calmette-Guérin interacts with the carboxyl-terminal heparin binding domain of fibronectin: implications for BCG-mediated antitumor activity; Cheng DL et al.; Intravesical bacillus Calmette-Guerin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma of the bladder . The mechanisms by which BCG achieves this effect remain unclear . Reports have attributed an important role to fibronectin both in the initial attachment of BCG to bladder surfaces and in the limitation of tumor cell motility . In the present study, using limited protease cathepsin B degradation followed by Western blot analyses with antibodies to various domains of the fibronectin molecule, we showed that BCG appears to bind to fibronectin near the carboxyl terminal and adjacent to the heparin binding domain . Furthermore a 51-chromium release assay with human bladder cancer cell line T24 as target cells and lymphokine activated killer (LAK) cells as effector cells showed that fibronectin was needed for tumor cytotoxicity by the LAK cells . By using antibodies and peptides to various domains of the fibronectin molecule, the heparin binding domain, but not the cell binding domain, carboxyl terminal region, or the amino terminal region of the fibronectin molecule, was identified as essential to tumor cell lysis by the LAK cells . Flow cytometric analysis showed that both peripheral blood lymphocytes and the LAK cells express fibronectin receptors VLA-3, VLA-4 and VLA-5 on their surfaces . However, the numbers of receptors are not significantly different in the two cell populations . We conclude that, by binding near the carboxyl terminal region and adjacent to the heparin-binding domain of the fibronectin molecule, BCG may protect this region of the molecule from tumor proteases, and may thus allow the antitumor activity of the host immune cells to take place.

Planta Med, 1994 Oct, 60(5), 414 - 6
Suppression of chemically and immunologically induced hepatic injuries by gentiopicroside in mice; Kondo Y et al.; Gentiopicroside (GPS), a main bitter secoiridoid constituent of roots of Gentiana macrophylla Pall., was tested for therapeutic effects on the two hepatic injury models, the CCl4-induced and lipopolysaccharide (LPS)/bacillus Calmette-Guerin (BCG)-induced hepatitides . An increase in serum level of hepatic aminotransferases (GOT: EC 2.6.1.1 . and GPT: EC 2.6.1.2.) induced by a p.o . treatment of CCl4 was suppressed by pretreatment with GPS at 30-60 mg/kg/day for 5 consecutive days . An increase of these enzymes triggered by an i.v . treatment with LPS in mice primed with bacillus Calmette-Guerin (BCG) was also inhibited by GPS pretreatment at the same dose of GPS . In the BCG/LPS model, tumor necrosis factor (TNF), a major inflammatory mediator, was increased in serum with a peak at 90-120 min, followed by an increase of serum transaminase activities . GPS treatment significantly suppressed the increase of TNF in serum at the therapeutic doses, suggesting that GPS protected against hepatitis by inhibiting the production of TNF.

Hinyokika Kiyo, 1994 Oct, 40(10), 873 - 7
{Long-term results and complications of intravesical instillation of bacillus Calmette-Guerin for prophylaxis of bladder cancer recurrence}; Irie A et al.; Since 1982, we treated 22 patients with superficial bladder cancer via intravesical bacillus Calmette-Guerin (Tokyo 172 strain) for prophylaxis against tumor recurrence . To determine the long-term efficacy and complications of BCG therapy, retrospective analysis was performed . The BCG therapy was initiated one to two weeks after complete transurethral resection of visible tumors . One hundred twenty mg or 80 mg of BCG suspended in 40 ml of 0.9% NaCl solution was instilled into the bladder once a week for 6 times, then monthly for 10 times . Of 22 patients, 10 tumor recurrences were recognized in 4 patients (mean followup 74 months) . Recurrent free rate at 5 years was 78.5% . Common side effects were low grade fever and bladder irritability . Although these side effects were self-limiting, 15 patients (68%) refused instillation of BCG before completion of our protocol because of the bladder irritation . No relationship was observed between total dose of BCG instilled and tumor recurrence . Severe side effects such as permanent structural or functional alterations of the bladder or mycobacterial infection of other organs were not observed . Intravesical administration of Tokyo 172 strain of BCG seemed to be safe and useful for long-term prophylaxis of superficial bladder cancer recurrence.

Plant Physiol, 1994 Oct, 106(2), 643 - 50
Purification and partial characterization of NADPH-cytochrome c reductase from Petunia hybrida flowers; Menting JG et al.; NADPH-cytochrome c reductase was solubilized from the microsomal fraction of Petunia hybrida flowers by 3-{(3-cholamidopropyl)dimethylammonio}-1-propane sulfonate detergent and purified by adenosine 2',5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography . Two proteins with molecular sizes of 75 and 81 kD were detected in the purified preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Western blot analysis showed that both purified proteins cross-reacted with two different monoclonal antibodies raised against P . hybrida NADPH-cytochrome c reductase and rabbit anti-Jerusalem artichoke NADPH-cytochrome P450 reductase antibodies . Only one 84-kD protein was detected by western blot analysis of fresh microsomal extracts . Amino acid sequence analysis of tryptic peptides revealed significant similarity to the NADPH binding region of plant and animal NADPH-cytochrome P450 reductases and Bacillus megaterium cytochrome P450:NADPH-cytochrome P450 reductase . The pH optimum for reduction of ferricytochrome c was 7.4 and the Km values for the binding of NADPH and ferricytochrome c were 9.2 and 2.8 microM, respectively . We believe that the purified enzyme is a P . hybrida NADPH-cytochrome P450 reductase (EC 1.6.2.4).

FEMS Microbiol Lett, 1994 Oct 1, 122(3), 203 - 10
Membrane-bound Bacillus cytochromes c and their phylogenetic position among bacterial class I cytochromes c; Sone N et al.; Gram-positive bacteria lack a periplasmic compartment and contain only membrane-bound cytochromes c . There are at least two types . One is found in subunit II of cytochrome oxidase, and the other is small cytochrome c which is also membrane-bound because of an unprocessed signal sequence or post-translational acylation at the N-terminal end of the protein . These Bacillus cytochromes c are compared with known class I cytochromes c, and a phylogenetic tree has been constructed by the neighbour-joining method.

Plant Mol Biol, 1994 Oct, 26(1), 51 - 9
Insect resistance of transgenic plants that express modified Bacillus thuringiensis cryIA(b) and cryIC genes: a resistance management strategy; van der Salm T et al.; Tobacco and tomato plants were generated exhibiting insect resistance due to the introduction of modified cryIA(b) and cryIC genes of Bacillus thuringiensis . Limited modifications at selected regions of the coding sequences of both genes are sufficient to obtain resistance against Spodoptera exigua, Heliothis virescens and Manduca sexta . The criteria used to modify both genes demonstrate that the removal of sequence motifs potentially resulting in premature polyadenylation and transcript instability causes increased insect resistance . The expression of a cryIC-cryIA(b) fusion resulting in protection against S . exigua, H . virescens and M . sexta demonstrates the potential of expressing translational fusions, not only to broaden the insect resistance of transgenic plants, but also to simultaneously employ different gene classes in resistance management strategies.

Plant Mol Biol, 1994 Oct, 26(1), 285 - 90
Biosynthesis of lysine in plants: the putative role of meso-diaminopimelate dehydrogenase; Chatterjee SP et al.; Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway . Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all . However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean . Moreover, it was not found in Chlamydomonas, corn and tobacco as well . In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus . The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants.

Epidemiol Infect, 1994 Oct, 113(2), 297 - 306
Contamination of hospital linen by Bacillus cereus; Barrie D et al.; An investigation into two cases of post-operative Bacillus cereus meningitis revealed that hospital linen laundered by a batch continuous washing machine was heavily contaminated by B . cereus spores . The washing machine, detergents, other chemical additives and the water supply were eliminated as the source of contamination . It was found that the linen introduced into the washing machine had a high B . cereus spore content and that this was still present after the wash process . The spores were not killed by either the heat disinfection stage of the wash or the addition of chemical disinfectants and were not removed by the dilution in the process . The multiplication of B . cereus was thought to have occurred on used, damp linen stored in plastic bags, particularly when ambient temperatures were high . An increase in the water flow through the washing machine was the only measure associated with a decrease in B . cereus on laundered linen.

Cancer Res, 1994 Oct 1, 54(19), 5178 - 85
Establishment, molecular rescue, and expression of 123AV16-1, a tumor-reactive human monoclonal antibody; Hall BL et al.; The human monoclonal antibody (mAb) 123AV16-1 was generated by Epstein-Barr virus transformation of peripheral blood lymphocytes from a colorectal patient undergoing active specific immunotherapy with an autologous tumor cell-Bacille Calmette-Guerin vaccine . Direct immunohistochemical staining of tumor and normal pairs of tissues indicated that this human IgA1, lambda 2 mAb preferentially reacted with colon tumor epithelium . To generate a recombinant derivative of this Epstein-Barr virus-transformed cell line, we isolated the expressed complete heavy and light chain genes by a novel strategy and cloned them into modified pSV2-neo and pSV2-gpt expression vectors . The recombinant 123AV16-1 human mAb was expressed in both a murine myeloma and a human-murine heteromyeloma and was secreted as both monomers and dimers . The recombinant 123AV16-1 mAb expressed by both cell lines reacted with human colon tumor xenografts in a manner similar to the mAb derived from the Epstein-Barr virus-transformed cell line, indicating that the antibody specificity was not appreciably altered during the molecular rescue, cloning, or expression.

J Biochem (Tokyo), 1994 Oct, 116(4), 931 - 6
Construction and characterization of chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase; Kataoka K et al.; Phenylalanine dehydrogenase of Thermoactinomyces intermedius acts preferentially on L-phenylalanine and L-tyrosine, whereas leucine dehydrogenase of Bacillus stearothermophilus acts almost exclusively on L-leucine and some other branched-chain L-amino acids . The two enzymes share a sequence similarity (47%) . Aiming at elucidation of the mechanism of substrate recognition by the two amino acid dehydrogenases, we have genetically constructed a chimeric enzyme consisting of an N-terminal domain of phenylalanine dehydrogenase containing the substrate-binding region and a C-terminal domain of leucine dehydrogenase containing the NAD(+)-binding region . The chimeric enzyme purified to homogeneity acted on phenylalanine with a specific activity of 6% of that of the parental phenylalanine dehydrogenase and showed a broad substrate specificity in the oxidative deamination, like phenylalanine dehydrogenase . However, it acted much more effectively than phenylalanine dehydrogenase on isoleucine and valine . Its Km values for L-phenylalanine and L-leucine were similar to those of phenylalanine dehydrogenase . The substrate specificity of the chimeric enzyme in the reductive amination was an admixture of those of the two parent enzymes . These results suggest that the two domains of phenylalanine dehydrogenase and leucine dehydrogenase probably can fold independently . Accordingly, their chimera forms a new active enzyme which consists of their N- and C-terminal domains containing the substrate- and coenzyme-binding regions, respectively . However, the two domains of chimeric enzyme interact and communicate with each other to form a new active site and consistently show the new substrate specificity.

Protein Eng, 1994 Oct, 7(10), 1255 - 9
C-terminal truncations of a thermostable Bacillus stearothermophilus alpha-amylase; Vihinen M et al.; A series of truncated proteins from a thermostable Bacillus stearothermophilus alpha-amylase was prepared to study the importance of the extension in the C-terminus compared with other liquefying Bacillus alpha-amylases . The mutations introducing new translation termination sites shortened the 515 amino acid residue-long wild type enzyme by 17, 32, 47, 73 or 93 residues . The longer the truncation, the lower the specific activity of the enzyme . Only the two longest mutant proteins were active: the specific activity of the 498 residue variant was 97% and protein 483 was 36% that of the parental enzyme . The Km values of starch hydrolysis changed from 1.09 for wild type enzyme to 0.35 and 0.21 for mutants 498 and 483, respectively, indicating altered substrate binding . The mutant enzymes had almost identical pH and temperature optima with the wild type amylase, but enhanced thermal stability and altered end product profile . The consequences of the truncation to the structure and function of the enzymes were explored with molecular modeling . The liquefying amylases seem to require approximately 480 residues to be active, whereas the C-terminal end of B.stearothermophilus amylase is required for increased activity.

Pharm Res, 1994 Oct, 11(10), 1485 - 91
Gamma sterilization of a semi-solid poly(ortho ester) designed for controlled drug delivery--validation and radiation effects; Merkli A et al.; Radiation sterilization is becoming increasingly popular for the sterilization of many pharmaceutical products . Although this technique is not limited to the sterilization of polymers, it is probably the most suitable method for such materials . This method however suffers several drawbacks . The sterilization of a product must lead to a safety level of 10(-6), i.e . one chance in a million to find a contaminated sample . In many cases, this assurance of sterility can be achieved by using a uniform treatment dose of 2.5 Mrad, recommended by the pharmacopeia . We investigated the possibility of using doses of radiation inferior to 2.5 Mrad to sterilize a semi-solid poly(ortho ester) (POE) developed for use as carrier in controlled drug delivery . After determination of the initial bioburden, the polymer was intentionally contaminated with the bioindicator Bacillus pumilus E 601 . Following exposure to gamma irradiation, the D10 value of the radio resistant bioindicator was determined . Using the initial contamination value, the reduction factor D10 and the safety level, it is possible to calculate an optimal sterilizing dose for POE . All polymers are affected by ionizing radiation and the amount of radiation which produces a significant change in properties may vary from one polymer to the other . A molecular weight and dynamic viscosity decrease resulting from backbone cleavage was observed for this POE at a dose lower than 2.0 Mrad . Evaluation of the structure using 1H-NMR, 13C-NMR and IR analysis shows that for doses higher than 2.0 Mrad, another degredation process takes place.(ABSTRACT TRUNCATED AT 250 WORDS)

In Vitro Cell Dev Biol Anim, 1994 Oct, 30A(10), 690 - 5
Cytolytic differences among lepidopteran cell lines exposed to toxins of Bacillus thuringiensis subsp . kurstaki (HD-263) and aizawai (HD-112): effect of aminosugars and N-glycosylation; McCarthy WJ; Comparison of lytic-dose response behavior of seven lepidopteran cell lines to the activated delta-endotoxin polypeptides of Bacillus thuringiensis subspecies kurstaki (HD-263) and aizawai (HD-112) indicated distinct differences among the lines . The lines derived from Spodoptera species S . exigua (URC-SE-1A) and S . littoralis (UIV-SL-575) were more susceptible to lysis by aizawai toxin (Bta) than kurstaki toxin (Btk) as were cells from the Lymantria dispar line (IPLB-LD652Y) . However, the concentrations of Bta required for lysis of 50% of URC-SE-1A and IPLB-LD652Y cells (LC50) were 0.2 to 0.8 micrograms/ml compared to 5 to 9 micrograms/ml for UIV-SL-575 cells . In comparison, Btk LC50 concentrations for the three lines were similar (14 to 19 micrograms/ml) . Cells from S . frugiperda (IPLB-SF-21AE) and Trichoplusia ni (TN368) were similar in their response to Bta (LC50 = 2.5 to 3.7 micrograms/ml) and Btk (LC50 = 1.0 to 2.8 micrograms/ml) whereas the lines derived from Heliothis spp . were the least susceptible to both toxins . The LC50 concentrations for Bta with the H . zea line (IPLB-HA-1075) and H . virescens line (BCIRL-HV-AM1) were > 50 micrograms/ml and for Btk were > 50 micrograms/ml and 42 to 50 micrograms/ml, respectively, yet for both lines Btk was the more cytolytic . Cytolysis of TN368 cells could be inhibited to varying extents by preincubation of the toxins with the aminosugars of galactose, mannose, and glucose and their N-acetyl derivatives . The unsubstituted hexoses were not inhibitory.(ABSTRACT TRUNCATED AT 250 WORDS)

Tuber Lung Dis, 1994 Oct, 75(5), 334 - 40
Empiric antituberculosis treatment: benefits for earlier diagnosis and treatment of tuberculosis; Anglaret X et al.; SETTING: Tuberculosis may be diagnosed too late, especially in HIV-infected patients, with consequences on bacillus transmission and survival . Empiric antibuberculosis treatment (EATT) may be started before diagnosis of tuberculosis is confirmed . As rifampicin is a broad spectrum antibiotic, EATT including rifampicin may be effective in infections other than tuberculosis, leading to misdiagnosis . OBJECTIVE: To define the efficiency criteria of EATT with or without rifampicin . DESIGN: Between 1988 and 1991, 20 febrile patients with suspected tuberculosis (including 15 who were HIV-positive) were started on EATT in the absence of bacteriological or histological proof of tuberculosis . 10 patients (50%) received a 4-drug non-specific EATT including rifampicin, isoniazid, pyrazinamide and ethambutol, and 10 (50%), received a 3-drug specific EATT without rifampicin . RESULTS: In 10 patients (50%), the diagnosis of tuberculosis was confirmed by positive cultures within a mean of 32 days (15-57 days) after the beginning of EATT (group TB 1) . Of the 10 patients whose cultures remained negative, 4 (20%) became afebrile and showed improvement under EATT (group TB 2), and 6 (30%) remained febrile and did not improve (group No TB) . Patients from groups TB 1 and TB 2 became afebrile within a mean of 11 days (1-54 days) . This delay was not different between patients receiving specific or non-specific EATT . In patients receiving specific EATT, rifampicin was added to the initial 3-drug treatment after resolution of fever . CONCLUSION: EATT appears to be a useful method for rapid presumptive diagnosis and treatment of tuberculosis.

Immunology, 1994 Oct, 83(2), 227 - 31
Mycobacteria precipitate an SLE-like syndrome in diabetes-prone NOD mice; Baxter AG et al.; Non-obese diabetic (NOD) mice spontaneously develop organ-specific autoimmunity and are widely used as a model for diabetes . Aged NOD mice also exhibit some features of non-organ-specific autoimmune rheumatic disease such as anti-nuclear antibodies and late-onset haemolytic anaemia . Here, we report that a single dose of 2.6 x 10(7) heat-killed bacillus Calmette-Guerin (BCG) i.v . in 8-week-old NOD mice prevented diabetes but precipitated a syndrome similar to systemic lupus erythematosus (SLE) . Treated mice developed haemolytic anaemia, anti-DNA and anti-Sm anti-nuclear autoantibodies and an increased severity of sialadenitis . Perivascular lymphocytic infiltration in the kidneys and glomerular immune complex deposition were also found . The action of BCG appeared to be mediated by an adjuvant-like activity as treated mice showed a substantial increase in reticuloendothelial cell function and enhanced antigen presentation capacity.

Mol Microbiol, 1994 Oct, 14(2), 381 - 9
Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var . tenebrionis; Adams LF et al.; NB176 is a Bacillus thuringiensis mutant derived by gamma-irradiation of NB125 Bacillus thuringiensis var . tenebrionis (Krieg) . It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein . Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment . Each cryIIIA gene-bearing HindIII fragment was cloned from NB176 . The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers . Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn1000 . These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by gamma-irradiation . Integration of additional copies of the cryIIIA gene into the native 90 MDa plasmid of the wild-type B . thuringiensis var . tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.

Mol Microbiol, 1994 Oct, 14(1), 41 - 50
Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins; Domenighini M et al.; Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity . This cavity consists of 18 contiguous amino acids that form an alpha-helix bent over a beta-strand . The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence . Catalysis is supported by two spatially conserved amino acids, each flanking the NAD-binding site . These are: a glutamic acid that is conserved in all toxins, and a nucleophilic residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat-labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus . The latter group of toxins presents an additional histidine that appears important for catalysis . This structure suggests a general mechanism of ADP-ribosylation evolved to work on different target proteins.

Mol Microbiol, 1994 Oct, 14(1), 131 - 9
IS231A insertion specificity: consensus sequence and DNA bending at the target site; Hallet B et al.; In its natural host, Bacillus thuringiensis, the insertion sequence IS231A is preferentially inserted into the terminal inverted repeats of the transposon Tn4430 . Using a novel transposition assay, we demonstrate that the Tn4430 ends behave as insertion hot spots for IS231A in Escherichia coli . Sequence analysis reveals that IS231A insertion sites match the 5'-GGG(N)5CCC-3' consensus . However, this consensus is not the only determinant of IS231A insertion specificity . Although both Tn4430 ends have identical sequences, one is strongly preferred to the other and the orientation of insertion into this end is not random . We demonstrate that this preference is determined by the flanking regions of the site . These regions display a conserved periodic organization of their sequence which, by conferring anisotropic flexibility, would induce the DNA to bend in a roughly 'S'-shaped structure centered on the target consensus . DNA conformation analysis by polyacrylamide gel electrophoresis indeed shows that the preferred target site of IS231A is flanked by DNA segments curved in opposite directions . We present a model in which DNA bendability and curvature would contribute to the positioning of IS231A transposase on the target DNA.

Skeletal Radiol, 1994 Oct, 23(7), 569 - 71
Case report 865 . Bacillary angiomatosis; Standiford KN et al.; A case of bacillary angiomatosis was presented, characterized by subcutaneous lesions, systemic symptoms, and impressive periostitis which initially masked a small cortical lytic lesion . Review of the reported cases suggests that a diagnosis of bacillary angiomatosis should be strongly considered when periostitis is identified in an AIDS patient with skin lesions . Additionally, deep surgical biopsy of the skin lesion to include the subcutaneous tissue should be performed to confirm the diagnosis if the initial punch biopsies are unrevealing.

Pathologe, 1994 Oct, 15(5), 259 - 70
{Vascular tumors of the skin and soft tissue . Overview of newly characterized entities and variants}; Mentzel T et al.; This review summarizes vascular tumours of skin and soft tissues that have been characterised in recent years . Although most of them are very rare, knowledge about their reproducible clinicopathological features is important to avoid diagnostic pitfalls . These lesions include: bacillary angiomatosis, a vasoproliferative, pseudoneoplastic infection of immunocompromised patients caused by Rochalimaea henselae; tufted angioma, a variant of lobular capillary haemangioma characterised by a "cannon-ball" distribution of multiple lobules composed of packed capillaries and pericytes; microvenular haemangioma, a cutaneous haemangioma composed of thin-walled and irregularly branching blood vessels which dissect dermal collagen; sinusoidal haemangioma, a distinctive variant of cavernous haemangioma which may be located in the subcutaneous breast tissue and then may be confused with well-differentiated angiosarcoma; "hobnail haemangioma" (targetoid haemosiderotic haemangioma), a benign vascular tumour with a distinctive clinical targetoid appearance and a hobnail cytomorphology of the prominent endothelial tumour cells; retiform haemangioendothelioma, a very recently characterised low-grade angiosarcoma occurring most commonly in the extremities of adolescents which is characterised by arborising blood vessels arranged in a retiform pattern and lined by hobnail-like prominent endothelial cells; Kaposi-like infantile haemangioendothelioma, a borderline malignant tumour of infants mimicking Kaposi's sarcoma histologically; epithelioid angiosarcoma, a highly aggressive tumour in the spectrum of epithelioid vascular lesions which stains positively for endothelial and epithelial immunohistological markers; benign lymph-angioendothelioma (progressive lymphangioma), a benign, slowly growing macule or plaque which has to be distinguished from well-differentiated angiosarcoma and Kaposi's sarcoma; and lymphangiomatosis of the limbs, a poorly recognised angiomatosis occurring in young patients and limited mainly to the limbs with a favourable prognosis.

Clin Infect Dis, 1994 Oct, 19(4), 751 - 5
Lytic vertebral lesions: an unusual manifestation of AIDS-associated Kaposi's sarcoma; Isenbarger DW et al.; The differential diagnosis of neovascular skin lesions in patients with AIDS includes Kaposi's sarcoma and bacillary angiomatosis . It has been suggested that the radiographic presence of lytic bone lesions in association with these skin lesions supports a diagnosis of bacillary angiomatosis . We present a case of disseminated Kaposi's sarcoma in which evidence of lytic vertebral disease was seen on computed tomography; the histopathologic characteristics of the osseous lesions are described . Findings of magnetic resonance imaging implied more diffuse marrow involvement . Human immunodeficiency virus-associated osseous manifestations of rochalimaea infection and Kaposi's sarcoma are reviewed.

Appl Microbiol Biotechnol, 1994 Oct, 42(1), 78 - 84
Cloning and sequencing the degS-degU operon from an alkalophilic Bacillus brevis; Louw ME et al.; The sacU region from an alkalophilic Bacillus brevis was cloned and sequenced . The two open reading frames of the degS-degU operon encode polypeptides that gave calculated molecular masses of 43.8 kDa and 27.0 kDa, respectively . Sequence comparisons at the amino acid level to the B . subtilis degS-degU genes showed 74% and 84% similarity, respectively . On a multicopy vector the B . brevis degS-degU genes were found to cause hypersecretion of several extracellular enzymes in a B . subtilis rec- strain as well as in a B . subtilis sacU(HY) strain.

Immunobiology, 1994 Oct, 191(4-5), 564 - 8
Cytokine regulation of disease progression in leprosy and tuberculosis; Kaplan G; Studies in our laboratory have focussed on the role of cytokines in the regulation of the cellular immune response and disease progression in two important mycobacterial infection of man, namely leprosy and tuberculosis . Our studies in leprosy have involved the use of key regulatory cytokines such as IFN-gamma in the modulation of the cellular response of infected patients . We have investigated the effect of intradermal administration of low dose IFN-gamma on the lesions of anergic lepromatous patients and have reported an accelerated bacillary clearance from the skin . This was associated with the local accumulation of mononuclear cells and killing of infected macrophages . However, IFN-gamma administration also resulted in the induction of erythema nodosum leprosum, a toxic syndrome associated with excess TNF-alpha production . Both the toxic symptoms and the high levels of TNF-alpha production could be inhibited by thalidomide treatment, a drug we have shown reduces the half life of TNF-alpha mRNA . In preliminary clinical trials in tuberculosis patients we have attempted to use thalidomide to reduce TNF-alpha production and toxicities . These results are discussed.

Immunobiology, 1994 Oct, 191(4-5), 413 - 23
Transient inducible events in different tissues: in situ studies in the context of the development and expression of the immune responses to intracellular pathogens; Belkaid Y et al.; Intracellular pathogens whether facultative like Mycobacterium sp., e.g . Bacillus Calmette Guerin, Listeria monocytogenes or strictly intracellular like Leishmania sp . initiate either asymptomatic infectious processes or disease depending both on factors of the host (genetic as well as environmental ones) and the infectious/pathogenic agents . In this contribution, we first summarized informations which justify to develop in situ analysis to decipher the sequential events that result in different modes/classes of immune responses . How the mode of the immune response is determined remains a main question to address . Although it has recently become clear, in vitro, that immunocompetent cells and their cytokines are critical to set on a stable mode of immune response, acting on naive T cells, this area deserves more in vivo studies . Indeed, peripheral T cells, at different stages of differentiation, may exist in vivo (a) naive/virgin, (b) experienced, (c) effector T cells, depending on the level of stimulation of the immune system by either endogenous or exogenous (e.g . gut flora) signals . The three chosen examples illustrate our contributions in this field focusing on three different non-lymphoid tissues which may become infected: bone marrow (Bacille de Calmette Guerin), liver (Listeria monocytogenes), skin (Leishmania major) . These three illustrations also allow to attract attention on the interest of using mice of genetically different strains the immune response of which is set up under different modes.

Int J Biol Macromol, 1994 Oct, 16(5), 265 - 75
Gamma-poly(glutamic acid) formation by Bacillus licheniformis 9945a: physiological and biochemical studies; Birrer GA et al.; Cryogenically frozen vegetative cells of Bacillus licheniformis 9945a derived from young mucoid colonies were used to inoculate gamma-poly(glutamate) (gamma-PGA) production media containing L-glutamate, citrate and glycerol as carbon sources . A gel permeation chromatography (GPC) method was developed to determine gamma-PGA volumetric yield and molecular weight directly using culture filtrates . For GPC volumetric yield measurements, a calibration curve was generated using purified gamma-PGA to relate the gamma-PGA GPC peak area and polymer weight . Purified gamma-PGA was characterized by elemental analysis, 1H- and 13C-NMR spectroscopy . Cultures of B . licheniformis using all three carbon sources showed the following characteristics: cell growth mainly during the first 24 h; largest gamma-PGA volumetric productivity (approximately 0.12 gl-1 h-1) between 48 and 96 h; 11 g l-1 gamma-PGA volumetric yield by 96 h; reduction (utilization) of glycerol, glutamate and citrate during a 96 h cultivation time from 80 to 45 g l-1, 18 to 10 g l-1 and 12 to approximately 1 g l-1, respectively; a decrease in pH from 7.4 to approximately 5.5 by 42 h cultivation; acetic acid secretion into the medium at a maximum level of approximately 4.5 g l-1 and detection of the metabolite 2,3-butanediol (as acetoin) as a fermentation by-product at approximately 42 h and through a 96 h cultivation period . The presence of 2,3-butanediol indicated that the level of oxygen in the medium no longer supported a fully aerobic mode of metabolism . When the medium formulation was altered by removal of either citrate, L-glutamate or glycerol in shake flask experiments where pH was not controlled, 2.3, 9.0 and 4.0 g l-1, respectively, of gamma-PGA were formed . Variation of the medium ionic strength by the addition of up to 4% (w/v) NaCl led to the formation of gamma-PGA of relatively higher molecular weight but lower volumetric yield . Studies carried out on 5-day-old B . licheniformis cultures suggested that gamma-PGA depolymerase is intracellularly located or cell-bound . Culture filtrates showed no significant gamma-PGA depolymerase activity.

Appl Environ Microbiol, 1994 Oct, 60(10), 3711 - 7
Membrane-permeabilizing activities of Bacillus thuringiensis coleopteran-active toxin CryIIIB2 and CryIIIB2 domain I peptide; Von Tersch MA et al.; Bacillus thuringiensis toxin CryIIIB2 exhibits activity against two agriculturally important pests, the Colorado potato beetle, Leptinotarsa decemlineata, and the Southern corn rootworm, Diabrotica undecimpunctata . CryIIIB2 shows significant structural similarity to Colorado potato beetle-active toxin CryIIIA, whose crystal structure has been determined elsewhere {J . Li, J . Carrol, and D . J . Ellar, Nature (London) 353:815-821, 1991} . A clone limited to the putative 7-alpha-helical bundle domain I peptide of CryIIIB2 was constructed by PCR . The truncated protein was expressed at high levels in Escherichia coli . Domain I peptide was isolated and compared with native CryIIIB2 toxin in promoting ion efflux from synthetic phospholipid vesicles and formation of ion channels in black lipid membranes . The results showed that CryIIIB2 domain I peptide is sufficient for ion channel formation and promotes ion efflux . Both native CryIIIB2 toxin and domain I peptide were inefficient channel-forming proteins that produced noisy ion channels of various conductance states . In ion efflux assays, native toxin promoted greater ion efflux from synthetic vesicles than did the truncated peptide.

Biochem Biophys Res Commun, 1994 Sep 30, 203(3), 1745 - 9
Flavin supported fatty acid oxidation by the heme domain of Bacillus megaterium cytochrome P450BM-3; Gonvindaraj S et al.; Cytochrome P450BM-3 is a fatty acid hydroxylase that consists of a heme domain covalently attached to a diflavin (FMN+FAD) cytochrome P450 reductase domain . The heme and flavin domains can be separately expressed and purified from E . coli recombinant expression systems . Normally P450s require a protein redox partner as a source of electrons . We now have found that the P450BM-3 heme domain can be reduced by NADPH+FMN and that reduced FMN can support the P450 catalyzed hydroxylation of a fatty acid substrate, myristic acid . HPLC profiles show that the "artificial" FMN supported hydroxylation gives the same products as does holo-P450BM-3.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9544 - 8
Activation of (His)6-Raf-1 in vitro by partially purified plasma membranes from v-Ras-transformed and serum-stimulated fibroblasts; Dent P et al.; The serine-threonine protein kinase Raf-1 is an important signal transducer in mitogenesis, phosphorylating and activating mitogen-activated protein (MAP) kinase kinase . Raf-1 activation in vivo is dependent on Ras, but the mechanism of Raf activation is unknown . The ability of preparations of plasma membranes to activate exogenous (His)6-Raf-1 was studied . Plasma membranes of v-Ras-transformed NIH 3T3 cells, but not parental cells, enhanced MAP kinase kinase kinase (MAPKKK) activity dependent on addition of (His)6-Raf-1 and ATP/Mg . Treatment of membranes with concentrations of Bacillus cereus phosphatidylcholine-specific phospholipase C that activated Raf-1 in vivo failed to enhance MAPKKK activity in vitro . Activation of (His)6-Raf-1 in vitro by membranes was dependent on binding to Ras . Membranes from v-Src-transformed cells also activated (His)6-Raf-1 and synergized with v-Ras membranes . Serum-treatment of NIH 3T3 cells stimulated the ability of membranes to activate (His)6-Raf-1 . Activated (His)6-Raf-1 could be recovered on Ni(2+)-agarose, and this methodology was used to demonstrate that activation by membranes was ATP dependent . These findings demonstrate Ras- and ATP-dependent step(s) for Raf-1 activation by plasma membranes in vitro.

Proc Natl Acad Sci U S A, 1994 Sep 27, 91(20), 9407 - 11
Nitric oxide is an important mediator for tumoricidal activity in vivo; Farias-Eisner R et al.; When cultured in vitro, peritoneal macrophages, obtained from mice previously inoculated with bacillus Calmette-Guerin, release nitric oxide, which is cytostatic and/or cytolytic for tumor cells . However, it is not known whether nitric oxide has antitumor effects in vivo . Here we demonstrate that nitric oxide is an important mediator of host resistance to syngeneic and xenogeneic ovarian tumor grafts in C3HeB/FeJ mice . A murine ovarian teratocarcinoma cell line, utilized to study the mechanism of bacillus Calmette-Guerin-induced host resistance to a syngeneic ovarian tumor, proliferated when transplanted intraperitoneally . Marked tumoricidal activity was observed, however, when these murine ovarian teratocarcinoma cells were transplanted 8 days after intraperitoneal bacillus Calmette-Guerin inoculation . In studies related to xenogeneic ovarian tumor grafts, tumoricidal activity was observed after intraperitoneal transplantation of a human epithelial ovarian cancer cell line, NIH:OVCAR-3 . This cell line proliferates only in athymic nude (immunologically incompetent) mice . In both sets of experiments, tumoricidal activity was reduced by inhibition of nitric oxide synthesis . These results demonstrate the tumoricidal action of nitric oxide in vivo.

J Mol Biol, 1994 Sep 23, 242(3), 193 - 202
Cavity mutants of Savinase . Crystal structures and differential scanning calorimetry experiments give hints of the function of the buried water molecules in subtilisins; Pedersen JT et al.; The subtilisin molecule possesses several internal water molecules, which may be characterised as an integral part of the protein structure . We have introduced specific mutations (T71I, T71S, T71V, T71A and T71G) at position 71 in the subtilisin variant Savinase from Bacillus lentus . This position is involved in a hydrogen bonded network with several internal water molecules, forming a water channel . The water channel and most of the other internal water molecules are positioned in the interface between two half-domains of the subtilisin molecule . The data presented here indicate that the internal water molecules are structural, and may be the result of trapping during the folding process.

FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 43 - 8
Characterization of the type strain of Bacillus thuringiensis subsp . cameroun serotype H32; Juarez-Perez VM et al.; The strain 273 B, the type strain of a H serotype of Bacillus thuringiensis not yet characterized: B . thuringiensis subsp . cameroun, serotype H32, was isolated from soil samples collected in Cameroon . This strain produces cuboidal parasporal bodies composed of two major proteins of 53 kDa and 35 kDa . N-terminal sequences of the major proteins share no homology with published sequences . Only the 35 kDa protein is susceptible to digestion by trypsin . A complex array of 9 plasmids was revealed.

Gene, 1994 Sep 15, 147(1), 1 - 11
Biosynthesis of butirosin in Bacillus circulans NRRL B3312: identification by sequence analysis and insertional mutagenesis of the butB gene involved in antibiotic production; Aubert-Pivert E et al.; As an approach to an analysis of the biosynthesis of the aminoglycoside antibiotic butirosin (But), we investigated the chromosomal regions flanking the ButR gene (aphA4/butA) of Bacillus circulans NRRL-B3312, and have identified, by nucleotide sequence analysis, a large open reading frame (ORF; ButB) upstream from the ButR gene . Hybridization was detected between butB and chromosomal DNA from other Bacillaceae that produce But-like compounds (but not from non-producers) . Interruption of this sequence by insertion of an erythromycin-resistance-encoding gene (erm) at either of two distinct sites eliminated the production (biosynthesis or export) of But, thus indicating a role for butB in antibiotic production . Gene butB is transcribed in the same direction as butA and encodes a protein of 1616 amino acid (aa) residues with a 30-aa N-terminal signal peptide . Comparison of the sequence for the translation product (ButB) with the aa compositions and sequences of known bacterial surface proteins, such as S-layer proteins, suggests that this protein is cell-wall associated . It is proposed that ButB plays a role in the export of But from the producing organism.

Biochem J, 1994 Sep 1, 302 ( Pt 2), 611 - 6
Mutagenesis of two surface-exposed loops of the Bacillus thuringiensis CryIC delta-endotoxin affects insecticidal specificity; Smith GP et al.; Site-directed mutagenesis was used to determine the role of two surface-exposed loops (Gly-317-Phe-320 and Gln-374-Pro-377) in the insecticidal specificity of the Bacillus thuringiensis CryIC delta-endotoxin . Mutant toxins were generated by PCR using degenerate oligonucleotide primers, and expressed in Escherichia coli . More than 50 mutant toxins were screened for toxicity to the lepidopteran Spodoptera frugiperda Sf9 cell line using an in vitro lawn assay . A panel of these mutant toxins, which included toxic and non-toxic variants from both loops, was further screened for activity towards Aedes aegypti larvae . The activity of these mutants to Sf9 cells was quantified more precisely using a cell lysis assay . Three categories of mutants were identified: (1) those non-toxic to either Sf9 cells or Aedes aegypti larvae; (2) those fully toxic to both genera; and (3) those which were only toxic to Sf9 cells . For the first loop, the differential specificity was not restricted to any single residue . In the second loop, two mutant toxins with a Pro-377-->Ala substitution displayed this phenotype . The time dependence of toxicity towards Sf9 cells was examined using the same panel of mutants . All toxic mutants displayed an identical time course to the wild-type toxin, with the exception of the two Pro-377-->Ala mutants of the second loop . These toxins displayed a lower time dependence, no cell death occurring within the first hour of incubation . These results show that the two loops are important determinants of both the activity and specificity of the CryIC delta-endotoxin.

J Bacteriol, 1994 Sep, 176(18), 5654 - 64
Trehalose-6-phosphate hydrolase of Escherichia coli; Rimmele M et al.; The disaccharide trehalose acts as an osmoprotectant as well as a carbon source in Escherichia coli . At high osmolarity of the growth medium, the cells synthesize large amounts of trehalose internally as an osmoprotectant . However, they can also degrade trehalose as the sole source of carbon under both high- and low-osmolarity growth conditions . The modes of trehalose utilization are different under the two conditions and have to be well regulated (W . Boos, U . Ehmann, H . Forkl, W . Klein, M . Rimmele, and P . Postma, J . Bacteriol . 172:3450-3461, 1990) . At low osmolarity, trehalose is transported via a trehalose-specific enzyme II of the phosphotransferase system, encoded by treB . The trehalose-6-phosphate formed internally is hydrolyzed to glucose and glucose 6-phosphate by the key enzyme of the system, trehalose-6-phosphate hydrolase, encoded by treC . We have cloned treC, contained in an operon with treB as the promoter-proximal gene . We have overproduced and purified the treC gene product and identified it as a protein consisting of a single polypeptide with an apparent molecular weight of 62,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme hydrolyzes trehalose-6-phosphate with a Km of 6 mM and a Vmax of at least 5.5 mumol of trehalose-6-phosphate hydrolyzed per min per mg of protein . The enzyme also very effectively hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, but it does not recognize trehalose, sucrose, maltose, isomaltose, or maltodextrins . treC was sequenced and found to encode a polypeptide with a calculated molecular weight of 63,781 . The amino acid sequence deduced from the DNA sequence shows homology (50% identity) with those of oligo-1,6-glucosidases (sucrase-isomaltases) of Bacillus spp . but not with those of other disaccharide phosphate hydrolases . This report corrects our previous view on the function of the treC gene product as an amylotrehalase, which was based on the analysis of the metabolic products of trehalose metabolism in whole cells.

Ann Emerg Med, 1994 Sep, 24(3), 418 - 21
Exposure of emergency department personnel to tuberculosis: PPD testing during an epidemic in the community; Sokolove PE et al.; STUDY OBJECTIVE: The present epidemic of tuberculosis has increased the risk of transmission of tuberculosis to health care workers in general and emergency department staff in particular, who often treat patients with tuberculosis before their diagnosis . The purpose of this study was to determine the risk of tuberculosis exposure among the nursing and physician staff of an urban ED . DESIGN: Observational study of self-reported purified protein derivative (PPD) skin test results and tuberculosis exposure . SETTING: Urban, public ED . PARTICIPANTS: Attending physicians, resident physicians, and registered nurses . INTERVENTIONS: None . RESULTS: Questionnaires were sent to all attending physicians, resident physicians, and registered nurses in the ED at Harbor-UCLA Medical Center requesting information on the subject's present and prior PPD status, known exposure to tuberculosis, and duration of time and average number of hours worked in the ED . Ninety-six of 129 questionnaires (74%) were returned . Five of the respondents had been immunized with Bacillus of Calmette and Guerin vaccine (BCG) and 10 of the respondents were PPD positive before beginning work in the ED . Of the other 81 respondents, 31% (25 of 81) had become PPD positive while working in the ED . The majority of these conversions (15 of 25) occurred in the first 6 months of 1993 . A Kaplan-Meier survival analysis revealed nearly a 40% risk of PPD conversion after 60 months of full-time work in the ED . CONCLUSION: As measured by self-reported PPD status, a high rate of exposure to tuberculosis has been observed among the ED staff at Harbor-UCLA Medical Center . The highest rate of PPD conversion has been noted most recently, suggesting that there has been a significant increase in staff exposure to tuberculosis during 1992 and the beginning of 1993 . Systematic monitoring of PPD conversion rates among ED staff is necessary to determine the adequacy of ED respiratory isolation procedures during the current tuberculosis epidemic.

J Bacteriol, 1994 Sep, 176(17), 5571 - 3
The zymogen of the protease that degrades small, acid-soluble proteins of spores of Bacillus species can rapidly autoprocess to the active enzyme in vitro; Illades-Aguiar B et al.; The zymogen of the protease (GPR) that initiates protein degradation during spore germination in Bacillus species is not activated in vitro under normal physiological conditions . However, there is rapid, acid-pH-dependent, zero-order, proteolytic activation of the purified zymogen in high concentrations of dimethyl sulfoxide . These findings provide further evidence that GPR activates itself during sporulation.

J Bacteriol, 1994 Sep, 176(17), 5554 - 9
Identification of amino acid residues of Bacillus thuringiensis delta-endotoxin CryIAa associated with membrane binding and toxicity to Bombyx mori; Lu H et al.; Alanine substitution (A3) or deletion (D3) of residues 365 to 371 of Bacillus thuringiensis CryIAa insect toxin removed nearly all toxicity for Bombyx mori (> 1,000-fold less active than the wild type) . The loss of larvicidal activity in the mutants was not caused by increased sensitivity to larval gut enzymes but could be attributed to significantly reduced binding to B . mori brush border membrane vesicles . Some or all of the affected amino acid residues may interact directly or indirectly with the B . mori membrane receptor(s) . Such receptor binding appears to be directly correlated with insect toxicity.

J Appl Bacteriol, 1994 Sep, 77(3), 256 - 63
Studies on the Bacillus flora of milk and milk products; Crielly EM et al.; Bacillus licheniformis and B . cereus were the most commonly isolated species of Bacillus found in milk at all stages of processing . Bacillus licheniformis was ubiquitous in the farm environment and counts in raw milks heat-treated in the laboratory were higher during the winter months, whilst B . cereus was associated with cattle feed throughout the year, and tended to be more common in raw milks during the summer months . Although B . licheniformis was usually isolated in larger numbers than B . cereus, this pattern changed after raw and pasteurized milks and reconstituted milk powders were pre-incubated at ambient temperatures, and B . cereus came to dominate the Bacillus population, reaching levels associated with enterotoxin production . Investigation of the growth kinetics of strains of both species showed that B . cereus grew faster than B . licheniformis at ambient temperatures . It is suggested that post-pasteurization contamination, which is commonly blamed for spoilage of milk and milk products by B . cereus, is not necessarily the most important source of this organism.

Acta Urol Belg, 1994 Sep, 62(3), 63 - 8
Evolution of cellular and humoral response against Tuberculin and antigen 85 complex during intravesical treatment with BCG of superficial bladder cancer; Zlotta A et al.; Prophylactic treatment with Bacillus Calmette-Guerin (BCG) is an established and effective therapy of bladder cancer . The antitumor effect of BCG seems to be largely related to cellular immunological mechanisms, although its precise mode of action is unknown . Antitumor response of BCG seems to be initiated by the attachment of BCG to bladder wall via Fibronectin (FN) . The cellular immune response against Tuberculin PPD and the major secreted BCG antigen (Fibronectin-binding AG 85 complex) has been tested in a control group of 20 untreated bladder tumor patients and before and after 6 weekly intravesical BCG instillations in a group of 20 superficial bladder tumor patients . A major increase in the lymphoproliferative response against PPD and AG 85 was observed in respectively 66% and 57% of the treated patients . In contrast, no detectable antibody response (IgA, IgM, IgG) was observed against AG 85 complex after BCG treatment . On the other hand, antibodies against Tuberculin increased in 13 of 20 patients . This study seems to demonstrate a specific cellular immune activation against AG 85 Fibronectin-binding complex during BCG treatment of superficial bladder tumors . Humoral response against the AG 85 is not activated after BCG treatment . Further studies are needed to elucidate the role of AG 85 in the cellular intravesical penetration of BCG . Presence or absence of cellular response against this antigen could be of clinical value.

Trends Genet, 1994 Sep, 10(9), 328 - 33
Cell polarity in yeast; Chant J; Cell polarity is fundamental to the development and functioning of all organisms, from bacteria to humans . Examples of processes that involve cell polarity include the growth of axons, the interaction between T cells and their targets, the formation of buds by yeast, and sporulation in Bacillus spp . Recent work on budding yeast has provided valuable insights into the molecular machinery responsible for establishing and orienting cell polarity . Comparisons of the DNA sequences of genes involved in such pathways have raised the possibility that these mechanisms are conserved in all eukaryotic cells.

Can J Microbiol, 1994 Sep, 40(9), 782 - 6
Transformation of Bartonella bacilliformis by electroporation; Grasseschi HA et al.; Bartonella bacilliformis is a member of the order Rickettsiales, family Bartonellaceae . The bacterium is an intracellular parasite of human erythrocytes . To date, members of the family Bartonellaceae have not been transformed by standard chemical methods . We report the first successful transformation of a member of the Bartonellaceae family, B . bacilliformis, by the method of electroporation . The optimal conditions for electroporation of B . bacilliformis include a field strength of 12.5 kV/cm and a time constant of 5 ms using 0.2-cm cuvettes . With these parameters and the cosmid pEST (RK2 replicon), a transformation efficiency of 7.8 x 10(5) was obtained . Transformants were readily cultured on medium containing kanamycin sulfate at concentrations ranging from 15 to 600 micrograms/mL . Bacterial survival was approximately 31% under optimal electroporation conditions, and the maximal number of transformants was obtained with 80 ng of pEST DNA . Bartonella bacilliformis was verified as the transformed organism by a comparison of transformant protein profiles with those of wild-type B . bacilliformis using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and detection of the exogenous plasmid in DNA from the transformed bacteria by DNA hybridization . Transformations using the plasmids pMK20, pML31, and pUCK18 (containing the replicons ColE1, F, and pMB1, respectively) were not successful.

Appl Environ Microbiol, 1994 Sep, 60(9), 3096 - 104
Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus; Takata H et al.; Although the branching enzyme (EC 2.4.1.18) is a member of the alpha-amylase family, the characteristics are not understood . The thermostable branching enzyme gene from Bacillus stearothermophilus TRBE14 was cloned and expressed in Escherichia coli . The branching enzyme was purified to homogeneity, and various enzymatic properties were analyzed by our improved assay method . About 80% of activity was retained when the enzyme was heated at 60 degrees C for 30 min, and the optimum temperature for activity was around 50 degrees C . The enzyme was stable in the range of pH 7.5 to 9.5, and the optimum pH was 7.5 . The nucleotide sequence of the gene was determined, and the active center of the enzyme was analyzed by means of site-directed mutagenesis . The catalytic residues were tentatively identified as two Asp residues and a Glu residue by comparison of the amino acid sequences of various branching enzymes from different sources and enzymes of the alpha-amylase family . When the Asp residues and Glu were replaced by Asn and Gln, respectively, the branching enzyme activities disappeared . The results suggested that these three residues are the catalytic residues and that the catalytic mechanism of the branching enzyme is basically identical to that of alpha-amylase . On the basis of these results, four conserved regions including catalytic residues and most of the substrate-binding residues of various branching enzymes are proposed.

J Antibiot (Tokyo), 1994 Sep, 47(9), 959 - 68
Bacithrocins A, B and C, novel thrombin inhibitors; Kamiyama T et al.; Novel thrombin inhibitors, bacithrocins A, B and C, have been isolated from the culture broth of Bacillus laterosporus Laubach NR 2988 . The structures of these inhibitors have been determined to be N-acyl-L-phenylalanyl-DL-arginal by the 2D-NMR experiments on their oxidation products and by amino acid analysis . Bacithrocin A inhibits thrombin, factor Xa and trypsin with IC50s of 48, 13 and 0.65 microM, respectively, which are similar to those of bacithrocins B and C . Bacithrocins prolong the clotting time induced by thrombin and factor Xa.

Res Microbiol, 1994 Sep, 145(7), 503 - 18
Analysis of a cis-active sequence mediating catabolite repression in gram-positive bacteria; Hueck CJ et al.; One form of catabolite repression (CR) in the Gram-positive genus, Bacillus, is mediated by a cis-acting element (CRE) . We use here a consensus sequence to identify such elements in sequenced genes of Gram-positive bacteria . These are analysed with respect to position and type of gene in which they occur . CRE sequences near the promoter region are mainly identified in genes encoding carbon catabolic enzymes, which are thus likely to be subject to CR by a global mechanism . Functional aspects of CREs are evaluated.

Mol Microbiol, 1994 Sep, 13(6), 965 - 72
Synergism of mosquitocidal toxicity between CytA and CryIVD proteins using inclusions produced from cloned genes of Bacillus thuringiensis; Wu D et al.; The toxicity to mosquito larvae of the parasporal body produced by Bacillus thuringiensis subsp . israelensis and the PG-14 isolate of B . thuringiensis subsp . morrisoni is at least 20-fold greater than any of the four mosquitocidal proteins of which it is composed (CytA, CryIVA, B, and D) . This high toxicity is postulated to be due to synergistic interactions among parasporal proteins . However, this remains controversial because values reported for the specific toxicity of individual proteins, especially the CytA protein, vary widely owing to the methods used to purify and assay toxins against larvae . In an attempt to resolve questions of purity, specific toxicity, and synergism, individual genes encoding the CytA and CryIVD toxins were cloned and expressed in acrystalliferous B . thuringiensis subsp . israelensis cells using the shuttle vector pHT3101 . CytA and CryIVD inclusions were purified and their toxicity was determined alone and when combined at different ratios using bioassays against first instars of Aedes aegypti . The LC50 for the CytA inclusion was 60 ng ml-1, whereas the LC50 for the CryIVD was 85 ng ml-1 . In comparison, the LC50s for different combinations of CytA and CryIVD inclusions ranged from 12-15 ng ml-1, 4-5 times higher than the toxicity of either protein alone, demonstrating marked synergism between these two proteins . These results suggest that the high toxicity of the wild-type parasporal bodies of B . thuringiensis subspp . israelensis and morrisoni is due to synergism among three or four of their major proteins.

Pept Res, 1994 Sep-Oct, 7(5), 238 - 41
A general approach for identifying and cloning peptide synthetase genes; Turgay K et al.; A wide variety of bioactive peptides are synthesized nonribosomally by large multienzyme complexes employing the thiotemplate mechanism . Based on the known and highly conserved structures of several genes encoding multifunctional peptide synthetases, we developed a universal polymerase chain reaction (PCR) approach for amplifying, cloning and identifying parts of putative peptide synthetase genes . We showed, by cloning fragments of peptide synthetase genes from the phaseolotoxin-producing strain Pseudomonas syringe pv . phaseolica and from Bacillus licheniformis, which produces the branched peptide antibiotic bacitracin, that this approach is applicable . It gives a new and potentially general access to the biosynthetic genes of many nonribosomally synthesized peptides.

J Gastroenterol Hepatol, 1994 Sep-Oct, 9(5), 433 - 6
Spontaneous bacterial peritonitis and culture negative neutrocytic ascites in patients with non-alcoholic liver cirrhosis; al Amri SM et al.; Medical records of 18 patients with spontaneous bacterial peritonitis (SBP) and 19 patients with culture negative neutrocytic ascites (CNNA) were reviewed . The diagnosis of SBP was based on a positive ascitic fluid culture, a polymorphonuclear cell count (PMN) greater than 250 cells/mm3 and the absence of an intra-abdominal source of infection . The diagnosis of CNNA was based on a PMN count greater than 250 cells/mm3, a negative ascitic fluid culture, the absence of an intra-abdominal source of infection and no antibiotic treatment in the preceding 30 days . All patients in both groups had liver cirrhosis, which was mainly (62.2%) due to HBV infection . A single strain, mostly 'a Gram-negative' bacillus, was recovered from the ascitic fluid culture in the vast majority of patients (83%) with SBP . There were no significant differences between the clinical data of both groups . However, the CNNA group had a significantly better Pugh score (P value = 0.01) with a mean score of 9.42 +/- 2.24, compared to the SBP group (10.94 +/- 2.88) . The only significant difference in the laboratory data was that the total bilirubin was higher in the SBP group (P < 0.01) . Hospital mortality was significantly higher in the SBP patients compared to those with CNNA, 50 and 16%, respectively (P < 0.03) . Recurrent ascitic fluid infection occurred in one of five patients who initially presented . In contrast no recurrence was documented in 12 patients with CNNA . Spontaneous bacterial peritonitis is a serious complication of liver cirrhosis with significantly higher mortality than CNNA . A single organism, usually enteric, is the most common causative agent.

Biokhimiia, 1994 Sep, 59(9), 1393 - 400
{Secreted serine proteinase from the spore-forming bacteria Bacillus intermedius 3-19}; Balaban NP et al.; Extracellular serine proteinase has been isolated from the cultural medium of Bacillus intermedius 3-19 using CM-cellulose chromatography and affinity chromatography on bacitracin-Sepharose . The specificity of the proteinase with a wide range of natural and synthetic substrates has been investigated . The greatest activity was observed with tripeptides containing C-terminal Leu or Phe . The enzyme was completely inhibited by diisopropylfluorophosphate and partly inhibited by thiol-specific reagents . It is concluded that B . intermedius proteinase is a thiol-dependent serine proteinase pertaining to the subtilisin group . The amino acid composition of the enzyme has been determined . The enzyme contains one to three 1/2 cysteine residues, one of which is supposedly a Cys residue . The N-terminal amino acid sequences of the protein is AQTVPYGIPQIKAPA-.

Med Hypotheses, 1994 Sep, 43(3), 135 - 7
Bartonella bacilliformis or a similar organism and cardiovascular disease; Sood FH et al.; Bartonella bacilliformis invades the endothelial lining of the cardiovascular system . Damage to the red blood cells and white blood cells, the effects of the toxins, invasion of the brain and electrical charges induced by the organism so interfering with normal electrical stimulation of the heart may explain many of the features of cardiovascular disease (1-5).

Clin Infect Dis, 1994 Sep, 19(3), 528 - 40
The International Tuberculosis Campaign: a pioneering venture in mass vaccination and research; Comstock GW; If an American pediatrician's conversation with Dr . Johannes Holm, a Danish pathisiologist and future director of the International Tuberculosis Campaign, had not been interrupted, the campaign would probably not have become a monumental precedent for world health activities . The International Tuberculosis Campaign was conducted under the auspices of the United Nations International Children's Emergency Fund and three Scandinavian voluntary organizations . In a program that started in the war-torn areas of Europe, nearly 30 million persons underwent tuberculin testing, and almost 14 million were given BCG (bacille Calmette-Guerin) vaccine . In addition, a postgraduate school for physicians was initiated, new laboratories were established and old ones were improved, hundreds of young doctors and nurses were introduced to international public health, and, perhaps most important, research and service were successfully integrated . The success of the campaign led to its becoming the first major disease control and research activity of the World Health Organization.

Int J Food Microbiol, 1994 Sep, 23(1), 99 - 109
Characteristics of Bacillus cereus related to safe food production; Dufrenne J et al.; Thirty Bacillus cereus strains, isolated from different sources, were characterized in relation to safe food production . The minimal growth temperatures of the strains varied from < or = 5 degrees C to 11 degrees C . Generation times at 7 degrees C of strains capable of growing at temperatures < or = 5 degrees C were approximately 8.2 h . The D90 degrees C-values of spores of strains with a minimal growth temperature of 11 degrees C determined in phosphate buffer at pH 7.0 ranged from 4.8 to > 200 min . Strains with the capacity to grow at temperatures < or = 9 degrees C, had a D90 degrees C value ranging from 4.6 to 14 min . Addition of either nisin (250 micrograms/ml) or diacetyl (1500 micrograms/ml) to the heating menstruem at the single concentrations investigated seemed not influence the thermal destruction of spores . Germination of spores of almost all strains occurred in all three media tested (Brain Heart Infusion, rice extract and milk) even at temperatures below the minimal growth temperature . All B . cereus strains tested yielded positive results with a commercial test kit for diarrhoeal enterotoxin . The results indicate that strains with the capacity to grow at temperatures < or = 7 degrees C are not essentially different from those with minimal growth temperatures of > 10 degrees C.

Int J Food Microbiol, 1994 Sep, 23(1), 111 - 6
Stability of spores of Bacillus cereus stored on silicagel; Dufrenne J et al.; Spores of four different strains of Bacillus cereus were stored on silicagel at 22 degrees C and in physiological saline solution at -20 degrees C for a period of 260 days . At different intervals the spores were tested for survival, heat sensitivity and capacity to germinate . There was no clear change in any of the parameters tested, so storage on silicagel can be a good alternative for storage as a frozen suspension . Spores stored in this way can easily be exchanged for reference material and used for microbiological challenge testing.

Int J Food Microbiol, 1994 Sep, 23(1), 1 - 15
Bacillus cereus in infant foods and dried milk products; Becker H et al.; Dried milk products and infant food are known to be frequently contaminated with Bacillus cereus . Sources of the organism and its behaviour in the product and in the equipment during processing are discussed . With regard to the incidence of B . cereus in infant food, 261 samples distributed in 17 countries were collected and examined for its B . cereus content . Fifty-four percent of the samples were contaminated with B . cereus reaching levels from 0.3 to 600/g . Counts higher than 10/g were found in only 27 samples (10%) . Most of the positive samples (44%) contained 0.3 to 10 B . cereus/g . Four samples (1.5%) were contaminated with more than 100 organisms/g reaching a maximum level of 600 B . cereus/g . When classified into different types of products about 50% of the infant formulae based on milk, the follow-on formulae and the weaning foods were contaminated with B . cereus as well as 69% of those based on soy protein and 92% of the special dietetic foods . Compared to our earlier studies from 1982/83, the percentage of contaminated samples from Germany increased from 31% to 70% in the case of infant formulae, from 28% to 55% in the case of follow-on formulae, and from 40% to 100% in the case of special dietetic food . The percentage of weaning food contaminated with B . cereus remained nearly unchanged . It should be stressed, however, that the numbers of B . cereus were almost the same in both studies with the highest count in 1982/83 being 460 and in 1992 600/g . Samples naturally contaminated with counts of about 100 B . cereus/g were reconstituted and incubated at a room temperature of 27 degrees C . Levels of 10(5) B . cereus/g were reached after 7-9 h . Toxigenicity of B . cereus in dried milk products and infant food as well as food poisoning outbreaks attributed to B . cereus are discussed.

J Am Mosq Control Assoc, 1994 Sep, 10(3), 403 - 6
Emergency control of Aedes aegypti in the Dominican Republic using the Scorpion 20 ULV forced-air generator; Tidwell MA et al.; In an effort to develop a more effective measure for use in emergency control of the dengue vector Aedes aegypti . applications of a combination of a larvicide (Bacillus thuringiensis israelensis {B.t.i.}) and an adulticide (permethrin) were made using a truck-mounted forced-air generator (Scorpion 20) and evaluated in the Dominican Republic . This method has the potential to simultaneously control adults and larvae . In bioassay cages placed in household water containers at the time of application, larval mortalities were 95.1 and 100% for 2 application rates of permethrin mixed with B.t.i . Adult mortalities were not as impressive, probably because of resistance to permethrin . Higher adult mortality in caged specimens (78.5%) and a substantial reduction in the natural population (68.4%) of Ae . aegypti were obtained following a 2.1-g AI/ha application of deltamethrin alone.

J Am Mosq Control Assoc, 1994 Sep, 10(3), 363 - 73
Integrated management of waste tire mosquitoes utilizing Mesocyclops longisetus (Copepoda: Cyclopidae), Bacillus thuringiensis var . israelensis, Bacillus sphaericus, and methoprene; Tietze NS et al.; This study evaluated the compatibility and efficacy of using a predatory copepod, Mesocyclops longisetus in concert with 3 "biorational" compounds for mosquito control in waste tires . The toxicity of Bacillus thuringiensis var . israelensis (B.t.i), Bacillus sphaericus, and methoprene to Mesocyclops longisetus was assessed in the laboratory using concentrations 10 times the maximum labeled or suggested rate and based on a water depth of 7.6 cm . Microbials were tested using mature copepods exposed for durations of 24, 48, and 72 h . Methoprene bioassays consisted of individually exposing newly hatched copepods (i.e., nauplius larvae) and monitoring their development to maturity . The toxicity tests indicated B.t.i., B . sphaericus, and methoprene were not deleterious to copepods at concentrations exceeding those expected in the field . Copepods exposed to methoprene matured normally, and when mated, 50% developed egg sacs . A 5-month field test, integrating the copepod and B.t.i., B . sphaericus, and methoprene provided better mosquito reduction together than either copepods or control agents alone . When copepods were combined with B.t.i . or methoprene, overall reduction of 3rd- and 4th-instar larvae during the 5-month interval was equal to or greater than 90% . Bacillus thuringiensis var . israelensis alone temporarily produced a high degree of larval reduction (up to 100%), however reapplications were necessary to maintain that level of control . Of all the treatments, B . sphaericus alone produced the lowest degree of mosquito suppression due to lack of toxicity to Aedes albopictus, the predominant species during the study . It is recommended that mosquito control managers consider integrating M . longisetus and B.t.i . or methoprene against mosquitoes in waste tires.

Toxicon, 1994 Sep, 32(9), 1125 - 36
Biochemical and morphological changes in rat muscle cultures caused by 28,000 mol . wt toxin of Bacillus thuringiensis israelensis; Cahan R et al.; The 28,000 mol . wt protein of Bacillus thuringiensis israelensis showed a high degree of toxicity to rat muscle in culture . Application of 1 microgram/ml to the culture medium completely inhibited cell fusion . Reversibility of this effect was demonstrated by replacement of the culture medium with fresh medium, and the consequence was that cell fusion was resumed . When differentiated myotubes were treated with 1 microgram/ml of the toxin, the spontaneous contractile activity was abolished within 20 min . Cytotoxic effects were observed 1 hr after treatment was initiated, as manifested by creatine kinase (CK) release to the medium . Two hours after toxin was applied to the muscle culture, the myotubes were deteriorated whereas the mononucleated cells were not affected . Six or 7-day-old cultures which were treated by 1 microgram/ml of 28,00 mol . wt toxin revealed a change in the levels of Na+ and K+ within the fibres as analysed by X-ray microanalysis (XRMA) . Preincubation of the toxin for 20 min with phospholipids before application to the cells reduced the cytotoxic effect . Phosphatidylinositol and phosphatidylserine were the most efficient inhibitors, whereas phosphatidylcholine, sphingomyelin and phosphatidylethanolamine were less effective in protecting cultures from the cytotoxic effects of the 28,000 mol . wt protein.

Biosci Biotechnol Biochem, 1994 Sep, 58(9), 1738 - 41
Enzymatic conversion of dethiobiotin to biotin in cell-free extracts of a Bacillus sphaericus bioB transformant; Ohshiro T et al.; The activity of biotin synthase, responsible for biotin synthesis from dethiobiotin, was demonstrated in a completely defined reaction mixture with cell-free extracts of a Bacillus sphaericus bioB transformant . Among the sulfur compounds tested, only S-adenosyl-L-methionine was active, while L-methionine and L-cysteine had no significant effect . Protein concentrations higher than 15 mg/ml in the reaction mixture were needed to detect biotin synthase activity . When dialyzed cell-free extracts were used for the reaction, NADH, NADPH, or FAD among the well-known cofactors tested enhanced the activity, and Fe2+, Mn2+, and Ca2+ among the metal ions tested also had some effects.

Biotechnology (N Y), 1994 Sep, 12(9), 915 - 8
Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management; Bosch D et al.; To obtain Bacillus thuringiensis crystal proteins with new properties and to identify the regions involved in insecticidal activity, we generated hybrid genes composed of cryIC and cryIE by in vivo recombination . Analysis of the hybrid proteins showed that domain III of CryIC is involved in the toxicity towards Spodoptera exigua and Mamestra brassicae . Transfer of this domain to CryIE, which is not active against these insects, resulted in a new protein with a broader activity . This hybrid protein binds to different receptors than CryIC, suggesting its use as an alternative for CryIC in resistance management programs.

Biochem Cell Biol, 1994 Sep-Oct, 72(9-10), 397 - 402
Effect of the antirheumatic agent Tenidap on CD3, CD4, and CD8 expression and interleukin-1 and leukotriene B4 secretion in human peripheral blood mononuclear cells; Conti P et al.; Thymocytes that express the complete CD3-T-cell receptor (TCR) complex are CD4- and CD8- . The CD4+ T-cell population can be subdivided into at least two quite distinct subsets, TH1 and TH2 cells, based upon cytokine expression . Interleukin-1 (IL-1) appears to be required for optimal proliferation of T cells in response to antigen and it seems that in the absence of IL-1, TH2 clones proliferate less in response to antigen . Tenidap is an antirheumatic agent that has an inhibitory effect on IL-1 production . In these studies, we show that isolated human peripheral blood mononuclear cells (PBMCs) treated in vitro with Tenidap (15 micrograms/mL) for 48-h incubations significantly (p < 0.05) enhanced the present of CD4+ expression compared with untreated cells (control), as determined by cytofluorimetric analysis . Lipopolysaccharide and Bacillus Calmette-Guerin were used as positive controls . When the cells were tested for CD3 or CD8 receptor expression, no differences were found between the untreated PBMCs and the treated (15 micrograms/mL Tenidap) cells . No change was found when cells were incubated for 72 h . Moreover, our data show a strong dose-dependent inhibitory effect of Tenidap (15 micrograms/mL) on IL-1 alpha, IL-1 beta, and leukotriene B4 secretion in PBMCs treated overnight . The increased CD4+ expression by Tenidap in PBMCs may suggest an important role for this new antirheumatic agent in immunity and may hold future therapeutic promise for diseases involving IL-1 and leukotriene B4 as mediators.

Eur J Immunol, 1994 Sep, 24(9), 2237 - 42
Response of interleukin-6-deficient mice to tumor necrosis factor-induced metabolic changes and lethality; Libert C et al.; Whether interleukin (IL)-6 contributes to tumor necrosis factor (TNF)-induced lethal shock or whether, on the contrary, it is part of a protective feedback system, remains unresolved . Here, we report experiments with IL-6 gene-disrupted mice (IL-6(0/0)) . We have tested the susceptibility of these to TNF-induced metabolic changes and lethality in different models, and compared the results with those obtained with IL-6+/+ wild-type mice . We studied the response to TNF in three different models: (i) murine TNF administration; (ii) TNF in galactosamine (GalN)-sensitized mice; (iii) TNF in Bacillus Calmette-Guerin-sensitized mice . We observed no significant difference between the two types of mice in any of the three models . Furthermore, IL-6(0/0) mice could be equally well desensitized (by IL-1) to TNF/GalN-induced lethality and tolerized to TNF-induced shock as IL-6+/+ mice . We also observed that, in response to turpentine, TNF or IL-1, IL-6(0/0) mice produced significantly less acute phase proteins (APP) than IL-6+/+ mice . In IL-6(0/0) mice, less corticosterone was induced by TNF than in the control mice, while the response to adrenocorticotropic hormone was the same . The results indicate that IL-6 is not contributing in a major way to the pathogenesis leading to TNF-induced shock, and that neither IL-6 nor the APP studied are essential for a protective feedback system.

Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 162 - 8
On the domain structure of cytochrome P450 102 (BM-3): isolation and properties of a 45-kDa FAD/NADP domain; Black SD; Cytochrome P450 102 is a catalytically self-sufficient monooxygenase isolated from barbiturate-induced Bacillus megaterium . The enzyme contains FAD, FMN, and heme in a single polypeptide chain of 1048 residues, and each of the cofactors is believed to be located in a separate domain . In the present study we have used exhaustive endogenous proteolysis to produce a 45 kDa fragment of the cytochrome . This fragment bound the 2',5'-adenosine diphosphate moiety of NADP(H) strongly, with approximately the same dissociation constant as in the native enzyme, and contained only FAD (0.93 equivalents per polypeptide, epsilon 453nm = 11,200 M-1cm-1) . Reduction of the flavin by sodium dithionite proceeded quite slowly to yield FADH2, but no stable semiquinone species was produced upon air re-oxidation . In contrast, NADPH rapidly reduced this FAD/NADP(H) domain aerobically to produce the FADH . semiquinone radical . At a 75:1 molar ratio of the FAD/NADP(H) domain to the P450 102 heme domain, no laurate hydroxylase activity was observed . Gas-phase sequence analysis showed the presence of two major sequences beginning at Phe646 (403 residues, MW 45,033) and Asp652 (397 residues) . These data are in agreement with the crystal structures of related enzymes and closely define the boundary of the FAD/NADP+ domain in P450 102.

J Biol Chem, 1994 Aug 26, 269(34), 21576 - 82
Oxidative phosphorylation by ADP + P(i)-loaded membrane vesicles of alkaliphilic Bacillus firmus OF4; Guffanti AA et al.; ATP synthesis in ADP + P(i)-loaded membrane vesicles of the facultative alkaliphile Bacillus firmus OF4 at an external pH of 10.5 did not depend upon the presence of cell wall polymers, e.g . as a proton barrier or sequestration device . Upon energization with ascorbate plus phenazine methosulfate, vesicles at pH(out) = 7.5 generated an electrochemical proton gradient (delta p) of -160 mV, acid and positive out, whereas at pH(out) = 10.5, the delta p was -40 mV, alkaline and positive out . Nonetheless, ATP synthesis was more rapid at the more alkaline pH value, especially in the presence of 200 mM K2SO4, which markedly lowered the surface potential . No synthesis was observed upon abolition of the delta p . Respiration-derived transmembrane potentials (delta psi) energized ATP synthesis much better than an equally large diffusion potential . The diffusion potential failed to energize ATP synthesis above pH 9.5 . When delta p, all in the form of a delta psi, was titrated downward at either pH 7.8 or 9.5, ATP synthesis by the latter vesicles was much less adversely affected in the delta p range of -150 to -50 mV, supporting the existence of a sparing, non-chemiosmotic energy component at high pH.

Biochemistry, 1994 Aug 23, 33(33), 9929 - 36
Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp . 1011: effects of replacements on substrate binding and cyclization characteristics; Nakamura A et al.; Three-dimensional structures of cyclodextrin glucanotransferases (CGTases) have revealed that four aromatic residues, which are highly conserved among CGTases but not found in alpha-amylases, are located in the active center . To analyze the roles of these aromatic residues, Phe-183, Tyr-195, Phe-259, and Phe-283 of Bacillus sp . 1011 CGTase were replaced by site-directed mutagenesis, and the effects of this procedure were examined . Y195L-CGTase, in which Tyr-195 was replaced by a leucine residue, underwent a drastic change in its cyclization characteristics: it produced considerably more gamma-cyclodextrin than the wild-type enzyme and virtually no alpha-cyclodextrin . Y195L-CGTase had increased Km values for cyclodextrins, whereas the values for a linear maltooligosaccharide donor were insignificantly changed . Taken together with the structural information of CGTase crystals soaked with substrates, we propose that Tyr-195 plays an important role in the spiral binding of substrate . Replacing either Phe-183 or Phe-259 with leucine induced increased Km values for acceptors . Furthermore, the double mutant F183L/F259L-CGTase had considerably decreased cyclization efficiency, but the intermolecular transglycosylation activity remained normal . These results indicated that Phe-183 and Phe-259 are cooperatively involved in acceptor binding, and that they play a critical role in cyclization when the nonreducing end of amylose binds to the active center of CGTase . Replacing Phe-283 with a leucine residue induced a decrease in kcat and in affinity for acarbose, suggesting that Phe-283 is involved in transition-state stabilization.

Biochim Biophys Acta, 1994 Aug 17, 1207(2), 143 - 51
A new enzyme, maltobionate alpha-D-glucohydrolase, from alkalophilic Bacillus sp . N-1053; Shirokane Y et al.; A new enzyme, maltobionate alpha-D-glucohydrolase, was purified to apparent homogeneity from a cell-free extract of alkalophilic Bacillus sp . N-1053 about 930-fold with a yield of 18% and some of its properties were investigated . The enzyme showed optimum activity at about pH 7.0, and was stable over the range of pH 6.0-9.5 . The molecular weight was estimated to be 152,000 and 71,000 by HPLC gel filtration on TSKgel G3000SWXL and SDS-polyacrylamide gel electrophoresis, respectively . The enzyme hydrolyzed maltobionate more effectively than disaccharides such as maltose and maltitol or trisaccharides such as maltotrionate, maltotriose and maltotriitol, but showed no activity toward polysaccharides such as amylose, amylopectin and soluble starch . The reaction products from 1 mol of maltobionate were found to be 1 mol of beta-D-glucose and 1 mol of D-gluconate . The Km value for maltobionate was 1.63 mM and the Vmax/Km value for maltobionate was the largest among the substrates tested . The enzyme activity was almost completely inhibited by Hg2+, Ag+, iodine and N-bromosuccinimide, and also inhibited by p-nitrophenyl alpha-D-glucoside, maltose and maltitol.

Biochem Biophys Res Commun, 1994 Aug 15, 202(3), 1227 - 34
Production of antibodies against Bacillus thuringiensis delta-endotoxin by injecting its plasmids; Pang AS; Bacillus thuringiensis insecticidal crystalline delta-endotoxin proteins are plasmid encoded . Specific antibodies against the delta-endotoxin were obtained in mice and rabbits by injecting isolated B . thuringiensis DNA plasmids into their muscles . Antibodies could be detected by ELISA as early as two weeks after the first injection . They formed immunoprecipitin lines with the toxin in the Ouchterlony agarose plate and neutralized the toxicity of the toxin . Because no special equipment, delivery mechanism or purification of the protein is required, this technique may provide a way to raise polyclonal antibodies to proteins encoded by bacterial plasmids and recombinant products.

Am J Ophthalmol, 1994 Aug 15, 118(2), 152 - 7
Bacillary angiomatosis of the conjunctiva; Lee WR et al.; A 70-year-old man had unilateral congestion of the right upper eyelid, which contained a nodular mass . A biopsy was performed, and histologic, immunocytochemical, and ultrastructural studies disclosed a pseudoneoplastic proliferation of endothelial cells and pericytes in a region containing clumps of bacteria . This combination of histologic features is characteristic of bacillary angiomatosis, which has been described in the skin, particularly in association with immunodeficient states, especially acquired immunodeficiency syndrome, but not in the conjunctiva . A second biopsy contained a diffuse polyclonal lymphocytic infiltrate in which large lymphocytes with irregular nuclei and mitotic figures were prominent . Systemic examination disclosed mild splenomegaly and a benign paraproteinemia . Treatment with topical gentamicin and systemic erythromycin brought about a complete resolution of the symptoms and signs within eight weeks, and there has been no sign of recurrence for the past two years.

Am J Ophthalmol, 1994 Aug 15, 118(2), 145 - 51
Ophthalmic manifestations of Rochalimaea species; Golnik KC et al.; Rochalimaea henselae and R . quintana belong to the order Rickettsiales and are thought to be responsible for trench fever, bacillary angiomatosis, and cat scratch disease . We recently examined four patients with intraocular inflammation of unknown origin . Each patient had either unilateral or bilateral moderate loss of visual acuity ranging from 20/25 to counting fingers . Bilateral intraocular inflammation manifested by anterior and posterior segment cells, retinal lesions, macular exudate, and optic nerve head swelling was present to varying degrees . The R . henselae to R . quintana antibody titers were greater than or equal to 1:256 in each case . Marked improvement in vision occurred after treatment with either oral ciprofloxacin hydrochloride and prednisone or doxycycline hyclate . Rochalimaea species should be considered in the differential diagnosis of intraocular inflammation and inflammatory optic neuropathy . Appropriate treatment may result in marked improvement in visual acuity.

FEMS Microbiol Lett, 1994 Aug 15, 121(2), 147 - 52
Isolation and characterization of Tn917-generated bacitracin deficient mutants of Bacillus licheniformis; Herzog-Velikonja B et al.; Two Tn917-generated bacitracin deficient mutants of Bacillus licheniformis were isolated . Southern blot analysis of chromosomal DNA extracted from both insertional mutants showed that Tn917 inserted in the vicinity of the gene coding for the enzyme BA2 of the bacitracin synthetase enzyme complex . Measurements of bacitracin synthetase activity in cell-free extracts and positive hybridization signals in the vicinity of the BA2 gene indicate that in both bacitracin deficient mutants Tn917 could be inserted in the BA1 gene or in segments involved in regulation . Thus, it could be possible that the genes for bacitracin synthetase are clustered in the B . licheniformis genome.

FEBS Lett, 1994 Aug 8, 349(3), 420 - 3
Altered chemotaxis of a Bacillus sphaericus L-ethionine-resistant sporulation mutant . A probable link between chemotaxis and sporulation; Andreev J et al.; A UV irradiation-induced mutant of B . sphaericus 2362 whose sporulation was inhibited neither by natural amino acids nor by L-ethionine was selected . The mutant (A61) grew slowly in rich amino acid medium and contained increased concentrations of heat-resistant spores throughout the growth . Slow growth of A61 was related to continuous presence of aging and sporulating cells even when the medium was rich in nutrients . The ability of the mutant to sense nutrient presence in the environment and to relate this information to systems regulating the switch from vegetative growth to sporulation seem to be damaged . A61 also demonstrated impaired chemotaxis . In contrast to the parent strain, only few amino acids elicited chemotactic response in A61 . Methylation of the A61 methyl-accepting chemotaxis protein(s) was lower than that of the parent strain by one order of magnitude . Spontaneous fast-growing phenotypic revertants of A61 displayed sporulation behavior characteristic of B . sphaericus 2362 . Their chemotaxis to amino acids was considerably improved . To some amino acids, it proved to be even stronger than in the original strain, B . sphaericus 2362 . It is suggested, that methyl transfer events originating in the chemotactic system are involved in the triggering of sporulation, the A61 mutation being located in this signalling pathway.

FEBS Lett, 1994 Aug 8, 349(3), 411 - 5
Motility and chemotaxis in Bacillus sphaericus . Dependence upon stage of growth; Andreev J et al.; Chemotaxis and motility of B . sphaericus 2362 were monitored as the function of a batch culture age . It was found that both functions changed independently during growth of the culture . Motility was low until the late logarithmic stage ensued, whereafter it increased sharply . The ability of cells to respond to chemoeffectors peaked at the mid-logarithmic phase . A major methyl-accepting chemotaxis protein (P53, M(r) = 53 kDa) was identified . The extent of label incorporation in this protein from L-{methyl-3H}methionine was maximal in mid- and late logarithmic phases of the growth . Cells in stationary cultures incorporated very low amounts of the label . At any stage, the labeling was maximal in starved cells; it was almost abolished in cells pre-incubated with amino acids . Although extents of P53 labeling in mid- and late logarithmic cells were similar, late logarithmic cells demonstrated a considerably impaired chemotaxis . Supermotile sporulating cells were practically insensitive to environmental stimuli . The difference in development of sensory and locomotive functions may be interpreted as an adaptive response . A well developed sensory apparatus would allow vegetative cells to adapt efficiently to fluctuating attractant gradients . Insensitive sporulating cells would tend to disperse randomly from the nutrient-exhausted area . Thus, spore formation would occur in larger volume of the habitat, increasing the chance of the microbial population to survive.

J Biol Chem, 1994 Aug 5, 269(31), 20167 - 71
Streptomyces griseus protease C . A novel enzyme of the chymotrypsin superfamily; Sidhu SS et al.; In this report we describe a novel chymotrypsin-like serine protease produced by Streptomyces griseus . The enzyme has been tentatively named S . griseus protease C (SGPC) . The gene encoding the enzyme (sprC) was identified and isolated on the basis of its homology to the previously characterized S . griseus protease B (SGPB) . The sprC gene encodes a 457-amino acid prepro-mature protein of which only the 255 carboxyl-terminal amino acids are present in the mature enzyme . Mature SGPC contains two distinct domains connected by a 19-amino acid linker region rich in threonines and prolines . While the amino-terminal domain is homologous to S . griseus proteases A, B, and E and the alpha-lytic protease of Lysobacter enzymogenes, the carboxyl-terminal domain is not homologous with any known protease . However, the carboxyl-terminal domain shares extensive homology with chitin-binding domains of Bacillus circulans chitinases A1 and D, suggesting that the enzyme is specialized for the degradation of chitin-linked proteins . Recombinant expression and preliminary characterization of the catalytic properties of the enzyme are also reported . The primary specificity of SGPC is similar to that of SGPB; both enzymes preferentially cleave peptide bonds following large hydrophobic side chains.

Biochim Biophys Acta, 1994 Aug 2, 1218(3), 432 - 4
Gene structure and amino acid sequences of alcohol dehydrogenases of Bacillus stearothermophilus; Robinson GA et al.; Partial amino acid sequences of the two alcohol dehydrogenases of Bacillus stearothermophilus and the oligonucleotide sequence of a cloned fragment containing the gene for ADH 2334 were determined and compared with the known, derived ADH 1503 amino acid sequence . The two proteins are identical at 244 of 349 positions . ADH 2334 is encoded in a transcription unit containing an aldehyde dehydrogenase.

Appl Environ Microbiol, 1994 Aug, 60(8), 2911 - 5
Purification and characterization of thermostable beta-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost; Sakai K et al.; Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs . The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY . The enzyme specifically hydrolyzed C-4 beta-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives . The enzyme also hydrolyzed pNP-beta-N-acetyl-D-galactosaminide (26% of the activity of pNP-beta-N-acetyl-D-glucosaminide) . These results indicated that the enzyme is a beta-N-acetylhexosaminidase (EC 3.2.1.52) . Kms for acetylchitooligosaccharides were 1 x 10(-4) to 6 x 10(-4) M, while those for the pNP derivatives were 4 x 10(-3) to 8 x 10(-3) M . The optimum temperature of the enzyme was 75 degrees C, and it retained 100 and 28% reactivity after heating at 60 and 80 degrees C, respectively . The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea . The optimum and stable pH was around 6.5 . Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory . N-Acetyl-D-glucosamine inhibited the enzyme competitively (Ki = 4.3 x 10(-4) M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.

FEMS Microbiol Lett, 1994 Aug 1, 121(1), 31 - 4
A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereus causes vacuole formation in HEp-2 cells; Agata N et al.; A HEp-2 cell-vacuolation factor was extracted and purified from the culture supernatant of a Bacillus cereus strain which caused emetic-syndrome food poisoning . The final preparation was chemically pure, and the toxin was named as cereulide . Mass spectrometry, NMR studies and chemical degradation revealed that the cereulide is a cyclic dodecadepsipeptide, (D-O-Leu-D-Ala-L-O-Val-L-Val)3, which is closely related to the potassium ionophore, valinomycin.

Clin Exp Immunol, 1994 Aug, 97(2), 334 - 7
Bacillus Calmette-Guérin (BCG) and immunoglobulins synergistically enhance mineral dust-induced production of reactive oxygen metabolites by human monocytes; Nyberg P et al.; The modulating effect of BCG and polyclonal immunoglobulin on mineral dust-induced production of reactive oxygen metabolites (ROM) by human monocytes was studied using luminol-dependent chemiluminescence . BCG and immunoglobulin synergistically amplified the ROM production induced by chrysotile asbestos and quartz particles, and BCG caused a sharper dose response for poly-immunoglobulin added to the mineral dusts . Immunoglobulins did not affect zymosan yeast-induced ROM production, which was enhanced strongly by BCG . As there is evidence that phagocyte-derived ROM are of importance in mineral dust-induced lung injury, we suggest that the observed synergism between host response inflammatory mediators (poly-immunoglobulin) and exogenic irritants (BCG) may contribute to the outcome of exposure of mineral dusts, and thus in part explain the individual variations in susceptibility to mineral dust-induced diseases.

J Bacteriol, 1994 Aug, 176(15), 4750 - 3
Tn5401 disruption of the spo0F gene, identified by direct chromosomal sequencing, results in CryIIIA overproduction in Bacillus thuringiensis; Malvar T et al.; The Bacillus thuringiensis spo0F gene was identified by chromosomal DNA sequencing of sporulation mutants derived from a B . thuringiensis transposon insertion library . A spo0F defect in B . thuringiensis, which was suppressed by multicopy hknA or kinA, resulted in the overproduction of the CryIIIA insecticidal crystal protein.

J Bacteriol, 1994 Aug, 176(15), 4465 - 72
The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation; Watanabe T et al.; The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme . In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed . The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography . Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides . Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity . Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity . Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions . Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin . Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.

Cell Immunol, 1994 Aug, 157(1), 70 - 80
Interleukin-1 (IL-1) is an important cytokine in granulomatous alveolitis; Denis M; We examined the role of interleukin-1 (IL-1) in promoting an immune-induced lung fibrotic response in a mouse model of granulomatous alveolitis caused by exposure to heat-killed bacillus Calmette-Guerin . Instillation of the material induced an elevated production of IL-1 in the lungs of challenged mice . Lung homogenates from challenged mice contained high levels of antigenic IL-1, and alveolar macrophages from challenged mice released elevated levels of IL-1 . Treatment of mice with a specific monoclonal antibody directed against type-1 IL-1 receptor quite significantly reduced the initial influx of cells, especially neutrophils into the bronchoalveolar lavage . Type-1 IL-1 receptor blockade did not directly alter the levels of tumor necrosis factor alpha in the lung homogenates, but it did lead to an interleukin-6 superinduction in the lungs . IL-1 receptor blockade quite significantly diminished lung tissue damage and granuloma formation, judging from morphometric index and lung hydroxyproline measurements . This diminished tissue damage was also evident on tissue sections stained with Masson's trichrome, as seen by fewer granulomas and less collagen deposition . These data suggest that IL-1 plays an important role in determining a lung granulomatous response.

J Infect Dis, 1994 Aug, 170(2), 476 - 9
Bacille Calmette-Guérin immunization in normal healthy adults; Brewer MA et al.; Bacille Calmette-Guerin (BCG) vaccine was administered to 20 purified protein derivative (PPD) skin test-negative and human immunodeficiency virus serology-negative, healthy adults 18-65 years old . Local adverse reactions were monitored and intermediate-strength PPD skin test responses were evaluated 2 months and 1 year after vaccination . All vaccinees developed erythema, induration, and tenderness at the vaccination site . Muscular soreness was reported by 15 subjects, local ulceration with drainage by 14, and tender regional adenopathy by 2 . BCG organisms were isolated from the ulcer of the only vaccinee who was cultured . PPD skin tests revealed induration in all subjects . Normal healthy adults at risk of occupational exposure to tuberculosis and considering BCG immunization should be fully informed about the local reactions seen after BCG administration and the high rate of persistent PPD skin test responses . Since viable organisms can be recovered from ulcer drainage, vaccination sites should be covered to reduce nosocomial transmission of the vaccine strain.

Virology, 1994 Aug 1, 202(2), 904 - 11
The nucleotide sequence and genome organization of mushroom bacilliform virus: a single-stranded RNA virus of Agaricus bisporus (Lange) Imbach; Revill PA et al.; Mushroom bacilliform virus (MBV) is found in association with spherical virus-like particles in cultivated mushrooms (Agaricus bisporus) afflicted with La France disease . MBV possesses a monopartite ssRNA genome of positive sense and differs from the majority of characterized mycoviruses, which contain segmented dsRNA genomes . We have cloned and sequenced the MBV genome and determined that its length is 4009 nucleotides . The MBV genome contains four major and three minor open reading frames and has 5' and 3' noncoding regions of 60 and 250 nucleotides, respectively . The putative RNA-dependent RNA polymerase and the coat protein display homology with corresponding proteins encoded by certain plant viruses, particularly luteoviruses and carmoviruses.

J Urol, 1994 Aug, 152(2 Pt 1), 562 - 6
Effect of deoxyspergualin on vascular rejection in canine kidney transplantation; Tanabe K et al.; Deoxyspergualin (DSG), an analogue of spergualin produced by Bacillus laterosporus, has a strong immunosuppressive effect in various transplantation models . In this study, we investigated the effect of DSG on vascular rejection in canine kidney transplantation . To enhance vascular rejection, donor-specific blood transfusion (DST) was carried out on days 28, 21 and 14 preceding kidney transplantation . After DST, the donor kidney was transplanted to the recipient iliac fossa . The recipient animals were divided into five groups: namely, Group 1 (n = 7), no treatment; Group 2 (n = 6), DST only; Group 3 (n = 5), DSG only (treated with DSG intravenously at 1.2 mg./kg./day for the first 3 days after transplantation, 1.0 mg./kg./day for the following 3 days and 0.8 mg./kg./day for the following 8 days); Group 4 (n = 6), DST and DSG treatment (same protocol as Group 3); and Group 5 (n = 5), DST and cyclosporine (CsA) (treated with CsA orally at 10 mg./kg./day for 14 days after transplantation) . In Group 2, DST treatment significantly reduced kidney graft survival time (8.6 +/- 2.2 days) compared with Group 1 (14.1 +/- 5.5 days) . Despite DST, DSG treatment (Group 4) significantly prolonged graft survival time (29.5 +/- 2.6 days), whereas treatment with CsA (Group 5) did not prolong survival time (14.1 +/- 5.5 days) (Group 4 versus 5, p < 0.01) . The onset of rejection was significantly delayed in Group 4 (22.1 +/- 2.7 days) compared with Groups 2 (5.7 +/- 2.4 days) and 5 (13.0 +/- 5.7 days) (p < 0.01) . In contrast, the interval between rejection onset and animal death was significantly reduced in Groups 2 (3.0 +/- 0.6 days) and 5 (2.4 +/- 1.0 days) compared with Group 4 (7.3 +/- 1.7 days) (p < 0.01) . These findings suggest that DSG successfully prevented humoral-type (accelerated acute-type) rejections . Histologically, nonDST groups (Groups 1 and 3) showed minimum vascular rejection . In contrast, all recipients in Group 2 showed severe vascular rejection, as did 80% of CsA treated-animals (Group 5) . Despite DST, however, 84% of DSG treated-animals (Group 4) showed minimal or mild vascular rejection and only 17% had severe rejection (Group 4 versus 5, p < 0.04) . These data suggest that both clinically and histologically, DSG has more potent immunosuppressive effects against humoral and vascular rejection than CsA.

J Urol, 1994 Aug, 152(2 Pt 1), 388 - 92
Association of P53 nuclear overexpression and tumor progression in carcinoma in situ of the bladder; Sarkis AS et al.; We investigated the prevalence and clinical relevance of p53 nuclear overexpression, as detected by antibody PAb1801 and immunohistochemistry, in 33 patients with carcinoma in situ of the bladder . Median followup was 124 months . Disease progressed in 16 patients (48%) during followup . The association between p53 nuclear overexpression and tumor progression was assessed by multivariate analysis, controlling for possible confounding variables, such as patient age and sex, presence of associated stage Ta bladder tumor and adjuvant bacillus Calmette-Guerin therapy . Patients were stratified into 2 groups according to the per cent of tumor cells displaying p53 nuclear overexpression: group 1-18 with less than 20% tumor cells positive and group 2-15 with 20% or more tumor cells positive . Disease progressed in 3 patients (16.7%) in group 1 and in 13 (86.7%) in group 2 (p < 0.0001) . Detection of p53 nuclear overexpression in 20% or more tumor cells was the only independent marker of tumor progression in univariate and multivariate analyses (p = 0.004, adjusted relative risk 8.6, 95% confidence interval 2 to 40) . Death specifically from bladder cancer was also associated with this altered pattern of p53 expression (p = 0.01, Fisher's exact test) . We conclude that p53 nuclear overexpression is an early event in bladder cancer, occurring in 48% of cases of carcinoma in situ of the bladder . Our results also suggest that p53 nuclear overexpression offers significant clinical information and may be a useful tool in the selection of therapy for patients with carcinoma in situ of the bladder.

J Urol, 1994 Aug, 152(2 Pt 1), 367 - 73
Durability of the tumor-free response for intravesical bacillus Calmette-Guerin therapy; Nadler RB et al.; The long-term efficacy of bacillus Calmette-Guerin (BCG) has not been established . We describe the tumor-free status of patients 11 years after BCG treatment . Long-term followup for the patient population (mean 74.3 +/- 3.5 months, range 6 to 129) yielded a 28% (29 of 104 patients) tumor-free status for a single 6-week course of BCG . Of 66 patients who received a second 6-week course of BCG for recurrent tumors after failing the initial 6-week course 27 (41%) have remained tumor-free . Overall, 56 of 104 patients (54%) remain tumor-free after 1 or 2 courses of BCG . Analysis of recurrences with respect to 3 intervals (2 or less, 2 to 5 and more than 5 years) revealed recurrence rates of 61% (63 of 104 patients), 23% (7 of 30) and 22% (5 of 23), respectively, after 1, 6-week course of BCG . Similarly, recurrence rates for the same periods for patients receiving a second 6-week course of BCG were 42% (28 of 66), 21% (6 of 28) and 23% (5 of 22), respectively . Patients receiving either 1 or 2, 6-week BCG courses who were tumor-free at 2 years experienced essentially identical recurrence rates during the 2 to 11-year followup (36% and 33%, respectively) . Overall, 23 of 66 patients (35%) who were tumor-free at 2 years had recurrent tumors during the 2 to 11-year followup . We conclude that while BCG is effective therapy for superficial bladder tumors, a continuous potential for tumor recurrence exists for responding patients necessitating life-long followup.

Mol Microbiol, 1994 Aug, 13(4), 569 - 75
Location of a lepidopteran specificity region in insecticidal crystal protein CryIIA from Bacillus thuringiensis; Liang Y et al.; The Bacillus thuringiensis insecticidal crystal protein CryIIA has both high mosquito activity and gypsy moth activity; in contrast CryIIB, which is 87% homologous, displays no mosquito activity and has a three-fold lower gypsy moth activity . The regions responsible for specificity against gypsy moth (Lymantria dispar) and mosquito (Aedes aegypti) larvae were located by introducing MluI and XhoI sites into homologous positions within the putative domain II of both cryIIA and cryIIB genes, which divided almost equally the respective second domains into three regions . Taking advantage of naturally occurring NheI and NarI sites that border the putative domain II, a set of seven chimeric proteins were produced by exchanging all combinations of those regions between CryIIA and CryIIB . Analysis of the toxicity of these chimeric proteins demonstrated that the lepidopteran and dipteran specificity regions of CryIIA were not colinear . While the specificity region of CryIIA against mosquito larvae involved region 1 and probably also region 2, the specificity region of CryIIA against gypsy moth larvae was located within region 2.

Mol Microbiol, 1994 Aug, 13(3), 505 - 12
Regulation of xylose utilization in Bacillus licheniformis: Xyl repressor-xyl-operator interaction studied by DNA modification protection and interference; Scheler A et al.; Xylose utilization in Bacillus licheniformis is inducible by xylose . We establish here that the Xyl repressor recognizes and binds an xyl operator sequence located 12 nucleotides downstream from the transcription start site of the xyl operon . DNA-retardation experiments employing xyl regulatory DNA and soluble protein extracts indicate complex formation in the presence of Xyl repressor . Two repressor-operator complexes are distinguished by different gel mobilities . They yield the same in situ copper-phenanthroline footprint . This result suggests that a single xyl operator may be bound by different oligomers of Xyl repressor . Methylation and hydroxyl radical cleavage protection of the xyl operator by Xyl repressor binding and ethylation interference of Xyl repressor binding to the xyl operator reveals symmetrical interaction of the repressor with two half sites of the operator, which show palindromic symmetry and are located on the same side of the B-form DNA structure.

J Endod, 1994 Aug, 20(8), 377 - 80
Evaluation of the antibacterial effects of intracanal Nd:YAG laser irradiation; Hardee MW et al.; Fifty canals of extracted single-rooted teeth were prepared to a size 50 master apical file, sterilized in ethylene oxide, and inoculated with a known quantity of Bacillus stearothermophilus spores . Five groups of 10 canals each were used . The control group received no treatment . The four treatment groups were exposed to pulsed Nd:YAG laser radiation or 0.5% NaOCl alone and in combination . The root canals were flushed with sterile distilled water to recover spores, and serial dilutions were incubated on blood agar and the number of colony-forming units recovered was determined . Analysis of the data indicated a 2-log reduction in colony-forming units among the four treatment groups as compared with the controls; however, no significant differences were observed among the treatment groups . In none of the treatment groups were the root canals sterilized.

Skeletal Radiol, 1994 Aug, 23(6), 478 - 81
Case report 860: Bacillary angiomatosis of the calcaneum; Omarini LP et al.; Bacillary angiomatosis (BA) is newly reported infectious disease observed mainly in HIV-infected patients, caused by a small gram-negative bacillus of the Rochalimea genus . From a purely dermatological presentation similar to that of Kaposi's sarcoma, it may evolve into a systemic disease . Bone lesions seem fairly frequent . We report a case of an isolated osteolytic lesion due to the BA bacillus.

J Clin Microbiol, 1994 Aug, 32(8), 1930 - 4
Detection of cilia-associated respiratory bacillus by PCR; Cundiff DD et al.; The cilia-associated respiratory (CAR) bacillus is an unclassified, gram-negative, motile bacterium that has been implicated as an etiologic agent of respiratory disease in laboratory rodents . In the present study, approximately 1,200 bases of the 16S rRNA gene from three CAR bacillus isolates were sequenced . CAR bacillus-specific primers were designed on the basis of the 16S rRNA gene sequence and used in a PCR assay . The PCR assay detected as little as 500 fg of purified CAR bacillus DNA . The expected 267-bp DNA fragment was amplified from respiratory tissue of frozen, formalin-fixed, and paraffin-embedded samples from experimentally and naturally infected rats and mice . In contrast, no product was amplified from respiratory tissues of sham-infected experimental animals or animals that were serologically or histopathologically negative for the CAR bacillus . Our findings indicate that this PCR assay is a rapid, specific, and sensitive detection method for the diagnosis of CAR bacillus infection in rats and mice.

Protein Sci, 1994 Aug, 3(8), 1329 - 40
The sequence of a subtilisin-type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability; Volkl P et al.; The hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 degrees C and pH 7.0 . Cell homogenates exhibit strong proteolytic activity within a temperature range of 80-130 degrees C . During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacillus sp . The total DNA sequence of the gene encoding the protease (named "aerolysin") was determined . Multiple sequence alignment with 15 different serine-type proteases showed greatest homology with subtilisins from gram-positive bacteria rather than archaeal or eukaryal serine proteases . Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P . aerophilum subtilisin . This allowed identification of sites potentially contributing to the thermostability of the protein . One common transition put alanines at the beginning and end of surface alpha-helices . Aspartic acids were found at the N-terminus of several surface helices, possibly increasing stability by interacting with the helix dipole . Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.

Antimicrob Agents Chemother, 1994 Aug, 38(8), 1820 - 3
Biological activity of amoebicin m4-A from Bacillus licheniformis M-4; Lebbadi M et al.; Amoebicin m4-A from Bacillus licheniformis M-4 exerts a bactericidal and bacteriolytic action on Bacillus megaterium GR10 . Protein, DNA, and RNA synthesis are inhibited, and the membrane electrical potential of this bacterium is depleted by amoebicin . Synthesis of DNA and RNA by Naegleria fowleri HB-1 is also inhibited . Liposomes constructed from L-alpha-phosphatidylcholine become permeable to ions, low-molecular-weight solutes, and high-molecular-weight polymers after treatment with amoebicin.

Lab Anim Sci, 1994 Aug, 44(4), 305 - 12
Characterization of cilia-associated respiratory bacillus isolates from rats and rabbits; Cundiff DD et al.; Isolates of the cilia-associated respiratory (CAR) bacillus were harvested from the trachea of three naturally infected rats and five naturally infected rabbits and were grown on 3T3 mouse fibroblast cells . Isolates were compared by growth characteristics in mammalian cell culture, by use of protein and DNA analyses, and by experimental infections of BALB/c mice . Examination of CAR bacillus isolates by transmission electron microscopy indicated that organisms from rats and rabbits were similar in appearance and had an acidic mucopolysaccharide layer . In culture, the isolates from rats appeared larger than the rabbit isolates and formed large multiorganism aggregates, whereas isolates obtained from rabbits did not . Protein and antigenic analyses and DNA ribotyping revealed minor differences between isolates but could not be used to distinguish the rat from the rabbit isolates . Mice experimentally inoculated with CAR bacillus of rat origin developed interciliary colonization, seroconverted, and developed microscopic pulmonary lesions . Mice inoculated with isolates of rabbit origin did not display intraciliary colonization, seroconvert, or develop pulmonary disease . The findings of this study indicate that CAR bacillus isolates of rat and rabbit origins may be distinct strains and suggest that, in mice, isolates of rat origin may be more virulent than those of rabbit origin.

Int J Pept Protein Res, 1994 Aug, 44(2), 123 - 9
Peptide synthesis catalyzed by the Glu/Asp-specific endopeptidase . Influence of the ester leaving group of the acyl donor on yield and catalytic efficiency; Bongers J et al.; We recently described a two-step enzymatic semisynthesis of the superpotent analog of human growth hormone releasing factor, {desNH2Tyr1,D-Ala2,Ala15}-GRF(1-29)-NH2 (4), from the precursor, {Ala15,29}-GRF(4-29)-OH (1) . C-Terminal amidation of 1 to form {Ala15}-GRF(4-29)-NH2 (2) was achieved by carboxypeptidase-Y-catalyzed exchange of Ala29-OH for Arg-NH2 . The target analog 4 was then obtained by acylation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR (3) (R = CH3CH2- or 4-NO2C6H4CH2-) catalyzed by the V8 protease . In this paper we report on the use of the recently isolated Glu/Asp-specific endopeptidase (GSE) from Bacillus licheniformis, which is shown to be an efficient catalyst for the segment condensation of 2 and 3 . GSE is more stable than the V8 protease under the conditions employed (20% DMF, pH 8.2, 37 degrees C) . The extent of conversion of 2 into 4 is limited by proteolyses at Asp3-Ala4 and Asp25-Ile26 . However, this proteolysis is virtually eliminated by use of the appropriate ester leaving group, R . A systematic study of the kinetics of the GSE-catalyzed segment condensations of 2 and a series of tripeptide esters, desNH2Tyr-D-Ala-Asp(OH)-OR (3) {R = CH3CH2- (3a), CH3- (3b), ClCH2CH2- (3c), C6H5CH2- (3d), 4-NO2C6H4CH2- (3e)} revealed that rate of aminolysis versus proteolysis, and hence the conversion of 2 into 4, increase with increasing specificity (Vmax/Km) of GSE for the tripeptide ester.(ABSTRACT TRUNCATED AT 250 WORDS)

Anat Rec, 1994 Aug, 239(4), 349 - 59
Porphyrin accumulation in the harderian glands of female Syrian hamster results in mitochondrial damage and cell death; Antolin I et al.; BACKGROUND: The Harderian glands of female Syrian hamsters contain very high concentrations of protoporphyrin (in the range of micrograms per mg of tissue) which accumulate in the tubulo-alveoli of the gland . We have studied the process of synthesis, accumulation, and secretion of this cyclic compound by the secretory cells of the hamster Harderian glands . METHODS: The animals used were female Syrian hamster of 15, 35, 75, 180, and 360 days of age . Items first examined were (1) percentage of the "clear cells," (2) area occupied by intraluminal porphyrins, and (3) histological characteristics of "clear cells" by light and transmission electron microscopy (TEM) . In a second study the total content of porphyrins was determined . Finally, the levels of mRNA for the enzyme aminolevulinate synthase (ALV-S) were measured . RESULTS: In the glands of female hamsters, both the tissue concentration and the intraluminal area occupied by protoporphyrin correlate with the appearance of a special type of cell (clear cells) which show signs of cell degeneration . In addition, the expression of the gene for ALV-S, which is the limiting enzyme in porphyrin production, also parallels the relative number of clear cells . Analyzed under TEM, these clear cells display dilated mitochondria and short and swollen endoplasmic reticulum cisternae . In a late phase of necrosis, the nuclear envelope appears disorganized with scarce chromatin . The mitochondria undergo complete destruction, resulting in electron-dense bacillary formations which progressively coalesce in large and dense areas of protoporphyrin . The cell dies after this accumulation, being secreted by a "cytogen" mechanism . CONCLUSIONS: In view of our results, the Harderian gland of female Syrian hamster may provide a useful model for the study of the mechanism by which the anomalous accumulation of protoporphyrin induces cell damage in human protoporphyria.

Vaccine, 1994 Aug, 12(11), 1041 - 51
Immune response in healthy volunteers vaccinated with killed leishmanial promastigotes plus BCG . I: Skin-test reactivity, T-cell proliferation and interferon-gamma production; Castes M et al.; This study reports the results of a vaccine trial established to study the cellular immune responses in vivo (skin-test reactivity) and in vitro (T-cell proliferation and interferon-gamma production) to both leishmanial and mycobacterial antigens following vaccination of healthy volunteers from a leishmaniasis-endemic area with killed leishmanial promastigotes, with or without BCG (Bacille Calmette-Guerin) . Skin tests were performed using purified protein derivative of tuberculin (PPD) and leishmanial antigen in 692 volunteers, and 208 doubly negative subjects (< or = 7 mm induration) were selected to participate in the trial . The study subjects were divided into four vaccine groups: (A) killed promastigotes plus BCG, (B) BCG alone, (C) killed promastigotes alone, and (D) placebo . Three vaccine doses were administered at 6-10-week intervals . The skin-test responses to PPD and leishmanial antigen were reassessed at 4-6- and 12-18-month follow-ups . The results of this trial demonstrated that the combined vaccine, i.e . killed promastigotes of Leishmania plus BCG, results in the stimulation of an immune response to both leishmania and mycobacterial antigens in a high percentage of vaccines (> 85%), manifested either by skin-test conversion, lymphocyte proliferation and/or interferon-gamma production . This was evident after the first dose of vaccine for lymphocyte proliferation and interferon-gamma production and was maintained for a year after the three doses of vaccine . Group B (which received BCG alone), responded as well as group A to PPD but not as well to leishmanial antigen . The reverse was true for group C which received promastigotes alone . Group A attained a 38% leishmanin skin-test conversion at the 4-6-month follow-up, which was associated with double PPD/leishmanial antigen responder status . In contrast, a 35% skin-test conversion was found at the 12-18-month follow-up in group C (promastigotes alone), but this was not associated with responses to PPD . A significant percentage of conversion was observed in the placebo group at the 12-18-month follow-up, both to PPD (58%) and leishmanial (21%) antigens, which suggests either environmental exposure to mycobacterial or leishmanial antigens during the vaccine trial or, more probably, a response to the repeated leishmanial skin tests . Further studies are required to determine whether the presence of proliferative and/or interferon-gamma responses in the absence of a skin-test response are sufficient indicators of potential vaccine success.

Cardiovasc Surg, 1994 Aug, 2(4), 470 - 3
Surgical treatment of ventricular septal defect and ruptured sinus of Valsalva associated with infective endocarditis; Sugimoto T et al.; Two patients with ventricular septal defect of Kirklin type I and ruptured right coronary sinus of Valsalva associated with infective endocarditis were operated on . Both had bacillus vegetation clinging to the aortic and pulmonary valves and the right ventricular intimal wall around the septal defect . Aortic and pulmonary regurgitation were also found . The surgical approach included vertical incision of the right ventricular outflow tract and pulmonary trunk and transverse aortotomy . The right coronary sinus of Valsalva showed distinct aneurysmal change in one patient . The aortic valve and infected Valsalva sinus were excised in both cases, and the pulmonary valve and right ventricular wall where infection extended thoroughly debrided . The resulting defect, including the ventricular septal defect and excised right Valsalva sinus and aortic annulus, was closed with one patch, and the prosthetic valve inserted in the position of the original aortic valve using this patch as part of the annulus . Both patients had a good postoperative course and are doing well, although slight pulmonary regurgitation persists.

J Antibiot (Tokyo), 1994 Aug, 47(8), 909 - 16
A new methionine antagonist that has antifungal activity: mode of action; Aoki Y et al.; A new antifungal, azoxybacilin (an unusual amino acid with an azoxy moiety) was identified from Bacillus cereus, and its in vitro antifungal activity and mode of action were investigated . Azoxybacilin was active against a broad spectrum of fungi . It was especially active against mycelial fungi, such as Aspergillus, and did not show antibacterial activity . No cross-resistance with antifungals currently on the market was observed . The IC50 values of azoxybacilin antifungal activity against Saccharomyces cerevisiae were significantly greater when amino acids containing sulfur were added to the growth medium, whereas other amino acids were not effective at all . We, therefore, tested the effect of the intermediates involved in the synthetic pathway of these amino acids . The activity markedly diminished when one of the following four intermediates was present in the medium:homocysteine, cysteine, cystathionine or methionine . These four intermediates were the same as those required for the growth of the O-acetylhomoserine sulfhydrylase mutant, S . cerevisiae ONO726, indicating that azoxybacilin would inhibit a step or steps in the sulfur-fixation pathway.

Microbiology, 1994 Aug, 140 ( Pt 8), 1869 - 80
Biochemical characterization of Bacillus thuringiensis cytolytic delta-endotoxins; Koni PA et al.; The entomocidal delta-endotoxins CytA and CytB produced by Bacillus thuringiensis (Bt) subspecies israelensis and kyushuensis respectively showed a similar level of toxicity to mosquito larvae but were not toxic to the larvae of the lepidopteran Manduca sexta . CytA and CytB are also similar in sequence, predicted secondary structure and alpha-helical content, the only obvious difference being a C-terminal fifteen residue 'tail' on CytB . Investigations of the function, if any, of the CytB C-terminal 'tail' showed that this delta-endotoxin is highly expressed and forms inclusions in an acrylstalliferous Bt mutant without the aid of the 20 kDa 'helper' protein from Bt subspecies israelensis which is essential for CytA inclusion formation . After proteinase K treatment, CytA and CytB were processed to virtually the same points in a sequence alignment and were equally haemolytic in vitro . However, the results suggested that unprocessed CytB differs from unprocessed CytA in that the former is not haemolytic.

Immunology, 1994 Aug, 82(4), 584 - 90
Neutralization of transforming growth factor-beta 1 in a mouse model of immune-induced lung fibrosis; Denis M; We examined the contribution of the cytokine transforming growth factor beta 1 (TGF-beta 1) in the inflammatory response and fibrotic reaction in a mouse model of immune-induced lung fibrosis caused by repeated intranasal exposure to heat-killed bacillus Calmette-Guerin (BCG) . Mice received 200 micrograms of BCG 3 days/week for 4 weeks, and simultaneous intraperitoneal injections of a monospecific rabbit antiserum against mouse TGF-beta 1 or a preimmune serum (normal rabbit globulin) . BCG instillations generated a copious release of antigenic TGF-beta 1 in the lungs at 1, 2, 3 and 4 weeks (up to 15 ng/lungs/mouse) . Treatment with anti-TGF-beta 1 antiserum significantly diminished the number of free lung cells recovered by bronchoalveolar lavage (BAL), although the BAL cellular profile was not affected . Moreover, anti-TGF-beta 1 treatment of challenged mice diminished very significantly the total levels of interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) in the lungs of animals challenged with BCG . Histological examination and morphometric analysis of Masson's Trichrome-stained sections and measurements of total lung hydroxyproline levels showed a substantial decrease in lung fibrosis and granulomatous response of challenged mice given anti-TGF-beta 1 . These data argue for a role for TGF-beta 1 in inducing inflammation and lung fibrosis in response to an immune stimulus.

J Biochem (Tokyo), 1994 Aug, 116(2), 357 - 67
Entire nucleotide sequence for Bacillus brevis Nagano Grs2 gene encoding gramicidin S synthetase 2: a multifunctional peptide synthetase; Saito F et al.; Bacillus brevis Nagano grs2 gene, which encodes gramicidin S synthetase 2 (GS2) catalyzing activation and combination of four constituent amino acids of gramicidin S, namely, proline, valine, ornithine, and leucine, has been sequenced . The open reading frame of grs2 gene specifies a 4,450-amino acid protein with a calculated molecular weight of 508,658 . There are four domains with a mean of 1,042 amino acid residues containing a repeated sequence of about 600 amino acids, which is highly homologous to the amino-terminal half of gramicidin S synthetase 1 (GS1) (about 40-50% identity) . Three domains of grs2 protein, excluding the first one, show homology over the entire sequences of 1,042 amino acids, but the first domain only shows homology in the conserved 600-amino acid sequence . The last 300-amino acid sequence of grs2 protein following the fourt domain has no homology with any of the above sequences . Translation products of subcloned fragments containing the third or the fourth domain catalyzed ornithine- or leucine-dependent ATP-32Pi exchange, respectively . These results, together with a previous report on a proline-activation domain indicated that the repeated and conserved domains are the individual activation sites of the constituent amino acids; the activation sites are arranged in the order of peptide elongation on GS2 . Several motifs of grs2 protein are conserved among the multiple domains of peptide synthetases and aminoacyl or acyl adenylate-forming enzymes.

Biosci Biotechnol Biochem, 1994 Aug, 58(8), 1424 - 9
Molecular cloning of a fungal cDNA encoding protein disulfide isomerase; Kajino T et al.; Based on the partial amino acid sequences of a protein disulfide isomerase (PDI) from Humicola insolens, two primers were synthesized for reverse transcriptase mediated polymerase chain reaction (RT-PCR) of a fungal RNA . A 0.2-kbp fragment around the consensus sequence of PDIs was obtained and used as a probe for screening a fungal cDNA library . A cDNA clone of PDI from H . insolens was isolated and encoded a polypeptide consisting of 505 amino acids, which was characterized by a N-terminal signal sequence composed of 20 amino acids, a consensus sequence (WCGHCK) at two positions, and a C-terminal endoplasmic reticulum retention signal (HDEL) . Bacillus brevis harboring an expression plasmid bearing the fungal PDI cDNA was prepared and its culture supernatant showed a significant PDI activity . This indicates that glycosylation of a fungal PDI is not essential for the enzymatic activity related to an interchange of disulfide bonds.

Rev Biol Trop, 1994 Aug, 42 Suppl 2, 105 - 9
{Identification of the corn mosaic virus, a rhabdovirus, in Costa Rica}; Rivera C et al.; Maize mosaic virus (MMV), a rhabdovirus, was identified associated to maize field plants, showing stunting and continuous chlorotic stripes uniformly distributed over the leaf blade . The virus was detected in field samples by agar-gel immunodifussion . Enveloped, bacilliform virus particles were observed by electron microscopy in thin sections of naturally infected leaf tissue.

Am J Dermatopathol, 1994 Aug, 16(4), 355 - 63
On the discriminatory value of anti-HPCA-1 (CD-34) in the differential diagnosis of benign and malignant cutaneous vascular proliferations; Suster S et al.; The staining pattern of monoclonal antibody anti-HPCA-1 (CD-34) was studied in 95 cases of benign and malignant cutaneous vascular proliferations and compared with other vascular endothelium-associated antigenic markers in paraffin-embedded tissues . The proliferating vessels in 22 cutaneous capillary hemangiomas, 8 lobular capillary hemangiomas, and 1 case of papillary intravascular endothelial hyperplasia stained strongly positively for anti-HPCA-1, and the intensity of the reaction was paralleled by that of factor VIII-related antigen (FVIII), Ulex europaeus lectin-1 (UEA), and vimentin (VIM) . The vessels in 10 cases of granulation tissue, 6 cases of cavernous hemangioma, 6 cases of angiokeratoma, 5 cases of angiolymphoid hyperplasia with eosinophilia (epithelioid hemangioma), and 3 cases of bacillary angiomatosis showed a lack of reactivity with anti-HPCA-1 and staining of variable intensity with the other markers . Twenty cases of Kaposi's sarcoma (seven patch, five plaque, eight nodular stage) showed strong labeling with anti-HPCA-1 in small, well-formed vessels scattered among the spindle-cell proliferation, and four of these cases showed focal positivity of scattered spindle cells . Nine cases of cutaneous angiosarcoma, two cases of low-grade epithelioid angiosarcoma, and one case of spindle-cell hemangioendothelioma were negative for anti-HPCA-1 and showed variable reactivity for FVIII and UEA; all cases stained strongly positively for VIM . The results of this study indicate that although anti-HPCA-1 shows a high sensitivity for the staining of normal vascular endothelium, its specificity may be restricted to mature, well-formed vessels, therefore rendering its discriminatory value very limited for the identification of poorly differentiated vascular endothelial neoplasms.

Biochemistry, 1994 Jul 26, 33(29), 8712 - 8
Characterization of a medium wavelength type DNA photolyase: purification and properties of photolyase from Bacillus firmus; Malhotra K et al.; The gene for the apoenzyme of Bacillus firmus photolyase was cloned and sequenced . The enzyme was overproduced in Escherichia coli, purified, and characterized . It has the unique property of having the maximum activity over a wavelength range where all other known photolyases exhibit modest activity . The enzyme contains reduced FAD and methenyltetrahydrofolate and has an absorption and action spectrum peak at 410 nm, and it repairs DNA with a quantum yield of phi approximately 0.75.

Gene, 1994 Jul 22, 145(1), 115 - 20
Sequence analysis of the sbsA gene encoding the 130-kDa surface-layer protein of Bacillus stearothermophilus strain PV72; Kuen B et al.; Bacillus stearothermophilus (Bs) contains a surface-layer (S-layer) protein (SbsA), which forms a hexagonal array on the cell wall . In order to understand the structural/functional relationship of SbsA from Bs PV72, the entire nucleotide (nt) sequence of the sbsA gene was determined from three overlapping fragments . The 3'-end was cloned and expressed in Escherichia coli, whereas the 5'-region was amplified from the genome of Bs PV72 by the polymerase chain reaction using two overlapping fragments . The open reading frame (3684 nt) of sbsA is predicted to encode a protein of 1228 amino acids (aa) . The SbsA is synthesized with a leader sequence of 30 aa . The predicted SbsA aa profile was similar to most other sequenced S-layer proteins, containing more acidic than basic aa (pI 5.1) and a very low amount of sulfur-containing aa . Based on aa sequence data, SbsA has weak homology of with the S-layer proteins from B . sphaericus, Rickettsia rickettsii, B . brevis HPD31 and B . brevis 47 (OWP).

Biochemistry, 1994 Jul 19, 33(28), 8521 - 6
pH-induced conformational transitions of Cry IA(a), Cry IA(c), and Cry IIIA delta-endotoxins in Bacillus thuringiensis; Feng Q et al.; Three protoxins and corresponding delta-endotoxins from Bacillus thuringiensis (BT) were studied by means of circular dichroism spectroscopy and size-exclusion HPLC . At neutral pH, the Cry IIIA toxin exists only as a 65-kDa monomer . The toxins of Cry IA(a) and Cry IA(c) exist both as 66-kDa monomers and as oligomers with apparent molecular masses greater than 220 kDa . At neutral pH, interconversion between monomer and oligomer is slow, and the two, separate forms exist for several days . Equilibration between the monomer and oligomer in both Cry IA(a) and Cry IA(c) toxins is facilitated by increasing the pH of the solutions to above 10 . The relative amounts of monomer and oligomer depend upon temperature, pH, and buffer composition . CD spectra of the protoxins and toxins indicate a large helix content . The CD spectra of HPLC-isolated, monomeric Cry IA(a) and Cry IA(c) are quite similar, but are different from the spectrum of Cry IIIA . The Cry IA(a) and Cry IA(c) protoxins exhibit more helical CD spectra than the corresponding toxins . The CD spectra, with the pH titration of Cry IA(a), reveal that there is a significant increase in helical content as the pH is changed from neutral to alkaline values, but no decrease at low pH . A similar titration of Cry IIIA revealed no significant change in structure from pH 6 to pH 11 . The CD spectrum of Cry IIIA at pH 2 indicates that the helical content of the toxin has significantly decreased . The magnitude of the 222-nm signal decreases from pH 7 to 2, with a midpoint of approximately pH 4.5.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1994 Jul 18, 348(3), 249 - 54
Functional analysis of block 5, one of the highly conserved amino acid sequences in the 130-kDa CryIVA protein produced by Bacillus thuringiensis subsp . israelensis; Nishimoto T et al.; There are five amino acid sequences highly conserved among Bacillus thuringiensis delta-endotoxins . We have changed the amino acid residues in block 5, one of the conserved sequences, of CryIVA . When the amino acid residues with charged side chains were replaced by others, the amount of production of the altered CryIVA protein was markedly decreased . It is suggested that the decrease is caused by the unstable conformation of the altered CryIVA protein molecule, as judged by digestion with trypsin and thermolysin . On the other hand, the substitution of amino acid residues in block 5 did not affect the insecticidal activity of CryIVA . These results strongly suggest that block 5 of CryIVA is one of the stability-determining elements of the protoxin molecule.

J Mol Biol, 1994 Jul 15, 240(3), 267 - 70
Crystallization and preliminary X-ray diffraction analysis of a type I beta-glucosidase encoded by the bgIA gene of Bacillus polymyxa; Sanz-Aparicio J et al.; The enzyme encoded by the bgIA gene of Bacillus polymyxa, a type I beta-glucosidase belonging to family I of glycosyl hydrolases, has been purified to homogeneity from an Escherichia coli culture which overexpressed the gene, and crystallized . The crystals, which diffract to 3.0 A resolution, belong to the orthorhombic space group C222(1) . The cell dimensions are a = 155.4 A, b = 209.4 A, c = 209.7 A.

Biochemistry, 1994 Jul 12, 33(27), 8367 - 74
Are D- and L-chiro-phosphoinositides substrates of phosphatidylinositol-specific phospholipase C?
Bruzik KS, Hakeem AA, Tsai MD.
Derivatives of chiro-inositol have been recently shown to mediate many important biological processes . This work addresses the question of whether phosphatidylinositol-specific phospholipase C (PI-PLC) could be involved in the generation of these chiro-inositol derivatives . Two diastereomers of the analog of phosphatidylinositol containing 1D- and 1L-chiro-inositol have been synthesized . 1D-2-O-(1,2-O-Dipalmitoyl-sn-glycero-3-phospho)-chiro-inositol (1D-chiro-PI) was synthesized in 12 steps starting from 1D-2,3,4,5-O-tetrakis(methoxymethylene)-myo-inositol by the inversion of the hydroxyl group at the 1-position of inositol followed by several protection/deprotection and phosphorylation steps . IL-2-O-(1,2-O-Dipalmitoyl-sn-glycero-3-phospho)-chiro-inositol (1L-chiro-PI) was synthesized in eight steps starting from 1L-chiro-inositol using regioselective silylation of the hydroxyl group at the 2-position of chiro-inositol in a key synthetic stage . Both diastereomers were subjected to cleavage by PI-PLC from Bacillus thuringiensis . The reaction of 1L-chiro-PI produced chiro-inositol 1,2-cyclic phosphate, however, at the rate of 10(-3) of that attained with the natural substrate, phosphatidylinositol . On the other hand, 1D-chiro-PI was found to be resistant to PI-PLC . These results suggest that the natural chiro-inositol derivatives should have the 1L-configuration if they are produced by PI-PLC, which is in contrast to the 1D-configuration reported by others . We therefore have isolated chiro-inositol from the total bovine liver lipid and determined its absolute configuration . The obtained chiro-inositol was found to be exclusively of the 1L-configuration, with the enantiomeric purity exceeding 99%.

Hinyokika Kiyo, 1994 Jul, 40(7), 575 - 9
{Intravesical bacillus Calmette-Guerin therapy for superficial bladder cancer}; Yamada Y et al.; Intracavitary instillation of Tokyo 172 strain Bacillas Calmette-Guerin was performed on 39 patients with superficial bladder cancer after contact Nd:YAG laser irradiation for tumors . The BCG group received intravesical instillation of 80 mg BCG at two week intervals for 6 months . Recurrence occurred in 7 of the 39 patients . In the 7 recurrent cases instillation of BCG had been discontinued after 2-7 instillations due to bladder irritation, with recurrence seen 6-27 months later . The non-recurrence rate in the group (27 cases) instilled BCG more than seven times was 94.0% . The non-recurrence rate in this group was significantly higher than that in the group (12 cases) with less than six BCG instillations . The non-recurrence rate in the group (26 cases) without BCG therapy was not significantly different from that in the group (12 cases) with less than six BCG instillations . Our findings suggested that frequent (more than seven times) instillation of BCG increased the non-recurrence rate, and that less than six BCG instillations is not significantly effective for preventing the recurrence of superficial bladder cancer.

Appl Environ Microbiol, 1994 Jul, 60(7), 2304 - 10
Expression of the crystal protein gene under the control of the alpha-amylase promoter in Bacillus thuringiensis strains; Chak KF et al.; The expression of an insecticidal crystal protein gene of Bacillus thuringiensis under the control of the alpha-amylase gene promoter was investigated . The cryIC gene, which encodes a protein known to have a unique activity against Spodoptera (armyworm) species, was used in this investigation . The cryIC gene was placed, along with the alpha-amylase promoter from B . subtilis, in a B . thuringiensis-derived cloning vector, generating a pair of recombinant plasmids, pSB744 and pSB745 . The cloning vector that contains the minimal replicon of B . thuringiensis subsp . kurstaki HD73 is stably maintained in a variety of B . thuringiensis strains, as previously reported by Gamel and Piot (Gene 120:17-26, 1992) . The present study confirmed that the recombinant plasmids are also stably maintained in B . thuringiensis subsp . kurstaki Cry-B and HD73 growing in media without selection pressure for at least 48 h . The cryIC gene on the recombinant plasmids were notably expressed at high levels in both recombinant strains . Expression of the introduced cryIC gene on the recombinant plasmid in B . thuringiensis subsp . kurstaki HD73 did not impair expression of the resident cryIA(c) gene . The CryIA(c) protein is known to have a high level of activity against loopers such as Trichoplusia ni (the cabbage looper) . As a result of coexpression of the introduced cryIC gene and the resident cryIA(c) gene, recombinant strain HD73 acquired an additional insecticidal activity against Spodoptera exigua (the beet armyworm) whereas the original activity level against T . ni was maintained.

Cancer Immunol Immunother, 1994 Jul, 39(1), 49 - 52
Local bacillus Calmette-Guérin therapy for bovine vulval papilloma and carcinoma; Hill FW et al.; Thirty cows from a pedigree Friesian dairy herd with bovine vulva papilloma and carcinoma were treated by intralesional injections of live bacillus Calmette-Guerin (BCG) . This treatment induced total regression of all of six carcinomas . Whilst, after treatment, limited regression was also observed in advanced papillomas, BCG has little or no effect on the early stages of papillomas . This is the first study of BCG therapy in this type of cancer.

Br J Urol, 1994 Jul, 74(1), 44 - 6
Rapid tumour recurrence following cessation of long-term treatment with intravesical thiotepa; Mukamel E et al.; OBJECTIVE: To report tumour recurrence related to cessation of long-term therapy with thiotepa . PATIENTS AND METHODS: A group of 12 patients with low grade (I-II), low stage (TA-T1) transitional cell carcinoma of the bladder were included in the study . All patients had been treated with intravesical thiotepa for a period ranging from 24 to 71 months and none had had a recurrence for a period of between 15 and 51 months . After referral to this department all the patients were withdrawn from thiotepa therapy . RESULTS: All the patients developed bladder tumours within 6 months of cessation of therapy . The recurrent tumours were grade II in 10 patients and grade III in two patients . Eight patients had stage TA and four had stage T1 . All responded to bacille Calmette-Guerin therapy and none had tumour recurrence on follow up at 24 months . CONCLUSION: Meticulous follow-up of patients is indicated soon after the cessation of long-term therapy with thiotepa.

EMBO J, 1994 Jul 1, 13(13), 2976 - 84
Female sterile tobacco plants are produced by stigma-specific cell ablation; Goldman MH et al.; We identified a tobacco stigma-specific gene, designated STIG1 . The STIG1 gene is developmentally regulated and expressed specifically in the stigmatic secretory zone . We used a chimeric STIG1-GUS gene to show that the stigma-specific STIG1 gene expression pattern is controlled primarily at the transcriptional level . We constructed a stigma-specific cytotoxic gene by fusing the STIG1 gene 5' regulatory region with the coding sequence of the Bacillus amyloliquefaciens barnase gene, to assess the role of the stigmatic secretory zone in the pollination process . Pistils of transgenic STIG1-barnase tobacco plants undergo normal development, but lack the stigmatic secretory zone and are female sterile . Pollen grains germinate on the ablated 'stigmatic' surface, but are unable to penetrate the transmitting tissue of the style . Application of stigmatic exudate from wild-type pistils to the ablated surface increases the efficiency of pollen tube germination and growth and restores the capacity of pollen tubes to penetrate the style . Our data demonstrate the importance of the stigmatic secretory zone in the pollination process and provide an approach to identify compounds produced by the stigma that are critical for successful pollination and fertilization to occur.

Biochem J, 1994 Jul 1, 301 ( Pt 1), 199 - 203
Direct n.m.r . evidence for substrate-induced conformational changes in a beta-lactamase; Jamin M et al.; Cefoxitin and other beta-lactam antibiotics with a methoxy group on the alpha-face behave as very poor substrates of the Bacillus licheniformis beta-lactamase . The kinetic properties of the enzyme-cefoxitin system made it theoretically suitable for a detailed structural study of the acyl-enzyme . Unfortunately, soaking the crystals in cefoxitin solution did not allow detection of a crystalline acyl-enzyme complex . In contrast, direct observation by n.m.r . of the stable acyl-enzyme formed with cefoxitin and moxalactam indicated clear modifications of the enzyme structure, which were reflected in the aromatic and high-field methyl regions of the spectrum . The return to the initial free enzyme spectrum was concomitant with the hydrolysis of the acyl-enzyme, the process being slow enough to allow multidimensional n.m.r . experiments.

Neurology, 1994 Jul, 44(7), 1312 - 6
Rochalimaea antibodies in HIV-associated neurologic disease; Schwartzman WA et al.; Rochalimaea henselae, a recently described pathogen thought to cause syndromes as varied as bacillary angiomatosis, parenchymal bacillary peliosis, fever with bacteremia, and cat-scratch disease, is associated with CNS diseases including cerebral and retinal bacillary angiomatosis, as well as cat-scratch-related encephalitis, myelitis, cerebral arteritis, and retinitis . We used a newly developed enzyme immunoassay and the polymerase chain reaction to investigate the association of R henselae infection with HIV-related CNS disease and found that whereas seroprevalence rates in HIV-positive patients unselected for neurologic disease were 4% to 5.5%, those with neurologic disease had seroprevalence rates of 32% . The ratio of organism-specific antibodies in CSF compared with serum suggested intra-blood-brain-barrier synthesis of these antibodies . CSF specimens containing only R henselae IgM had 16S rDNA specific for R henselae . Stored serum from one of these patients indicated he had developed R henselae-reactive IgM antibodies 10 months prior to the onset of neurologic disease . In the 14 patients for whom clinical data were available, evidence of CNS invasion by R henselae was accompanied by acute and subacute mental status changes including hallucinations, disorientation, and rapidly progressive dementia.

J Leukoc Biol, 1994 Jul, 56(1), 10 - 4
Effect of thioglycollate and BCG stimuli on glucose and glutamine metabolism in rat macrophages; Costa Rosa LF et al.; The macrophage is a differentiated cell that takes part in a wide range of physiological and pathological processes, such as inflammatory and immunological responses . The stimulation of macrophages involves the acquisition of some functional characteristics such as phagocytosis, ability to kill tumor cells, and processing and presentation of antigens . In this study we have investigated the changes in macrophage glucose and glutamine metabolism as induced by stimulation by thioglycollate (inflammatory) and bacille Calmette-Guerin (BCG) (activated) . The results showed an increase in hexokinase and citrate synthase activities, due to both stimulatory processes . One difference between inflammatory and activated cells is the higher rate of glucose oxidation in the latter . The activation with BCG, however, also led to an enhancement in glutamine metabolism that is not observed in the inflammatory cell . These results suggest that glutamine metabolism might be important for macrophage function during immunological response.

Plasmid, 1994 Jul, 32(1), 10 - 8
Nucleotide sequence and analysis of an insertion sequence from Bacillus thuringiensis related to IS150; Smith GP et al.; A 5.8-kb DNA fragment encoding the cryIC gene from Bacillus thuringiensis (Bt) subsp . aizawai HD229 was subcloned into the pMex7 vector for expression in Escherichia coli . In addition to the 135-kDa CryIC delta-endotoxin, this DNA fragment also encoded a 30-kDa polypeptide whose open reading frame (orfX) was located less than 200 bp upstream of cryIC . Nucleotide sequencing showed that orfX was truncated at the 5' end, and full sequence was obtained from a second overlapping clone . Sequence analysis showed that orfX could encode a polypeptide closely related to the putative transposase from IS150 . OrfX was flanked by a 17-bp imperfect inverted repeat, defining the length of the element as 998 bp . Southern blot analysis revealed that the novel insertion sequence was present in a single copy and located in an identical position immediately upstream of cryIC in plasmid DNA from both Bt subsp . aizawai and entomocidus.

Mol Biochem Parasitol, 1994 Jul, 66(1), 105 - 10
Cloning and characterization of a cDNA encoding phosphofructokinase from Schistosoma mansoni; Ding J et al.; Schistosoma mansoni, a human parasitic worm, depends on anaerobic glycolysis as the main source of energy . Phosphofructokinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11; PFK) limits the rate of glycolysis in these organisms and it has been found to be a target for some antischistosomal agents . A cDNA clone from this parasite has been isolated and characterized . The cDNA is 3046 base pairs long, contains an open reading frame of 2346 bp and codes for a deduced protein of 781 amino acids . The putative protein encoded by the clone has an exact match with the human muscle PFK of 58% and a 73% match when conserved amino acid substitutions are considered . ATP and Fructose-6-P sites have been identified by crystallographic data in the Escherichia coli and Bacillus stearothermophilus PFKs . There is excellent homology between those PFKs and the schistosome PFK at those sites . The PFK-coding cDNA was expressed in insect cells and was shown to be enzymatically active . Western blot analysis of the recombinant protein in cell extracts gave a positive band with the expected molecular weight of 86 kDa.

Mol Microbiol, 1994 Jul, 13(1), 97 - 107
Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis; Agaisse H et al.; The promoter region of the cryIIIA toxin gene of Bacillus thuringiensis is composed of at least three domains: an upstream region extending from nucleotide positions -635 to -553 (with reference to the translational start codon of cryIIIA), an internal region extending from nucleotide positions -553 to -367, and a downstream region extending from nucleotide position -367 to +18 . Deletion analysis and transcriptional fusions to the lacZ gene indicate that full expression of cryIIIA requires the association of the upstream and the downstream region . Primer extension experiments reveal a major cryIIIA transcript (designated T-129) starting at nucleotide position -129 and another transcript (designated T-558) starting at nucleotide position -558 . Mutation in the -35 region of the promoter responsible for the initiation of T-558 indicates that the upstream promoter is essential for full expression of cryIIIA, although not sufficient . Deletion of the DNA region carrying the previously described cryIIIA promoter does not affect full expression of cryIIIA and does not modify the 5' end of T-129 . Taken together, these results indicate that the 5' end of T-129 is not a trnascriptional start site . Therefore, we propose that T-129 results from the processing of the mRNA initiated at the upstream promoter (T-558), generating a stable mRNA with a 5' extremity at nucleotide position -129 . From primer extension analysis and transcriptional fusions to lacZ, it appears that the upstream promoter is weakly but significantly expressed during the vegetative phase of growth, is activated at the onset of sporulation and remains active at least until t5 . However, unlike the promoters of other cry genes, this promoter is similar to sigma A-dependent promoters rather than sporulation-specific promoters . This promoter may therefore be transcribed by the E sigma A form of RNA polymerase . Activation at the onset of sporulation could result from the disappearance of a repressor, or the appearance of a stationary-phase-specific activator.

Mol Microbiol, 1994 Jul, 13(1), 161 - 9
A small (2.4 Mb) Bacillus cereus chromosome corresponds to a conserved region of a larger (5.3 Mb) Bacillus cereus chromosome; Carlson CR et al.; We have determined the sizes of the chromosomes of six Bacillus cereus strains (range 2.4-4.3 Mb) and constructed a physical map of the smallest B . cereus chromosome (2.4 Mb) . This map was compared to those of the chromosomes of four B . cereus strains and one B . thuringiensis strain previously determined to be 5.4-6.3 Mb . Of more than 50 probes, 30 were localized to the same half of the larger B . cereus and B . thuringiensis chromosomes . All 30 were also present on the small chromosome . Twenty of the probes present on the other half of the larger chromosomes were either present on extrachromosomal DNA, or absent from the B . cereus strain carrying the small chromosome . We propose that the genome of B . cereus/B . thuringiensis has one constant part and another less stable part which is more easily mobilized into other genetic elements . This part of the genome is localized to one region of the chromosome and may be subject to deletions or more frequent relocations between the chromosome and episomal elements of varying sizes up to the order of megabases.

Biochem Mol Biol Int, 1994 Jul, 33(4), 723 - 8
A study on the mechanism of polymerisation of Bacillus brevis flagellin; Novikov VV et al.; Optical properties of intact flagellin and of modified-on-tyrosine flagellin incapable of self-assembly have been studied by the circular dichroism (CD) method . The CD spectra of both flagellins do not change and virtually coincide within the pH range from 2.9 to 10.0 . In the presence of polyethylene glycol (PEG) and ammonium sulfate which accelerate flagellin polymerization, the character of the CD spectra changes and depends on pH . The increase in the PEG content to 20% and that in the ammonium sulfate content to 1 M over the pH range from 4.3 to 10.0 results in a significant rise of the molar ellipticity at 222 nm ({theta}222) of both flagellins . However, {theta}222 does not reach values typical for bacterial flagella . The results obtained are discussed with respect to conformational changes in the flagellin molecule during polymerization.

Antimicrob Agents Chemother, 1994 Jul, 38(7), 1651 - 4
Clinical trial of fusidic acid for lepromatous leprosy; Franzblau SG et al.; Fusidic acid was assessed for antileprosy activity in nine lepromatous leprosy patients . Patients received fusidic acid at either 500 mg/day for 12 weeks or 750 mg/day for 4 weeks followed by 500 mg/day for 8 weeks . All patients showed time-dependent clinical improvement and decreases in bacillary morphological index, radiorespirometric activity and PCR signal, and in serum phenolic glycolipid I . Fusidic acid appears to be a weakly bactericidal antileprosy agent which may have a role in the multidrug treatment of leprosy pending an evaluation of lepra-reaction-suppressive activity.

Infect Control Hosp Epidemiol, 1994 Jul, 15(7), 497 - 9
Pros and cons of BCG vaccination in countries with low incidence of tuberculosis; Tala EO et al.; Preventive bacille Calmette-Guerin (BCG) vaccination, together with case finding and effective chemotherapy, has formed an integral part of the tuberculosis (TB) control program in most countries . In some low-incidence countries the balance of prevention has been more on the side of chemoprophylaxis than of BCG vaccination . The time clearly has come when the strategy of mass BCG vaccination no longer is indicated medically, nor is it cost-effective . The pros and cons of the programs need to be critically evaluated against the present epidemiological background, taking into account the facts that TB, the killer disease, is recovering strength, human immunodeficiency virus infection is on the increase, and multidrug-resistant TB has changed the outcome of this previously fully curable disease . Although no longer appropriate for mass programs, BCG vaccination still should be considered for the protection of selected risk groups in low-incidence countries . The overall efficacy may be of the order 50% to 80%, but the variation is great . Therefore, further research urgently is needed on the effectiveness of BCG as an intervention in local TB programs.

Immunology, 1994 Jul, 82(3), 445 - 9
Antigen 85C on Mycobacterium bovis, BCG and M . tuberculosis promotes monocyte-CR3-mediated uptake of microbeads coated with mycobacterial products; Hetland G et al.; The uptake in monocytes of monodispersed latex microbeads precoated with whole bacillus Calmette-Guerin (BCG) cells, Mycobacterium tuberculosis sonicate or culture fluid, or antigen (Ag) from the culture fluid was examined by microscopy . There was a significantly higher cell association of beads coated with whole BCG cells, the secreted 85C component of the Ag 85 complex or M . tuberculosis sonicate than of phosphate-buffered saline (PBS)-treated control beads . Antibodies (Ab) to Ag 85 inhibited the uptake of BCG- and Ag 85C-treated beads . A monoclonal antibody (mAb) to complement receptor type 3 (CR3), but not mAb to CR1, inhibited the uptake of Ag 85C-coated beads, indicating that the mycobacterial Ag-dependent uptake of particles was mediated via CR3 on the monocytes . This points to the existence of a ligand on Ag 85C which may promote monocyte uptake of M . bovis, BCG and M . tuberculosis.

Immunology, 1994 Jul, 82(3), 361 - 4
Role of interleukin-6 in the induction of protective T cells during mycobacterial infections in mice; Appelberg R et al.; Interleukin-6 (IL-6) has been shown to regulate numerous functions of the immune system including the differentiation of T-cell subpopulations . Here we examined the involvement of this cytokine in the in vivo generation of a population of T cells able to protect mice against mycobacterial infections . BALB/c mice were infected intravenously with Mycobacterium avium 2447 and anti-IL-6 monoclonal antibodies were administered intraperitoneally throughout the course of the infection . Control mice were able to control the mycobacterial proliferation 1 month after inoculation, whereas mice whose IL-6 had been blocked showed progressive bacterial growth . To distinguish a role for IL-6 associated to the induction or expression of immunity mediated by T cells, we immunized mice with M . bovis bacillus Calmette-Guerin (BCG) Pasteur and challenged them 2 months later with M . avium . One group of mice received anti-IL-6 during the BCG vaccination and another during the M . avium challenge . When M . avium proliferation was assessed at day 30 of the challenge, it was found that the administration of anti-IL-6 during vaccination reduced the protection afforded by BCG compared to administration of the isotype control antibody . No difference in bacterial proliferation was observed at day 30 of challenge when antibodies were administered during M . avium challenge . Our results show that protective T cells arise during M . avium infections in mice after differentiating in the presence of IL-6.

Bioconjug Chem, 1994 Jul-Aug, 5(4), 357 - 63
Monoclonal antibodies to thioguanine: influence of coupling position on fine specificity; Nerstrom VM et al.; Thioguanine derivatives with reactive ester groups at positions 6, 7, or 9 of the purine ring were synthesized and coupled to a protein carrier . The purified protein derivative of tuberculin was used as the carrier for immunizing bacillus Calmette-Guerin primed mice . This led to high antibody titers against the homologously coupled hapten, and spleen cells from the immunized mice were used to produce monoclonal antibodies against thioguanine . All monoclonal antibodies were selected for their ability to recognize free thioguanine and were analyzed for their fine specificity by inhibition experiments with a panel of thiopurine derivates . The specificity of the monoclonal antibodies showed a strong dependence on the coupling position of the thioguanine . Within each group of monoclonal antibodies, raised against one of the three different conjugates, there was a high degree of heterogeneity, with antibodies differing in their binding according to the substitution on the thioguanine analogues used in the inhibition experiments . This panel of antibodies may be used for quantitative assays of thiopurines and their metabolites in patients undergoing treatment with thioguanine, 6-mercaptopurine, and azathioprine.

Pediatr Hematol Oncol, 1994 Jul-Aug, 11(4), 427 - 32
Pulmonary sequestration in a child with acute myeloid leukemia; Strada S et al.; The article describes a relatively rare congenital anomaly that was difficult to diagnose in a 10-year-old child with acute nonlymphoblastic leukemia . Just at diagnosis of leukemia, the patient showed a pathologic chest radiograph because of a parenchymal thickening at the right lung apex . The presence of bronchopneumonia was suspected, and broad-spectrum antibiotic therapy was started with subsequent antifungal treatment for persistent fever and concurrent chemotherapy-induced marrow aplasia, which did not favor pulmonary infiltrate recovery . Continuous culture tests, including bronchial swab, proved negative for Koch-Weeks bacillus, fungal organisms, and other pathogens . Computed tomography, however, was suggestive of Aspergillus lung involvement, and apical segmentectomy was performed . The anatomic pathologist suggested the diagnosis of intralobar sequestration . In summary, when pulmonary pathology with an excavation is found in a leukemic child, one must consider the possibility of pulmonary sequestration complicated by an infectious disease.

J Am Optom Assoc, 1994 Jul, 65(7), 472 - 9
Detection of the new tuberculosis: ocular examination as a diagnostic imperative; Frankel RM et al.; BACKGROUND: Until recently, tuberculosis in the U.S . had been considered a public health concern of the past, largely conquered by therapy devised in the 1950s . However, in the past several years, the incidence of tuberculosis has increased steeply and unexpectedly, owing to a conspiracy of new factors . These include: the epidemic of acquired immune deficiency, the emergence of drug-resistant strains, the confinement of susceptibles in crowded shelters, and the premature demolition of public health programs . METHODS: A dynamic interplay exists between the tubercle bacillus and the declining immune system in AIDS patients . Because of this, many atypical, clinical presentations of tuberculosis have emerged . RESULTS: The detection of ocular manifestations of tuberculosis has thus assumed increased importance . It can allow not only for an earlier diagnosis of TB, but an earlier diagnosis of AIDS, preventing spread of both diseases within the population . CONCLUSIONS: Evaluation of ocular signs of tuberculosis should now be a diagnostic imperative.

J Appl Bacteriol, 1994 Jul, 77(1), 9 - 13
A comparison of ELISA and RPLA for detection of Bacillus cereus diarrhoeal enterotoxin; Day TL et al.; Fourteen strains of Bacillus cereus isolated from different sources were examined for their ability to produce diarrhoeal enterotoxin by two commercial immunoassay kits (Oxoid BCET-RPLA and Tecra ELISA) and the microslide immunodiffusion assay . One strain that was positive in monkey feedings, as well as a number of other strains isolated from diarrhoeal outbreaks, gave positive results in the ELISA and negative results in the RPLA test systems . When tested in the microslide assay, these strains produced only one antigen which formed a line of identity with the reference toxin . The results of the control toxins provided with the kits substantiated that the two commercial assays did not detect the same antigen . Cultures positive with both assay kits were shown to produce diarrhoeal enterotoxin (by a line of identity) and other antigens in the microslide immunodiffusion assay.

Jikken Dobutsu, 1994 Jul, 43(3), 389 - 94
Detection of Bacillus piliformis by specific amplification of ribosomal sequences; Goto K et al.; In an effort to explore a sensitive species-specific detection system using the polymerase chain reaction (PCR) for B . piliformis, we sequenced 16S ribosomal DNA (rDNA) of the organism (MSK strain) isolated from the mouse and compared it with known rDNA sequences of the RJ strain isolated from the rat . Sequence homology between the MSK strain and the RJ strain was over 97%, but homology between the MSK strain and other bacterial species was less (70-83%) . The results indicated that the sequences included B . piliformis species-specific regions . On the basis of the sequences, we designed a PCR primer set which amplifies B . piliformis rDNA specifically . The PCR with the primer set detected not only these two strains but also an HN strain of hamster origin, although it did not detect other organisms . Therefore, this primer set was considered to be specific for B . piliformis species . More than one organism (RJ strain) could be detected by the PCR method . Nine Jcl:Wistar rats were infected perorally with 2x10(4) RJ strain organisms, and three rats each were sacrificed on days 1, 3 and 5 postinoculation (p.i.) to investigate the presence of the organism in the liver, heart, cecum, spleen and mesenteric lymph nodes by PCR and the immunofluorescence test . On days 1 and 3 p.i., B . piliformis was not detected in any tissues of the six rats, but B . piliformis was detected in two of the three rats sacrificed on day 5 p.i . The presence of the pathogen was seen in both liver and heart (1/3), or in the cecum (1/3) by both methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Bioorg Med Chem, 1994 Jul, 2(7), 691 - 5
Solvent isotope effects and the nature of electrophilic catalysis in the action of the lactate dehydrogenase of Bacillus stearothermophilus; Xie M et al.; Deuterium oxide at atom fractions of deuterium from 0.0 to 0.97 has an effect of less than 20% on the kinetic term kcat/KmB (believed to reflect the transition state for the hydride-transfer step) for the reduction of pyruvic acid by NADH at 55 degrees C, with catalysis by the tetrameric form of the lactate dehydrogenase of Bacillus stearothermophilus . This observation suggests that the hydride-transfer event is not assisted by protonic bridging to the carbonyl group being reduced . The results are consistent with protonic bridging only if an opposing isotope effect is present, for example from a generalized conformation or solvation change . The results are consistent with other forms of electrophilic catalysis.

Bioorg Med Chem, 1994 Jul, 2(7), 605 - 7
A simple method for determination of stereospecificity of aminotransferases for C-4' hydrogen transfer of the coenzyme; Nishimura K et al.; A simple method was established for determination of the stereospecificity of C-4' hydrogen transfer of the coenzymes (pyridoxal and pyridoxamine) . The method is based on the findings that aspartate aminotransferase of pig heart and D-amino acid aminotransferase of Bacillus sp . YM-1 catalyze the abstraction of the pro-S and pro-R proton at C-4' of pyridoxamine, respectively . Pyridoxal is a poor coenzyme, but readily released from the enzyme . It reacts in 3H2O with a substrate amino acid and an apo-aminotransferase whose stereospecificity for C-4' hydrogen transfer is to be determined . The resultant pyridoxamine which is tritiated at C-4' is incubated with an apo form of aspartate aminotransferase or D-amino acid aminotransferase and a substrate, alpha-keto acid . The stereospecificity for the C-4' hydrogen transfer examined is determined by measurement of radioactivity retained in the pyridoxal formed . We showed by means of this method that C-4' hydrogen transfer of coenzyme occurs on the si face of the external Schiff base in the transamination reactions of two aspartate aminotransferases of Bacillus sp . YM-2 and Escherichia coli, and aromatic amino acid aminotransferase of E . coli.

Rev Argent Microbiol, 1994 Jul-Sep, 26(3), 105 - 15
Study of Bacillus sp . culture conditions to promote production of unhairing proteases; Loperena L et al.; The substitution of chemical depilatory agents in the leather industry by proteolytic enzymes produced by Bacillus species has an important economical and environmental impact . In previous assays, a Bacillus sp . showing a promising depilatory activity was isolated . In this paper, a culture medium that stimulated the synthesis and segregation of depilatory proteases, was selected . The influence of pH, oxygen supply rate (KLaC*), and inoculum age was evaluated on cell growth and protease production . Assays were carried out in lab bioreactors (1.2-1.4 l) at 37 degrees C . Five different media that differed in carbon and nitrogen sources were tested . pH ranged from 4.0 to 8.5 . KLaC* varied between 40 and 470 mmol/lh . The best medium culture for protease production contained: nutrient broth (Britania) 8 g/l, yeast extract (Britania) 3 g/l, and mineral salts . Protease production was more effective at pH of 6.7, KLaC* of 360 mmol/lh, and inoculum age of 12 hours . These experimental conditions led to the following results: maximum proteolytic activity 2700 U/ml, overall volumetric protease productivity 300 u/ml-h, average specific growth rate 0.62 h-1, and average specific protease production rate 2.50 x 10(5) U/gh.

J Biochem (Tokyo), 1994 Jul, 116(1), 176 - 82
Role of the conserved glycyl residues located at the active site of leucine dehydrogenase from Bacillus stearothermophilus; Sekimoto T et al.; A tetrapeptide sequence, Gly-Gly-(Gly/Ala)-Lys, containing a catalytically important lysyl residue, is highly conserved in NAD(P)+-dependent amino acid dehydrogenases . To elucidate functional roles of the glycyl residues in this conserved sequence Gly-77, Gly-78, and Gly-79 of the recombinant leucine dehydrogenase from Bacillus stearothermophilus have been individually replaced with Ala by site-directed mutagenesis . All of the mutant enzymes had Michaelis constants for alpha-keto-iso-caproate and ammonia several times larger than the wild-type enzyme while retaining considerable catalytic activities . However, inhibition constants for a substrate analog without an alpha-carbonyl group were unchanged by the mutations . On the other hand, the rate of inactivation by pyridoxal 5'-phosphate and the microenvironment of aromatic residues, in particular of the sole tryptophanyl residue (Trp-46) located in the vicinity of the active site, were affected by the mutations of the glycyl residues . All of these results suggest that the conserved glycyl residues are important for fine-tuning of the position and/or orientation of the epsilon-amino group of Lys-80 at the active site to function efficiently as a general-base catalyst . Furthermore, the Gly-77 and Gly-78 mutant enzymes had markedly decreased thermal stabilities, showing that these two glycyl residues are also critical for the conformational stability of this thermostable enzyme.

Appl Microbiol Biotechnol, 1994 Jul, 41(5), 517 - 22
Thermostable alpha-amylase production by immobilized Bacillus licheniformis cells in agar gel and on acrylonitrile/acrylamide membranes; Tonkova A et al.; Immobilized cells of Bacillus licheniformis 44MB82-G were used for the production of thermostable alpha-amylase . The immobilization was carried out by entrapment in agar gel or by binding to formaldehyde-activated acrylonitrile/acrylamide membranes . The alpha-amylase production after 144 h of cultivation of membrane immobilized cells was 40% higher in comparison with the free cells . The respective value for the agar-entrapped cells was 22% . Similar trends were observed in the repeated batch fermentations performed with the immobilized cells . The scanning electron micrographs (SEM) of the immobilized cells gave additional information about their binding to the respective carriers.

Mol Microbiol, 1994 Jul, 13(1), 67 - 73
Bacteriophage-like particle of Rochalimaea henselae; Anderson B et al.; An extracellular particle approximately 40 nM in diameter was detected in culture supernatant from the fastidious bacterium Rochalimaea henselae . This particle has at least three associated proteins and contains 14 kbp linear DNA segments that are heterogeneous in sequence . The 14 kbp DNA was also present in R . henselae cells as an extrachromosomal element for all 14 strains tested . Despite attempts to induce lysis of R . henselae, plaque formation was not observed . A similar particle, also containing 14 kbp DNA, was observed in Bartonella bacilliformis, and may be analogous to a bacteriophage that has been described elsewhere for B . bacilliformis.

J Bacteriol, 1994 Jul, 176(14), 4409 - 15
Thiobacillus ferrooxidans tyrosyl-tRNA synthetase functions in vivo in Escherichia coli; Salazar O et al.; The tyrosyl-tRNA synthetase gene (tyrZ) from Thiobacillus ferrooxidans, an acidophilic, autotrophic, gram-negative bacterium that participates in bioleaching of minerals, was cloned and sequenced . The encoded polypeptide (TyrRZ) is 407 amino acids in length (molecular mass; 38 kDa) . The predicted protein sequence has an extensive overall identity (44%) to the sequence of the protein encoded by the Bacillus subtilus tyrZ gene, one of the two genes encoding tyrosyl-tRNA synthetases in this microorganism . Alignment with Escherichia coli TyrRS revealed limited overall identity (24%), except in the regions of the signature sequence for class I aminoacyl-tRNA synthetases . Complementation of an E . coli strain with a thermosensitive mutation in TyrRS showed that the protein encoded by the T . ferrooxidans tyrZ gene is functional and recognizes the E . coli tRNA(Tyr) as a substrate . TyrZ is a single-copy gene as revealed by Southern blot analysis . The gene was localized upstream from the putative promoters of the rrnT2 ribosomal RNA operon . Although no rho-independent transcription terminator was found between the two genes, a 1.3-kb RNA hybridized to a DNA probe derived from the tyrZ gene . The functional relationship between these two transcription units is discussed.

FEBS Lett, 1994 Jun 27, 347(2-3), 235 - 8
Altered chemotaxis of Bacillus sphaericus L-ethionine-resistant sporulation mutant . A probable link between chemotaxis and sporulation; Andreev J et al.; A UV irradiation-induced mutant of Bacillus sphaericus 2362 whose sporulation was inhibited neither by natural amino acids nor by L-ethionine was selected . The mutant (A61) grew slowly in rich amino acid medium and contained increased concentrations of heat-resistant spores throughout the growth . Slow growth of A61 was related to continuous presence of aging and sporulating cells even when the medium was rich in nutrients . Ability of the mutant to sense nutrient presence in the environment and to relate this information to systems regulating the switch from vegetative growth to sporulation seem to be damaged . A61 also demonstrated impaired chemotaxis . In contrast to the parent strain, only few amino acids elicited chemotactic response in A61 . Methylation of the A61 methyl-accepting chemotaxis protein(s) was lower than that of the parent strain by one order of magnitude . Spontaneous fast-growing phenotypic revertants of A61 displayed sporulation behavior characteristic of B . sphaericus 2362 . Their chemotaxis to amino acids was considerably improved . To some amino acids, it proved to be even stronger than in the original strain, B . sphaericus 2362 . It is suggested, that methyl transfer events originating in the chemotactic system are involved in the triggering of sporulation, the A61 mutation being located in this signalling pathway.

FEBS Lett, 1994 Jun 27, 347(2-3), 226 - 30
Motility and chemotaxis in Bacillus sphaericus . Dependence upon stage of growth; Andreev J et al.; Chemotaxis and motility of Bacillus sphaericus 2362 were monitored as a function of the batch culture age . It was found that both functions changed independently during growth of the culture . Motility was low until the late logarithmic stage ensued, whereafter it increased sharply . The ability of cells to respond to chemo-effectors peaked at the mid-logarithmic phase . A major methyl-accepting chemotaxis protein (P53, M(r) = 53 kDa) was identified . The extent of label incorporation in this protein from L-{methyl-3H}methionine was maximal in mid- and late-logarithmic phases of the growth . Cells in stationary cultures incorporated very low amounts of the label . At any stage, the labeling was maximal in starved cells; it was almost abolished in cells pre-incubated with amino acids . Although extents of P53 labeling in mid- and late logarithmic cells were similar, late logarithmic cells demonstrated a considerably impaired chemotaxis . Supermotile sporulating cells were practically insensitive to environmental stimuli . The difference in development of sensory and locomotive functions may be interpreted as an adaptive response . A well developed sensory apparatus would allow vegetative cells to adapt efficiently to fluctuating attractant gradients . Insensitive sporulating cells would tend to disperse randomly from the nutrient-exhausted area . Thus, spore formation would occur in larger volume of the habitat, increasing the chance of microbial population to survive.

J Mol Biol, 1994 Jun 24, 239(5), 689 - 97
Electron cryomicroscopy of Bacillus stearothermophilus 50 S ribosomal subunits crystallized on phospholipid monolayers; Avila-Sakar AJ et al.; 50 S ribosomal subunits from Bacillus stearothermophilus have been crystallized as 2-dimensional periodic arrays on phospholipid monolayer films at the water-air interface . These crystals were preserved in vitreous ice and imaged with 100 keV electrons under low dose and low temperature conditions . The unit cell parameters of the crystals are a = 371.3(+/- 3.8) A, b = 152.3(+/- 1.6) A, gamma = 96.3(+/- 1.0) degrees . Some of the image arrays of these crystals have twofold rotational symmetry with a phase residual of less than 25 degrees . The mean figure of merit of the merged structure factors from these image arrays out to 20 A resolution is higher than 0.87 . The 2-dimensional projection map shows a level of detail not seen in previous structural studies of the 50 S ribosome subunit . Some of these features may be related to the current 3-dimensional model of the subunit . This analysis illustrates the potential of using the electron crystallographic approach for determining the 3-dimensional structure of the 50 S ribosomal subunit crystallized on a monolayer surface . In addition, the structural information retrieved by electron crystallography might be useful for phasing X-ray data towards an atomic resolution model of the ribosome.

Biochemistry, 1994 Jun 21, 33(24), 7619 - 26
Site-directed mutagenesis of glutamate-166 in beta-lactamase leads to a branched path mechanism; Escobar WA et al.; Glutamate-166 of the Bacillus licheniformis beta-lactamase was specifically mutated to aspartate and cysteine in order to probe the function of this residue in catalysis . In both cases, a large decrease in activity (kcat/Km was 3.5 x 10(-5) smaller for E166C and 1 x 10(-3) smaller for E166D than for the wild-type) was observed, although the kinetics for the two mutants were very different . The pH-rate profiles for E166D and E166C reflected the ionization characteristics of the new residue at site 166 . This result indicates that the ionization of Glu-166 is responsible for the acidic limb of the kcat/Km-pH profiles, and suggests that the function of Glu-166 is that of a general base catalyst . The kinetics of the E166C mutant were investigated in detail . An initial burst was observed, whose amplitude was stoichiometric with the enzyme concentration, suggesting rate-limiting deacylation of the acyl-enzyme intermediate . However, further study revealed that in the presence of 0.5 M sodium sulfate, which stabilizes the native conformational state, the magnitude of the burst corresponded to 2 equiv of enzyme . This observation, in conjunction with the limited effect of the mutation on Km, indicated that the mutation resulted in a change in the kinetic mechanism from the linear, acyl-enzyme pathway to one with a branch leading to an inactive form of the acyl-enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 788 - 94
Intracellular proteolysis and limited diversity of the Bacillus thuringiensis CryIA family of the insecticidal crystal proteins; Almond BD et al.; The current concept of how the highly homologous Bacillus thuringiensis insecticidal crystal protein genes (cry genes) evolved is through recombination among themselves . The cryIA gene family, which is more than 80% identical, consists of only three known genes, even through they are often found together in the same bacterium . To examine the lack of diversity among these genes, recombinatorial chimeric protein toxin genes were constructed and transformed into E . coli, B . subtilis, and B . thuringiensis . Of the nine chimeric proteins examined in this work; three were degraded in E . coli, five in B . subtilis, and seven in B . thuringiensis, suggesting that most Cry proteins resulting from recombination events are degraded by intracellular proteases that are particularly prevalent in B . thuringiensis.

Biochem Biophys Res Commun, 1994 Jun 15, 201(2), 782 - 7
Ligand blot identification of a Manduca sexta midgut binding protein specific to three Bacillus thuringiensis CryIA-type ICPs; Martinez-Ramirez AC et al.; The CryIA(a), CryIA(b) and CryIA(c) Bacillus thuringiensis insecticidal crystal proteins (ICPs) were used in ligand-blot experiments to detect specific binding proteins in brush-border membrane vesicles (BBMV) of Manduca sexta . We identified a protein which binds these three CryIA-type ICPs . The apparent molecular mass of the protein, estimated on SDS-PAGE, was 210 kDa as was the CryIA(b) binding protein previously described by Vadlamudi and col . We have also demonstrated, in ligand blot experiments, that CryIA(a) and CryIA(c) compete with CryIA(b) for binding this 210 kDa protein . Properties of the binding molecule can be correlated with knowledge previously acquired through radiolabelled binding experiments.

Biochem J, 1994 Jun 15, 300 ( Pt 3), 737 - 42
The role of Glu-60 in the specificity of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) towards dinucleotides, poly(A) and RNA; Bastyns K et al.; A computer model of the complex between G2'p5'G and barnase, the recombinant ribonuclease of Bacillus amyloliquefaciens, was constructed, based on the known structure of the complex RNAase T1.G2'p5'G . This model suggests that the conserved residue Glu-60 plays an important role in the specificity of barnase for guanosine . A barnase mutant was therefore made in which Glu-60 was replaced by Gln . This mutation increases the Km for the dinucleotides GpC and GpA, by a factor of 10, but does not change the kcat . For ApA, the kcat/Km decreases by a similar factor, but the individual parameters could not be determined . The mutation, however, has no influence on the kcat and the Km of barnase action towards RNA and poly(A) . This demonstrates that the interactions between the substrate and the residue at position 60 must be different in the case of ApA and poly(A) . For RNA, this conclusion is also likely, but not absolutely certain, because barnase/RNA might be a Briggs-Haldane type enzyme/substrate pair . Therefore, if the effect of the mutation were limited to an increase of the dissociation rate constant of the substrate (k-1), this would not be evident in Km or kcat/Km . In view of the clear cut situation with poly(A), the pH profile for and the effect of salt concentration on the kinetic parameters of the mutant barnase were studied for this substrate . The influence of salt on the Km can be interpreted via the linked function concept and shows a cooperative dissociation of 7-10 counterions upon poly(A) binding . The binding of the substrate is strongly reduced at high pH, and the pKa involved decreases strongly at high salt concentrations . Poly(A) and RNA show a pH dependency of their absorbance spectrum, indicating a pH-dependent change of base stacking, which may influence the catalytic parameters.

J Mol Biol, 1994 Jun 10, 239(3), 430 - 2
Crystallization and preliminary X-ray analysis of restriction endonuclease BamHI-DNA complex; Strzelecka T et al.; Restriction endonuclease BamHI from Bacillus amyloliquefaciens has been co-crystallized with a 12 bp DNA fragment that encompasses its recognition site . The co-crystals diffract to at least 1.95 A resolution and belong to space group P2(1)2(1)2(1) . The unit cell parameters are a = 108.8 A, b = 81.9 A, c = 68.8 A, consistent with one complex in the crystallographic asymmetric unit . The direction of the DNA appears to be along the b axis . In order to achieve end to end stacking of DNA, the complex must lie on the screw axis along b . A self-rotation function has determined the directions of the non-crystallographic 2-fold axes.

Cancer Immunol Immunother, 1994 Jun, 38(6), 365 - 71
Bacillus Calmette-Guérin potentiates monocyte responses to lipopolysaccharide-induced tumor necrosis factor and interleukin-1, but not interleukin-6 in bladder cancer patients; Conti P et al.; During the past decade, particular attention has been focused on treatment of bladder cancer patients with the bacterial agent bacillus Calmette-Guerin (BCG) . In these studies, bladder cancer patients were instilled with BCG (75 mg/50 ml) once per week for 6 weeks, 1-2 weeks following trans-urethral resection of the bladder . Cystoscopy was performed after 6 weeks and, unless tumor progression was present, monthly treatments were given for 1 year . Blood was drawn 2 h after the last instillation, and monocytes were isolated (5 x 10(6) cells/ml) and treated, or not, with lipopolysaccharide (LPS) (20 microgram/ml) for tumor necrosis factor alpha (TNF alpha), interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6) release . The levels of monokines were determined by a monokine-specific enzyme-linked immunosorbent assay . Our results clearly show that, after 18 h incubation, macrophages from BCG-treated bladder cancer patients produced from 2.8- to 1.9-fold and from 2.0- to 1.3-fold greater amounts of TNF alpha and IL-1 alpha respectively, compared to macrophages from healthy controls, 5-fold higher than bladder cancer patients not treated with BCG . IL-6 was not affected . In another set of experiments macrophages (5 x 10(6) cells/ml) from healthy subjects were pretreated, or not, with BCG (100 micrograms/ml) overnight and treated, or not, with LPS 20 microgram/ml alone and in combination with interleukin-1 receptor antagonist (IL-1ra) 250 ng/ml . Macrophages treated with BCG had a strong stimulatory effect on IL-1 alpha release (9.45 ng/ml) while LPS was less effective (3.59 ng/ml) . The combination of BCG plus LPS produced an additive effect on IL-1 alpha release (13.71 ng/ml) compared to the effect of the compound alone . The addition of IL-1ra (250 ng/ml) to BCG was not effective, while when IL-1ra was added to BCG plus LPS only a partial inhibition of IL-1 alpha release was found (9.83 ng/ml), compared to BCG plus LPS without IL-1ra (13.71 ng/ml) . These effects seem to be related to the inhibition of IL-1 alpha stimulated with LPS, but not BCG . The priming effect of BCG exerted on LPS-stimulated monocyte production of TNF alpha and IL-1 alpha from bladder cancer patients led us to study the possible modulation of fibrinogen and C-reactive protein in the serum of BCG-treated cancer patients . The plasma levels of fibrinogen and C-reactive protein were higher (approximately twice) in BCG-treated patients compared to values obtained in untreated patients or healthy controls . We conclude that the beneficial immunotherapeutic effects of BCG in bladder cancer patients are related to its capacity to prime macrophages to enhance the release of TNF alpha and IL-1 alpha, but not IL-6 in response to physiological secondary stimuli, or through the direct stimulation of BCG on IL-1 alpha or TNF alpha, which are directly involved in the killing of cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochim Biophys Acta, 1994 Jun 1, 1192(1), 88 - 94
Phospholipid asymmetry in plasma membrane vesicles derived from BHK cells; Whatmore JL et al.; The transbilayer distribution of phospholipids in plasma membrane vesicles derived from BHK cells by treatment with iodoacetamide or fluoride and merocyanine 540 has been examined by exposing the vesicles to bee venom phospholipase A2 (PLA2) or to Bacillus cereus sphingomyelinase . The results show that almost all of the phosphatidylserine (PS) is on the inner lipid leaflet and most of the sphingomyelin is on the outer lipid leaflet . In contrast, about 50% of the phosphatidylcholine (PC) and 30-40% of the phosphatidylethanolamine (PE) is rapidly degraded by PLA2 and thus appears to be present on the surface of the vesicles . The pools of PC and PE which are accessible only slowly to PLA2 are degraded with halftimes of about 5 h and 2 h, respectively, and it is suggested that this rate reflects the rate of transbilayer migration of these lipids . We conclude that the profound energy depletion caused by treatment with iodoacetamide or fluoride does not alter the asymmetric distribution of PS across the plasma membrane but does have a marked effect on the transbilayer distribution of PE . Residual cells after treatment with fluoride and MC540 were also exposed to PLA2 . The results were broadly in agreement with those obtained with vesicles, suggesting that the vesicles were representative of the BHK cell plasma membrane in terms of phospholipid asymmetry . Fluoride or MC540 added separately caused little vesicle release but did lead to significant loss of phospholipid asymmetry . When centrifuged on a sucrose density gradient, vesicles were separated into two major fractions accounting for about two thirds and about 20%, respectively, of total phospholipid but no significant differences were seen in the transbilayer phospholipid asymmetry of the two fractions.

Dig Dis Sci, 1994 Jun, 39(6), 1197 - 209
Endothelial proliferation in experimental granulomatous colitis . Autoradiography and immunohistochemistry studies; Pooley N et al.; The time sequence and magnitude of endothelial cell proliferation was investigated in an experimental model of granulomatous colitis in rats, induced by intramural inoculations of mycobacterium Bacillus Calmette-Guerin . Colonic tissues were assessed by gross examination, histopathology, autoradiography, and immunohistochemistry . Gross examination of the colonic tissue showed thickening of the colonic wall, erythema, hemorrhage, and scattered ulcers . Histopathological findings were characterized by an acute transmural inflammation, progressing to chronic inflammation accompanied by regenerative changes in the glandular epithelium, goblet cell depletion, mucosal atrophy and fibrosis . Well-developed noncaseating granulomas were first observed at day 5 and were found to be a dominant feature up to day 17 . Autoradiographic studies showed increased endothelial cell labeling up to 17% at 48 hr, compared to less than 1% labeling in control animals . Immunostaining for factor VIII-related antibody, an endothelial cell marker, showed increased numbers of microvessels and individual positive cells located in areas of inflammation as early as 24 hr . At day 5 these individual cells along with dilated neocapillaries were found surrounding the granulomas . This model of granulomatous colitis mimics many features of the human disease state . The early increase in endothelial cell proliferation that precedes granuloma formation during the course of the inflammatory response may suggest that the events leading to the expression of granulomatous colitis are dependent on endothelial proliferation.

J Bacteriol, 1994 Jun, 176(11), 3111 - 6
Growth and bioenergetics of alkaliphilic Bacillus firmus OF4 in continuous culture at high pH; Sturr MG et al.; The effect of external pH on growth of alkaliphilic Bacillus firmus OF4 was studied in steady-state, pH-controlled cultures at various pH values . Generation times of 54 and 38 min were observed at external pH values of 7.5 and 10.6, respectively . At more alkaline pH values, generation times increased, reaching 690 min at pH 11.4; this was approximately the upper limit of pH for growth with doubling times below 12 h . Decreasing growth rates above pH 11 correlated with an apparent decrease in the ability to tightly regulate cytoplasmic pH and with the appearance of chains of cells . Whereas the cytoplasmic pH was maintained at pH 8.3 or below up to external pH values of 10.8, there was an increase up to pH 8.9 and 9.6 as the growth pH was increased to 11.2 and 11.4, respectively . Both the transmembrane electrical potential and the phosphorylation potential (delta Gp) generally increased over the total pH range, except for a modest fall-off in the delta Gp at pH 11.4 . The capacity for pH homeostasis rather than that for oxidative phosphorylation first appeared to become limiting for growth at the high edge of the pH range . No cytoplasmic or membrane-associated organelles were observed at any growth pH, confirming earlier conclusions that structural sequestration of oxidative phosphorylation was not used to resolve the discordance between the total electrochemical proton gradient (delta p) and the delta Gp as the external pH is raised . Were a strictly bulk chemiosmotic coupling mechanism to account for oxidative phosphorylation over the entire range, the deltaGp/deltap ration (which would equal the H+/ATP ratio) would rise from about 3 at pH 7.5 to 13 at pH 11.2, dropping to 7 at pH 11.4 only because of the rise in cytoplasmic pH relative to other parameters . Moreover, the molar growth yields on malate were higher at pH 10.5 than at pH 7.5, indicating greater rather than lesser efficiency in the use of substrate at the more alkaline pH.

J Urol, 1994 Jun, 151(6), 1634 - 7
T helper cell alveolitis after bacillus Calmette-Guerin immunotherapy for superficial bladder tumor; Reinert KU et al.; We report a case of T helper cell alveolitis after intravesical bacillus Calmette-Guerin (BCG) immunotherapy for a superficial bladder tumor . Despite the clinical appearance of miliary pulmonary tuberculosis no mycobacterial agent was found . Therefore, we concluded that the patient had a hypersensitivity reaction similar to sarcoidosis . The condition improved after immunosuppressive therapy with glucocorticosteroids . Differentiation of bronchoalveolar cells could be useful in distinguishing hypersensitivity to infection with BCG.

Infect Immun, 1994 Jun, 62(6), 2644 - 8
Identification of outer membrane proteins of Bartonella bacilliformis; Minnick MF; Purification of the outer membrane of Bartonella bacilliformis by sucrose step gradient centrifugation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) suggest that 14 proteins, ranging from 11.2 to 75.3 kDa, are located in the outer membrane of the pathogen . On the basis of M(r)s, eleven of these proteins have counterparts which are labeled by extrinsic radioiodination of intact bartonellae, and two of the proteins are visibly sensitive to extrinsic proteinase K digestion in analysis by SDS-PAGE . While nearly all the extrinsically radioiodinated proteins could be immunoprecipitated with rabbit antibartonella hyperimmune serum, proteins of 31.5, 42, and 45 kDa were prominent immunoprecipitants . Purified lipopolysaccharide from the outer membrane of B . bacilliformis produced a diffuse band of approximately 5 kDa on SDS-PAGE and was not detectable on immunoblots developed with rabbit antibartonella hyperimmune antiserum.

Antimicrob Agents Chemother, 1994 Jun, 38(6), 1314 - 9
Characterization and biological activity against Naegleria fowleri of amoebicins produced by Bacillus licheniformis D-13; Galvez A et al.; The strain Bacillus licheniformis D-13 produces three hydrophobic peptides (amoebicins d13-A, d13-B, and d13-C) that elicit antiamoebic activity against human-pathogenic and nonpathogenic species of Naegleria and have a broad spectrum of antibacterial activity . The three amoebicins have the same amino acid composition (three Asp, two Glu, two Val, and nine Leu residues) and molecular weight (1,870) . Amoebicin d13-B causes lysis of amoebae through disorganization of the cell membrane . It also induces permeability to 86Rb and membrane disruption in asolectin vesicles.

J Dairy Sci, 1994 Jun, 77(6), 1515 - 20
Detection of penicillin G in milk using a conductimetric method; Chen HC et al.; A highly sensitive method was developed that used conductance measurement for the detection of penicillin G in milk . The method is based on the inhibition by the antibiotic on the growth of Bacillus stearothermophilus ATCC 10149 . The conductance change in PM indicator agar containing the bacterial spores was continuously monitored at 55 degrees C by a microbiological analyzer, and the detection time was delayed when penicillin G was present in the samples . The detection limit of the method for penicillin G was .00016 IU/ml with a detection time of about 3.4 h, but only a narrow range (.00016 to .00062 IU/ml) of the antibiotic could be quantitatively analyzed . The conductimetric method is about 30 times more sensitive than several methods currently used . In addition, the conductance measurement is fully automatic, and multiple samples, 120 or 240, can be analyzed simultaneously.

Microbiology, 1994 Jun, 140 ( Pt 6), 1403 - 10
A spore-lytic enzyme released from Bacillus cereus spores during germination; Makino S et al.; The exudate of fully germinated spores of Bacillus cereus IFO 13597 in 0.25 M sodium phosphate buffer, pH 7.0, was found to contain a spore-lytic enzyme . This enzyme was found to cause loss of absorbance in coat-stripped spore suspensions and phase-darkening of the spores but had minimal activity on isolated peptidoglycan substrates . The enzyme was purified in an active form and identified as a 24 kDa protein which is either an amidase or a peptidase . The amino-terminal 19 residues had the following sequence: FSNQVIQRGASGEKVIELQ . The spore-lytic enzyme retained its activity in a medium of a relatively high ionic strength containing a non-ionic surfactant such as nonaethyleneglycol n-dodecyl ether . This activity was optimum at a salt concentration of about 30 mM in assay buffer at neutral pH . In contrast to the enzyme in a spore-bound form, the enzyme in solution was shown to be heat-sensitive and was readily inactivated by thiol reagents.

Microbiology, 1994 Jun, 140 ( Pt 6), 1337 - 40
The expression of the bstVIM gene from Bacillus stearothermophilus V is restricted to vegetative cell growth; Gonzalez E et al.; The activity of BstVI DNA methyltransferase was monitored during the sporulative cycle of Bacillus stearothermophilus V . Significant methylase activity was found only in bacteria growing vegetatively . This was confirmed by Northern hybridization, which indicated that the bstVIM gene was not transcribed in cells undergoing sporulation . Supporting evidence came from experiments which demonstrated that the RNA polymerase holoenzyme from these cells did not recognize the promoter elements upstream of the bstVIM gene.

Enferm Infecc Microbiol Clin, 1994 Jun-Jul, 12(6), 293 - 6
{Bacillary angiomatosis: report of 2 cases}; Salgado F et al.; BACKGROUND: Epithelioid bacillary angiomatosis (EBA) was studied as an infectious disease associated to immunosuppressive states, establishing the bases for performing differential diagnosis with other pathologic processes . METHODS: Two new cases of EBA in patients with human immunodeficiency virus infection (HIV) are presented . Diagnosis was performed by anatomopathologic study of the cutaneous lesions which had undergone biopsy . RESULTS: In one of the cases bacillary structures were observed under electron microscopy . This patient also presented Kaposi sarcoma (KS) with histologic study being therefore necessary to perform differential diagnosis between these pathologic processes . Both patients presented good response to treatment with erythromycin . CONCLUSIONS: 1) EBA is an infectious disease of good prognosis with antibiotic treatment which fundamentally affects severely immunosuppressed patients with human immunodeficiency virus infection . 2) Biopsy is the only differential diagnostic method for this disease with other processes with similar clinical appearance and different prognosis as in Kaposi sarcoma which may even coexist in these patients.

J Otolaryngol, 1994 Jun, 23(3), 216 - 20
Bacillary angiomatosis: a new entity in acquired immunodeficiency syndrome; Hnatuk LA et al.; Since the recognition of the acquired immunodeficiency syndrome (AIDS) in 1981, previously rare infections and neoplasms have become increasingly common . Bacillary angiomatosis, undescribed in the medical literature prior to 1983, is now second in frequency only to Kaposi's sarcoma with respect to the cutaneous manifestations associated with human immunodeficiency virus (HIV) infection . Caused by Rochalimaea henselae, bacillary angiomatosis is easily treated, when diagnosed early, with erythromycin . We present two cases of bacillary angiomatosis that presented to Toronto General Hospital and review this new and clinically interesting entity . The incidence of bacillary angiomatosis will undoubtedly increase as the HIV epidemic accelerates . Since bacillary angiomatosis commonly affects the head and neck region, it is important for the otolaryngologist to become increasingly proficient in its diagnosis and treatment . The current AIDS crisis demands that the otolaryngologist become aware not only of bacillary angiomatosis, but also of the other cutaneous head and neck manifestations of HIV infection.

Mol Microbiol, 1994 Jun, 12(5), 747 - 59
The efficiency of processing and secretion of the thermolysin-like neutral protease from Bacillus cereus does not require the whole prosequence, but does depend on the nature of the amino acid sequence in the region of the cleavage site; Wetmore DR et al.; Using deletion mutants, it is shown that part of the prosequence, the omega-peptide (-4, -24), of the thermolysin-like neutral protease (TNP) from Bacillus cereus, Cnp, is not required for efficient processing and secretion of fully functional mature protease . It is demonstrated that the rate and selectivity of proprotein processing is dependent on both the flexibility and primary sequence of the processing site . Processing is found to be particularly sensitive to the nature of the amino acid three residues upstream from the site of cleavage . A consensus sequence for TNP proprotein processing has been identified, which provides further insights . Finally, a larger deletion of a portion of the Cnp prosequence upstream from the omega-peptide that includes amino acids conserved among TNPs reduces the rate of processing and secretion of Cnp and results in the accumulation of export-incompetent pre-proprotein in the cell fraction.

FEMS Microbiol Lett, 1994 Jun 1, 119(1-2), 53 - 7
An improved method for detecting cytostatic toxin (emetic toxin) of Bacillus cereus and its application to food samples; Mikami T et al.; We developed an improved HEp-2 cell assay method for the detection of Bacillus cereus toxin, which affects the proliferation of HEp-2 cells . The cytostatic toxin was stable upon exposure to heat, pH 2, pH 11 and trypsin, which suggests it is an emetic . Using the HEp-2 cell assay, we examined the distribution and contamination of B . cereus strains that produced an emetic toxin in various foods . Although there were 228 enterotoxin producers among 310 B . cereus strains obtained from foods, 16 of them produced the cytostatic type (emetic toxin) . All of the strains that produced the cytostatic toxin were of the H.1 serotype.

Br J Urol, 1994 Jun, 73(6), 655 - 8
Intravesical bacille Calmette-Guérin in the treatment of superficial transitional cell carcinoma of the bladder; Rogerson JW; OBJECTIVE: To determine the efficacy of intravesical bacillus Calmette-Guerin (BCG) in the treatment of patients with superficial transitional cell carcinoma (TCC) of the bladder, and to assess the impact of fibrin clot inhibitors . PATIENTS AND METHODS: A retrospective review of 56 patients with superficial TCC of the bladder, treated with intravesical BCG after initial transurethral resection (TUR) of raised or papillary lesions or cold cup biopsy of areas of carcinoma in situ (CIS), was performed . Patient drug histories were reviewed for evidence of ingestion of medication known to inhibit fibrin clot formation . The impact of such medication was assessed using the Chi-square test . RESULTS: Fifty-six patients were treated between 1987 and 1991 of whom 52 were evaluable . Eighteen patients (35%) had a complete response with a mean follow-up of 19 months . Six patients (60%) in the group with CIS had a complete response rate with a mean follow-up of 28 months . Seven patients (13%) developed local progression and required cystectomy or external beam radiotherapy . Six patients (12%) died from metastatic disease . Three patients (6%) had significant complications . The adverse impact of fibrin clot inhibitors was found to be significant . CONCLUSION: Intravesical BCG is effective in the treatment of superficial TCC, especially CIS . A careful drug history is important to identify fibrin clot inhibitors so that, if possible, they may be withdrawn prior to intravesical BCG treatment.

Br J Urol, 1994 Jun, 73(6), 649 - 54
Immunotherapy with bacille Calmette-Guérin in patients with superficial transitional cell carcinoma of the bladder associated with bilharziasis; Wishahi MM et al.; OBJECTIVE: To test the effectiveness and possible side-effects that may result from intravesical immunotherapy with bacillus Calmette-Guerin (BCG) for superficial bladder cancer associated with bilharziasis . PATIENTS AND METHODS: Intravesical instillation of the Pasteur strain of BCG was carried out in 13 patients after transurethral resection of superficial transitional cell carcinoma (TCC) associated with bilharziasis . The recurrence rate in these patients was compared retrospectively with that of 17 patients with superficial TCC associated with bilharziasis in whom a recurrence had occurred previously and who had not received immunotherapy . RESULTS: There was a statistically significant decrease in the recurrence of tumours after intravesical BCG therapy . The tumour-free period after BCG treatment was 15.7 months compared with 6.7 months in the control group . CONCLUSION: The presence of bilharziasis does not appear to influence the effectiveness of immunotherapy with BCG for superficial bladder cancer.

Br J Urol, 1994 Jun, 73(6), 639 - 44
Marker tumour response to Evans and Pasteur bacille Calmette-Guérin in multiple recurrent pTa/pT1 bladder tumours: report from the Medical Research Council Subgroup on Superficial Bladder Cancer (Urological Cancer Working Party); Fellows GJ et al.; OBJECTIVE: To compare the efficacy of Evans bacille Calmette-Guerin (BCG) and Pasteur BCG in eradicating marker bladder tumours and to compare the toxicity of the two strains . PATIENTS AND METHODS: Ninety-nine patients with multiple recurrent pTa or pT1 bladder tumours were allocated at random to six instillations at weekly intervals of either Evans BCG or Pasteur BCG . All tumours were resected except one marker tumour . At cystoscopy 3 months after randomization all tumours including the marker tumour, if still present, were resected . RESULTS: The incidence of adverse events was similar in the two groups but numbers were small and only large differences would have been detected . No statistically significant difference in efficacy regarding the response of the marker tumour or the appearance of other tumours at 3 months was noted in the two groups . There was no evidence of stage progression of the marker tumours . CONCLUSIONS: In multiple recurrent pTa or pT1 bladder tumours clearing the bladder of all except one marker tumour provides a safe and convenient way of measuring the response to intravesical therapy . No significant difference in efficacy or toxicity was detected between Evans BCG and Pasteur BCG.

Appl Environ Microbiol, 1994 Jun, 60(6), 2164 - 7
Bacillus DNA in fossil bees: an ancient symbiosis?
Cano RJ, Borucki MK, Higby-Schweitzer M, Poinar HN, Poinar GO Jr, Pollard KJ.
We report here the isolation of DNA from abdominal tissue of four extinct stingless bees (Proplebeia dominicana) in Dominican amber, PCR amplification of a 546-bp fragment of the 16S rRNA gene from Bacillus spp., and their corresponding nucleotide sequences . These sequences were used in basic local alignment search tool searches of nonredundant nucleic acid data bases, and the highest scores were obtained with 16S rRNA sequences from Bacillus spp . Phylogenetic inference analysis by the maximum-likelihood method revealed close phylogenetic relationships of the four presumed ancient Bacillus sequences with Bacillus pumilus, B . firmus, B . subtilis, and B . circulans . These four extant Bacillus spp . are commonly isolated from abdominal tissue of stingless bees . The close phylogenetic association of the extracted DNA sequences with these bee colonizers suggests that a similar bee-Bacillus association existed in the extinct species P . dominicana.

Appl Environ Microbiol, 1994 Jun, 60(6), 2023 - 30
Biological activities of two fungistatic antibiotics produced by Bacillus cereus UW85; Silo-Suh LA et al.; Cultures and culture filtrates of Bacillus cereus UW85 suppress damping-off of alfalfa caused by Phytophthora medicaginis . We studied the role in disease suppression of two antibiotics from culture filtrates of UW85 that reversibly inhibited growth of P . medicaginis . We purified the two antibiotics by cation-exchange chromatography and high-voltage paper electrophoresis and showed that one of them, designated zwittermicin A, was an aminopolyol of 396 Da that was cationic at pH 7.0; the second, designated antibiotic B, appeared to be an aminoglycoside containing a disaccharide . Both antibiotics prevented disease of alfalfa seedlings caused by P . medicaginis . Purified zwittermicin A reversibly reduced elongation of germ tubes derived from cysts of P . medicaginis, and antibiotic B caused swelling of the germ tubes . Mutants generated with Tn917 or mitomycin C treatment were screened either for antibiotic accumulation in an agar plate diffusion assay or for the ability to suppress damping-off disease of alfalfa . Of 2,682 mutants screened for antibiotic accumulation, 5 mutants were substantially reduced in antibiotic accumulation and disease-suppressive activity . Of the 1,700 mutants screened for disease-suppressive activity, 3 mutants had reduced activity and they accumulated less of both antibiotics than did the parent strain . The amount of antibiotic accumulated by the mutants was significantly correlated with the level of disease suppression . Addition of either zwittermicin A or antibiotic B to alfalfa plants inoculated with a culture of a nonsuppressive mutant resulted in disease suppression . These results demonstrate that B . cereus UW85 produces two fungistatic antibiotics that contribute to suppression of damping-off disease of alfalfa.

Appl Environ Microbiol, 1994 Jun, 60(6), 1889 - 96
Cloning and DNA sequence of the gene coding for Bacillus stearothermophilus T-6 xylanase; Gat O et al.; Bacillus stearothermophilus T-6 produces an extracellular thermostable xylanase . Affinity-purified polyclonal serum raised against the enzyme was used to screen a genomic library of B . stearothermophilus T-6 constructed in lambda-EMBL3 . Two positive phages were isolated, both containing similar 13-kb inserts, and their lysates exhibited xylanase activity . A 3,696-bp SalI-BamHI fragment containing the xylanase gene was subcloned in Escherichia coli and subsequently sequenced . The open reading frame of xylanase T-6 consists of 1,236 bp . On the basis of sequence similarity, two possible -10 and -35 regions, a ribosome-binding site at the 5' end of the gene and a potential transcriptional termination motif at the 3' end of the gene, were identified . From the previously known N-terminal amino acid sequence of xylanase T-6 and the possible ribosome-binding site, a putative 28-amino-acid signal peptide was deduced . The mature xylanase T-6 consists of 379 amino acids with a calculated molecular weight and pI of 43,808 and 6.88, respectively . Multiple alignment of beta-glycanase amino acid sequences revealed highly conserved regions . Northern (RNA) blot analysis indicated that the xylanase T-6 transcript is about 1.4 kb and that the induction of this enzyme synthesis by xylose is on the transcriptional level.

EMBO J, 1994 Jun 1, 13(11), 2493 - 501
Three-dimensional structure of endo-1,4-beta-xylanase II from Trichoderma reesei: two conformational states in the active site; Torronen A et al.; The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 18.3% at 1.8 A resolution . The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel . The beta-sheet structure is twisted, forming a large cleft on one side of the molecule . The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase . The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues . The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft . One structure was determined at pH 5.0, corresponding to the pH optimum of XYNII . The second structure was determined at pH 6.5, where enzyme activity is reduced considerably . A clear structural change was observed, especially in the position of the side chain of Glu177 . The observed conformational change is probably important for the mechanism of catalysis in XYNII.

Scand J Immunol, 1994 Jun, 39(6), 602 - 6
Mycobacteria precipitate autoimmune rheumatic disease in NOD mice via an adjuvant-like activity; Baxter AG et al.; NOD mice spontaneously develop organ-specific autoimmunity and are widely used as a model for diabetes . NOD mice also exhibit some features of non-organ specific autoimmune rheumatic disease such as thymocytotoxic and anti-nuclear autoantibodies and they develop haemolytic anaemia in senescence . A single dose of 2.6 x 10(7) heat-killed Bacillus Calmette-Guerin (BCG) i.v . in 8-week-old NOD mice prevented diabetes but precipitated a syndrome similar to systemic lupus erythematosus (SLE), in which treated mice rapidly developed haemolytic anaemia, high titre anti-DNA and anti-Sm antinuclear autoantibodies, perivascular lymphocytic infiltration in the kidneys and glomerular immune complex deposition . Here, we examined the mechanism of action by which BCG precipitated rheumatic autoimmune disease in NOD mice . Two weeks after injection, reticuloendothelial cell function was dramatically increased in BCG-treated NOD mice . By 4 weeks, treated mice had a three- to four-fold increase in Mac-1+ and class-II+, B220-negative splenocytes and in vitro antigen-presentation capacity was enhanced two- to four-fold . In vivo responses to SRBC confirmed enhancement of DTH 4 weeks after BCG injection, consistent with an adjuvant-like activity.

Am J Respir Crit Care Med, 1994 Jun, 149(6), 1597 - 600
Boosting of tuberculin sensitivity among Southeast Asian refugees; Cauthen GM et al.; Following an initial negative Mantoux tuberculin skin test, a second test, given as soon as 1 wk later, has been shown to elicit markedly larger reactions (boosting) in 20 to 40% of refugees tested in the United States . We conducted a study to determine the explanation for this phenomenon . Using the Mantoux method of intradermal skin testing, 2,469 refugees from Southeast Asia were initially tested with tuberculin followed by sequential retesting 7 and/or 90 d later . They were also tested initially with nontuberculous mycobacterial antigens . A high proportion (35.5%) of Southeast Asian refugees had reactions (> or = 10 mm induration) to an initial tuberculin test, and 30.9% of the nonreactors exhibited boosting on a subsequent tuberculin test . Boosting, unlike reactivity to the initial tuberculin test, was not associated with exposure to a person with tuberculosis . However, boosting was associated with reactivity to nontuberculous mycobacterial antigens and a history of bacille Calmette-Guerin (BCG) vaccination . Boosting in this population is therefore attributable to environmental exposure to nontuberculous mycobacteria that are endemic in Southeast Asia or to BCG vaccination, rather than to remote infection with Mycobacterium tuberculosis . Sequential tuberculin screening and preventive therapy of persons with boosted reactions is not recommended as a tuberculosis prevention strategy in this population.

Biochem J, 1994 Jun 1, 300 ( Pt 2), 491 - 9
Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase; Nobbs TJ et al.; The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195 . This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein . A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior . In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H . The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143 . The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143 . Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme . However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change . Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes . The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme . As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Singapore Med J, 1994 Jun, 35(3), 283 - 5
Bacteroides fragilis meningitis; Ngan CC et al.; Bacteroides fragilis is an obligate anaerobic bacillus residing in the normal intestinal flora of the colon . Anaerobic bacterial meningitis due to this pathogen is rarely diagnosed and if present, a predisposing source of infection should be actively sought for . Anaerobic cultures of cerebrospinal fluids should be done for patients with meningitis, especially those with concomitant pathologies that predispose to anaerobic infections . Two cases of anaerobic meningitis due to Bacteroides fragilis, one associated with cholesteotoma and the other with nasopharyngeal carcinoma, are reported . Both were successfully treated with metronidazole.

J Commun Dis, 1994 Jun, 26(2), 82 - 7
Laboratory and field evaluation of Bacillus thuringiensis and B . sphaericus against mosquito larvae; Baruah I et al.; Laboratory and field trials were carried out with two formulations of Bacillus thuringiensis and four strains of Bacillus sphaericus (B 42, B 64, B 87 and B 33) against mosquito larvae in different breeding habitats of Tezpur, Assam . LC90 of B . thuringiensis var israelensis (formulation Teknar) against Ae . albopictus, Cx . quinquefasciatus and Cx . gelidus were recorded as 0.443, 0.453 and 2.15 ppm respectively and LC90 of B . thuringiensis (Deltox: VCRC B-17) against Ae . albopictus, Cx . quinquefasciatus, Cx . gelidus and Cx . malayi were 8.414, 11.22, 5.24 and 6.761 ppm respectively . LC90 of B . sphaericus strains B 42, B 64, B 87 and B 33 against Cx . quinquefasciatus were 0.055, 0.115, 0.046 and 0.257 ppm respectively . At the dosage of 1 l/ha 87 per cent mortality was achieved after 24 hrs with Bti and it increased to 90-95 per cent at 1.5 l/ha . In polluted cemented drains 93-97 per cent kill of Cx . quinquefasciatus was observed at 2.5 l/ha . Out of four strains of B . sphaericus evaluated, strain B 87 was found to be the most effective as 87-96 per cent kill was achieved with only 0.1 kg/ha for Cx . quinquefasciatus, Cx . vishnui and A . vagus . For others 0.2 kg/ha dosage, eliminated 96-100 per cent Cx . vishnui gr . for B 42, 92-93 per cent for B 64 and 90-93 per cent for B 33 strain.

C R Acad Sci III, 1994 Jun, 317(6), 485 - 8
On the mechanism of biotin synthase of Bacillus sphaericus; Florentin D et al.; A cell-free system of a bioB transformant of Bacillus sphaericus, effecting the last step of biotin biosynthesis, namely the introduction of sulfur into dethiobiotin has been recently described . S-adenosyl methionine (SAM) is absolutely necessary for activity . We show here, through experiments with {35S}SAM and {35S}Cys, that the sulfur donor is not SAM but probably cysteine (Cys) or a derivative . This finding together with the fact that NADPH and FAD are required for activity leads us to postulate some analogy between the biotin synthase system and other systems which use SAM as a source of desoxyadenosyl radical.

Int J Food Microbiol, 1994 Jun, 22(4), 297 - 9
Heat resistant fungi isolated from soil; Pieckova E et al.; The Thermal death time values (TDT) were estimated for Dichotomomyces cejpii, Gilmaniella humicola, Talaromyces avellaneus and Talaromyces bacillisporus isolated from soil . TDT values were compared with the TDT values of the known heat-resistant species, Byssochlamys nivea, Neosartorya fischeri and Talaromyces flavus . All species studied showed considerable heat resistance . The most resistant species Talaromyces avellaneus (172 cfu, initial concentration) of the isolates with unknown heat resistance withstood 90 degrees C for 10 min . Byssochlamys nivea appeared to be the most sensitive species under our experimental conditions.

Sheng Li Xue Bao, 1994 Jun, 46(3), 231 - 7
{Selective transport of Li+ and Na+ through lipid bilayers by nanhumycin}; Cui JJ et al.; Nanhumycin, a new polyether antibiotic, has potent growth inhibitory effect on hay bacillus and antagonistic activity against chicken coccidiosis . Previous experiments on nerve-muscle preparations have shown that all the effects of nanhumycin on biological membrane could be correlated with its ability to act as a Na+ carrier . In this paper, the effects of nanhumycin on the permeability of the lipid bilayers were characterized and the main results were as follows: Nanhumycin caused a concentration-dependent increase in membrane conductance (Gm) of the lipid bilayers . By measuring the reversal potential in an asymmetrical solution system, it was demonstrated that the changes of Gm were attributed to an increase in permeability of the lipid bilayers to cations (PLi/PNa = 0.02), especially to Li+ and Na+ . The PLi:PNa:PK = 4.55:1.00:0.03 . These results suggests that nanhumycin is a cation carrier with high permeability for Li+ and Na+.

Biol Pharm Bull, 1994 Jun, 17(6), 773 - 8
Purification and characterization of iron-containing urethanase from Bacillus licheniformis; Zhao CJ et al.; Urethane is potentially carcinogenic and teratogenic to human and has been reported to be a contaminant of various kinds of alcoholic beverages . Enzymatic removal of urethane is one possible approach to remove this cancer-causing chemical from alcoholic beverages . Among Bacillus licheniformis strains, IFO 12107 showed the highest urethane hydrolyzing activity when cultivated in a urethane-containing white medium . The enzyme was purified about 300-fold by means of several chromatographic steps to homogeneity . The enzyme activity was strongly inhibited by batho- and o-phenanthroline . The complete loss of enzyme activity following treatment with bathophenanthroline was fully restored by the addition of Fe3+ at a ratio of 4 iron atoms to 1 mol apoenzyme . This result was obtained by the incorporation of 59Fe3+ into apourethanase . ESR spectroscopy showed that the enzyme contained a typical high-spin Fe3+ . The urethanase hydrolyzed carbamyl ester derivatives more rapidly than amide derivatives . The molecular weight of the native enzyme was about 160 kDa (gel-filtration), and that of the subunit was 42 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE) . This shows the enzyme to be a homotetramer . The pI and Km values were 5.5 and 0.17 mM, respectively . The enzyme was considerably resistant to high concentrations of ethanol, which is a great advantage for the industrial removal of urethane from alcoholic beverages.

J Biomol Struct Dyn, 1994 Jun, 11(6), 1417 - 24
Microcalorimetric determination of the thermostability of three hybrid (1-3,1-4)-beta-glucanases; Welfle K et al.; Thermodynamic parameters of the three hybrid (1-3,1-4)-beta-glucanases H(A12-M), H(A12-M) delta Y13, and H(A16-M) composed of short N-terminal regions derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the homologous Bacillus macerans enzyme were determined in 2 mM sodium cacodylate pH 6.0, 1.5M guanidine hydrochloride, containing 1 mM CaCl2 or 1 mM EDTA . Melting of H(A12-M) delta Y13 and H(A16-M) in the presence of calcium ions is characterized by two subtransitions; only one transition is observed in the case of H(A12-M) . In calcium-free buffer each of the three hybrid enzymes melts in one two-state transition . Transition temperatures Tm and molar enthalpy changes delta H are reduced in the absence of calcium ions but the reduction is much more pronounced for H(A12-M) delta Y13 and H(A16-M) than for the less thermostable enzyme H(A12-M).

Arch Esp Urol, 1994 Jun, 47(5), 445 - 8
{Is urogenital tuberculosis a current disease still?}; Cabezudo Hernando IA et al.; In 1991, the epidemiology service of the health department of La Rioja announced that the number of deaths and new cases were higher than the national average (45/100,000 in 1991) . Our service detected 16 new cases of urogenital tuberculosis during 1990-1991 . The most common clinical symptoms being urgency and frequency . The bacteriological diagnosis using the Lowenstein-Jensen culture medium was positive in 87.50% of the cases . Anomalies were detected in 87.50% of the urographic analyses, the more frequently observed being infundibular involvement . Polychemotherapy consisting of rifampicin, ethambutol and isoniazid was administered for a period of 9 months in all of the cases, which resulted in negative cultures in 100% of the patients . Control evaluation was performed every 3 months, including CBK and serial bacilloscopy, biochemical assays, renal ultrasound, examination of eye grounds (1st trimester), chest X-ray and sputum bacilloscopy . Only endourological methods were utilized, except in one patient with coexisting congenital stricture of the pyeloureteric junction.

Immunology, 1994 Jun, 82(2), 244 - 8
A single mycobacterial protein (hsp 65) expressed by a transgenic antigen-presenting cell vaccinates mice against tuberculosis; Silva CL et al.; We used a retroviral shuttle vector {pZIPNeoSV(X)} to transfect a monocyte-like murine tumour cell line (J774.G8) with the Mycobacterium leprae gene encoding heat-shock protein (hsp) 65 . The antigen was expressed and presented on the surface of the transfected cell in association with major histocompatibility complex (MHC) class I and class II for recognition by T cells from specifically sensitized mice . We show here that when these transfected cells were used as a vaccine and introduced parentally into syngeneic (BALB/c) mice they conferred a remarkably high degree of protective immunity against subsequent challenge with either M . bovis bacillus Calmette-Guerin (BCG) or M . tuberculosis H37Rv.

Comput Appl Biosci, 1994 Jun, 10(3), 285 - 94
A comparison of Radial Basis Function and backpropagation neural networks for identification of marine phytoplankton from multivariate flow cytometry data; Wilkins MF et al.; Two artificial neural network classifiers, the well-known Multi-layer Perception (MLP) (also known as the 'backpropagation network'), and the more recently developed Radial Basis Function (RBF) network, were evaluated and compared for their ability to identify multivariate flow cytometric data from five North Sea plankton groups (Dinoflagellidae, Bacillariophyceae, Prymnesiomonadida, Cryptomonadida, and other flagellates) . RBF networks generally performed similarly to MLPs, and slightly better in cases where the data were markedly multimodal; RBF networks also have much shorter training times . The performance of MLPs was improved greatly by the use of a symmetrical bipolar 'transfer function' as opposed to the commonly-used asymmetric form . The issues of network optimisation and computational efficiency in use are discussed.

Infect Immun, 1994 Jun, 62(6), 2536 - 44
Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins; Andersen P; An experimental vaccine that was based on secreted proteins of Mycobacterium tuberculosis was investigated in a mouse model of tuberculosis . I used a short-term culture filtrate (ST-CF) containing proteins secreted from actively replicating bacteria grown under defined culture conditions . The immunogenicity of the ST-CF was investigated in combination with different adjuvants, and peak proliferative responses were observed when ST-CF was administered with the surface-active agent dimethyldioctadecylammonium chloride . The immunity induced by this vaccine was dose dependent, and, in the optimal concentration, the vaccine induced a potent T-helper 1 response which efficiently protected the animals against a subsequent challenge with virulent M . tuberculosis . Antigenic targets for the T cells generated were mapped by employing narrow-molecular-weight fractions of ST-CF . The experimental vaccine primed a broadly defined T-cell repertoire directed to multiple secreted antigens present in ST-CF . A vaccination with viable Mycobacterium bovis bacillus Calmette-Guerin (BCG), in contrast, induced a restricted T-cell reactivity directed to two secreted protein fractions with molecular masses of 5 to 12 and 25 to 35 kDa . The protective efficacy of the ST-CF vaccine was compared with that of a BCG standard vaccine, and both induced a highly significant protection of equal magnitude . The vaccination with ST-CF gave rise to a population of long-lived CD4 cells which could be isolated 22 weeks after the vaccination and could adoptively transfer acquired resistance to T-cell-deficient recipients . My results confirm the hypothesis that M . tuberculosis cells release protective antigens during growth . The high efficacy of a subunit vaccine observed in the present study is discussed as a possible alternative to a live recombinant vaccine carrier.

Infect Immun, 1994 Jun, 62(6), 2417 - 25
Chemical definition, cloning, and expression of the major protein of the leprosy bacillus; Rivoire B et al.; The decline in prevalence of leprosy is not necessarily matched by a fall in incidence, emphasizing the need for new antigens to measure disease transmission and reservoirs of infection . Mycobacterium leprae obtained from armadillo tissues was disrupted and subjected to differential centrifugation to arrive at preparations of cell wall, cytoplasmic membrane, and cytosol . By committing 0.3 g of M . leprae to the task, it was possible to isolate from the cytosol and fully define the major cytosolic protein . Amino-terminus sequencing and chemical and enzymatic cleavage, followed by more sequencing and fast atom bombardment-mass spectrometry of fragments, allowed description of the entire amino acid sequence of a protein of 10,675-Da molecular mass . The sequence derived by chemical means is identical to that deduced previously from DNA analysis of the gene of a 10-kDa protein, a GroES analog . The work represents the first complete chemical definition of an M . leprae protein . PCR amplification of the 10-kDa protein gene, when cloned into Escherichia coli with a pTRP expression vector, allowed production of the recombinant protein . Chemical analysis of the expressed protein demonstrated that it exactly reflected the native protein . The recombinant major cytosolic protein appears to be a promising reagent for skin testing, still probably the most appropriate and pragmatic means of measuring incidence of leprosy.

Zhonghua Jie He He Hu Xi Za Zhi, 1994 Jun, 17(3), 159 - 61, 190
{Infection of acid-fast bacterial L-forms}; Zhang SF et al.; One hundred and fifty five cases which pathological diagnosis were chronic lymphadenitis were studied in order to detect infection of acid-fast bacterial L-forms by Ziehl-Neelsen (ZN), intensified kinyoun (IK) and immunohistochemical staining (PAP) . The results showed that the expression of M-tuberculosis antibody was positive in 106 cases (68.4%) . Among them, 94 cases (60.6%) were positive in IK staining . The positive rate of acid-fast bacillus was 0.6% (1 case), of L-forms was 60% (93 cases) . L-forms are higher pleomorphic, spherical bodies, giant bodies or long filaments were shown . The L-forms mostly localised within macrophages, only a few distributed sporadically . The possible causes of misdiagnosis physician and pathologist were discussed . The prognostic value was also suggested.

Biodegradation, 1994 Jun, 5(2), 77 - 82
Cloning and expression of a pathway for benzene and toluene from Bacillus stearothermophilus; Natarajan MR et al.; Bacillus stearothermophilus strain BR325 demonstrating broad aromatic substrate capability was isolated from petroleum-contaminated soil . The chromosomally-located aromatic pathway from this isolate was cloned into Escherichia coli as a 32 kb insert in cosmid pHC79, conferring growth on benzene, phenol, and toluene as sole carbon sources.

Indian J Malariol, 1994 Jun, 31(2), 43 - 7
Comparative toxicity of certain mosquitocidal compounds to larvivorous fish, Poecilia reticulata; Mittal PK et al.; Toxicity of certain mosquitocidal compounds (both larvicides and adulticides) to the larvivorous fish Poecilia reticulata was determined in the laboratory . Among the various chemical insecticides tested, the synthetic pyrethroid deltamethrin was most toxic to fish (LC50 = 0.016 ppm), while the organophosphorus insecticide abate was least toxic (LC50 = 34 ppm) . The bioinsecticides Spherix (Bacillus sphaericus) and Bactoculicide (Bacillus thuringiensis H-14) showed highest safety for the fish (LC50 > 1000 mg/litre) . Integrated use of larvivorous fish and bioinsecticide in vector control has been suggested.

Immunol Cell Biol, 1994 Jun, 72(3), 215 - 21
Identification of a Mycobacterium leprae-specific T cell epitope on the 70 kDa heat shock protein; Roche PW et al.; A major antigen of the leprosy bacillus, Mycobacterium leprae, is the 70 kDa heat shock protein (Hsp70), which has significant sequence homology with Hsp70 from other mycobacterial species as well as Hsp70 from eukaryotes . A unique region of 70 amino acids at the C-terminus of the M . leprae Hsp70 has been previously identified . This study investigated whether mice immunized with the C-terminal fragment of M . leprae Hsp70 recognize T cell epitopes in this species-specific portion of the molecule . Murine lymphoproliferative responses to overlapping peptides spanning the C-terminal 70 amino acids were restricted to mice of an H-2b haplotype and identified the presence of a determinant in sequence 567-591 . Lymph node cells from mice immunized with this peptide recognized both the C-terminal fragment and the whole Hsp70 molecule . Moreover, mice immunized with the same peptide responded to the whole Hsp70 molecule in a delayed-type hypersensitivity reaction . The significance of M . leprae-specific T cell epitopes in the host response to mycobacterial infection is discussed.

Clin Infect Dis, 1994 Jun, 18(6), 958 - 62
Detection of culture-resistant bacterial pathogens by amplification and sequencing of ribosomal DNA; Wilson KH; Molecular phylogeny is profoundly influencing the field of bacterial evolution . New knowledge in this area has led to an exciting ability to detect and classify bacteria without culturing them . The process involved consists of either amplification or cloning of ribosomal DNA from a bacterial population, sequencing of this ribosomal DNA, and phylogenetic analysis of the sequences obtained . This approach has so far been applied successfully to four infectious diseases: bacillary angiomatosis, human ehrlichiosis, Whipple's disease, and Tyzzer's disease . Interpretation of data obtained by this method has been straightforward.

FEBS Lett, 1994 May 30, 345(2-3), 135 - 8
Purification and properties of a novel enzyme from Bacillus spp . T-3040, which catalyzes the conversion of dextran to cyclic isomaltooligosaccharides; Oguma T et al.; A novel enzyme, cycloisomaltooligosaccharide glucanotransferase (CITase), catalyzes the conversion of dextran to cyclic isomaltooligosaccharides by intramolecular transglucosylation (cyclization reaction) . CITase was purified to homogeneity from the culture filtrate of Bacillus sp . T-3040 isolated from soil . The Mr of the enzyme was estimated to be 98,000 by SDS-PAGE . The enzyme catalyzed the cyclization reaction and gave three cyclic isomaltooligosaccharides (cycloisomalto-heptaose, -octase, and -nonaose) at a total yield of about 20% . Coupling and disproportionation reactions were also observed . These results showed that this enzyme is a multi-functional enzyme which catalyzes intramolecular and intermolecular transglucosylation.

Gene, 1994 May 27, 143(1), 149 - 50
Characterization of the 16S-23S rRNA intergenic spacer of Bartonella bacilliformis; Minnick MF et al.; The 16S-23S intergenic spacer region from the ribosomal RNA (rRNA) operon of Bartonella bacilliformis was cloned and characterized . The spacer is 906 nucleotides (nt) in length and contains the genes encoding isoleucine-tRNA (tRNA(Ile)) and alanine-tRNA (tRNA(Ala)) . The tRNA-encoding genes are separated by 122 nt and are centrally located in the 16S-23S spacer region, with approx . 300 flanking nt . Genes encoding tRNA(Ile) and tRNA(Ala) have 88.3 and 93.4% sequence identity, respectively, to the homologous genes of Rhodobacter sphaeroides.

Biochemistry, 1994 May 24, 33(20), 6110 - 20
Regulation and phase equilibria of membrane lipids from Bacillus megaterium and Acholeplasma laidlawii strain A containing methyl-branched acyl chains; Rilfors L et al.; Phosphatidylethanolamine (PE) was isolated from Bacillus megaterium grown at 20 and 55 degrees C (PE-20 and PE-55) . Iso and anteiso methyl-branched, saturated acyl chains are predominant in B . megaterium, and the value of the molar ratio of iso/anteiso acyl chains is more than 20-fold higher in PE-55 than in PE-20 . Moreover, about 21 mol% of the acyl chains of PE-20 are monounsaturated . The phase equilibria differ between the two PE preparations: (1) PE-20 is more prone to form reversed nonlamellar phases than PE-55; (2) PE-20 forms both reversed cubic (I2) and reversed hexagonal (H(II)) phases while PE-55 forms only an HII phase; and (3) the lamellar liquid-crystalline (L alpha) phase of PE-20 takes up about 70% more water than the L alpha phase of PE-55 . These differences can be explained by the differences in the acyl chain composition . When the growth temperature is raised, PE molecules with a reduced tendency to form nonlamellar phases are probably synthesized by B . megaterium in order to counteract the bilayer destabilizing effect of the temperature . The regulation of the acyl chain composition is not needed in order to regulate the temperature for the transition between gel/crystalline and L alpha phases of the membrane lipids . Acholeplasma laidlawii strain A-EF22 was grown at 37 degrees C on 15-(1,1,1(-2) H3)methylhexadecanoic acid, 14-(1,1,1(-2)H3)methylhexadecanoic acid or 13-(1,1,1(-2)H3)methylhexadecanoic acid, and these acids constituted 84-89 mol% of the acyl chains in the membrane lipids . The molar ratio between the two dominating lipids, monoglucosyldiacylglycerol (MGLcDAG) and diglucosyldiacylglycerol (DGlcDAG), decreased, and the molar fraction of the anionic lipids increased, when the methyl branch was moved from position 15 to position 13 . Concomitantly, the order of the methyl branch increased in cells as well as in total lipid extracts . The phase equilibria of total lipid extracts (neutral lipids removed) were studied with 20 wt % of water, and HII and I2 phases were formed above 63-67 degrees C . These results indicate that the regulation of the polar head-group composition compensates for the difference in acyl chain packing introduced into the bilayer by the three branched-chain fatty acids . The regulation of the polar head-group composition of the A . laidlawii lipids cannot regulate the temperature for the transition between gel/crystalline and L alpha phases of the lipids, i.e . the transition to fluid acyl chains.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1994 May 20, 269(20), 14530 - 5
Identification of active site carboxylic residues in Bacillus licheniformis 1,3-1,4-beta-D-glucan 4-glucanohydrolase by site-directed mutagenesis; Juncosa M et al.; Active site residues of 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from Bacillus licheniformis have been identified by site-directed mutagenesis . Previous work revealed that Glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaciens enzyme . To search for the general acid catalyst, the Asp and Glu residues conserved among the Bacillus isozymes have been mutated to Asn and Gln, respectively . Out of the 14 positions studied, only the E138Q mutation yielded an inactive enzyme, whereas the E134Q and D136N mutants retained less than 0.5% of the wild type activity . Based on the three-dimensional structure of a hybrid B . amyloliquefaciens-Bacillus macerans 1,3-1,4-beta-D-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly located into the active site cleft to participate in catalysis . Glu-138 appears as the most likely candidate to function as the general acid catalyst, while Asp-136 may affect the pK alpha of the catalytic residues.

Biochemistry, 1994 May 17, 33(19), 5728 - 38
A structure-based analysis of the inhibition of class A beta-lactamases by sulbactam; Imtiaz U et al.; From the crystal structure of the Bacillus licheniformis 749/C beta-lactamase, energy-minimized structures for the precatalytic, the acyl-enzyme intermediate, and the acylated linear inactivating species for sulbactam--a clinically useful mechanism-based inactivator for class A beta-lactamases--were generated . The effect of individual Ser-235-Ala and Arg244-Ser point mutations on the inactivation and turnover processes was consistent with the existence of hydrogen bonds between the side chains of these residues and the sulbactam species . The departure of the sulfinate leaving group from the acyl-enzyme intermediate of sulbactam is believed to be a prerequisite for the inactivation process . In order to explore the influence of the leaving group, penicillanic acid (2), penicillanic acid alpha-S-oxide (3), and penicillanic acid beta-S-oxide (4) were synthesized and studied in kinetic experiments with the TEM-1 beta-lactamase . Penicillanic acid is only a substrate, but penicillanic acid S-oxides were both substrates and inactivators for the enzyme . An argument is presented to rationalize these observations on the basis of the leaving ability of thiolate, sulfenate, and sulfinate from the acyl-enzyme intermediates of penicillanic acid (2), the penicillanic acid S-oxides (3 and 4), and sulbactam, respectively . The departure of the leaving group does not appear to be rate limiting in the inactivator process, but is an indispensable component of the irreversible inactivation of the enzyme . Molecular dynamics calculations of the putative inactivating species suggest that Lys-73, Lys-234, and Ser-130 are three likely residues that may be modified in the course of the inactivation chemistry . A discussion is presented of the mechanism of formation of the transiently inhibited enzyme species, which comes about as a consequence of the tautomerization of the double bond of the inactivating iminium moiety . In addition, the mechanistic details presented for sulbactam are compared and contrasted with those of clavulanic acid, another clinically used inactivator for class A beta-lactamases.

Eur J Biochem, 1994 May 15, 222(1), 203 - 14
Cation binding to a Bacillus (1,3-1,4)-beta-glucanase . Geometry, affinity and effect on protein stability; Keitel T et al.; The hybrid Bacillus (1,3-1,4)-beta-glucanase H(A16-M), consisting of 16 N-terminal amino acids derived from the mature form of the B . amyloliquefaciens enzyme and of 198 C-proximal amino acids from the B . macerans enzyme, binds a calcium ion at a site at its molecular surface remote from the active center {T . Keitel, O . Simon, R . Borriss & U . Heinemann (1993) Proc . Natl Acad . Sci . USA 90, 5287-5291} . X-ray diffraction analysis at 0.22-nm resolution of crystals grown in the absence of calcium and in the presence of EDTA shows this site to be occupied by a sodium ion . Whereas the calcium ion has six oxygen atoms in its coordination sphere, two of which are from water molecules, sodium is fivefold coordinated with a fifth ligand belonging to a symmetry-related protein molecule in the crystal lattice . The affinity of H(A16-M) for calcium over sodium has been determined calorimetrically . Calcium binding stabilizes the native three-dimensional structure of the protein as shown by guanidinium chloride unfolding and thermal inactivation experiments . The enhanced enzymic activity of Bacillus beta-glucanases at elevated temperatures in the presence of calcium ions is attributed to a general stabilizing effect by the cation.

CMAJ, 1994 May 15, 150(10), 1561 - 71
Essentials of tuberculosis control for the practising physician . Tuberculosis Committee, Canadian Thoracic Society; Molecular cloning of a maltose transport gene from Bacillus stearothermophilus and its expression in Escherichia coli K-12; Department of Microbiology, University of Sydney, NSW, AustraliaGenes responsible for maltose utilization from Bacillus stearothermophilus ATCC7953 were cloned in the plasmid vector pBR325 and functionally expressed in Escherichia coli . The 4.2 kb Bacillus DNA insert in clone pAM1750 suppressed the growth defects on maltose caused by mutations in E . coli maltose transport genes (malE, malK or complete malB deletion) but not mutations in genes affecting intracellular maltose metabolism (malA region) . Transport studies in E . coli and B . stearothermophilus suggested that pAM1750 codes for a high affinity transport system, probably one of two maltose uptake systems found in B . stearothermophilus ATCC7953 . Nucleotide sequence analysis of a 3.6 kb fragment of pAM1750 revealed three open reading frames (ORFs) . One of the ORFs, malA, encoded a putative hydrophobic protein with 12 potential transmembrane segments . MalA showed amino acid sequence similarity to proteins in the superfamily containing LacY lactose permease and also some similarity to MalG protein, a member of a binding protein-dependent transport system in E . coli . The products of two other ORFs were not hydrophobic, did not show similarity to other known sequences and were found not to be essential for maltose utilization in transport-defective E . coli mutants . Hence MalA protein was the only protein necessary for maltose transport, but despite giving a detectable but low level of transport function in E . coli, the protein was very poorly expressed and could not be identified.

Proc Natl Acad Sci U S A, 1994 May 10, 91(10), 4120 - 4
Reversal of resistance to Bacillus thuringiensis in Plutella xylostella; Tabashnik BE et al.; Continued success of the most widely used biopesticide, Bacillus thuringiensis, is threatened by development of resistance in pests . Experiments with Plutella xylostella (diamondback moth), the first insect with field populations resistant to B . thuringiensis, revealed factors that promote reversal of resistance . In strains of P . xylostella with 25- to 2800-fold resistance to B . thuringiensis compared with unselected strains, reversal of resistance occurred when exposure to B . thuringiensis was stopped for many generations . Reversal of resistance was associated with restoration of binding of B . thuringiensis toxin CryIA(c) to brush-border membrane vesicles and with increased biotic fitness . Compared with susceptible colonies, revertant colonies had a higher proportion of extremely resistant individuals . Revertant colonies responded rapidly to reselection for resistance . Understanding reversal of resistance will help to design strategies for extending the usefulness of this environmentally benign insecticide.

J Mol Biol, 1994 May 6, 238(3), 346 - 65
Quantitative analysis of crystal growth . Tryptophanyl-tRNA synthetase crystal polymorphism and its relationship to catalysis; Carter CW Jr et al.; We show that quantitative analysis of replicated, full-factorial crystal growth experiments and, by implication, similar studies of a wide variety of other phenomena, can be a powerful tool for analyzing macromolecular systems with complex, interacting dependencies on functionally significant factors . Bacillus stearothermophilus tryptophanyl-tRNA synthetase crystallizes in three different crystal forms depending on the ligands present under otherwise identical conditions . Comparison of crystallographic space groups for complexes with different ligands reveals that the three forms entail at least two very different families of packing arrangements that are correlated with specific changes in the enzyme ligation state . One is associated with the ligand-free enzyme, substrate ligands, and the binding of the activated amino acid; the other results from the presence of high ATP concentrations and/or the synthesis of the unusual acyl-transfer product, tryptophanyl-2'(3') ATP . Together with previous physico-chemical studies of aminoacyl-tRNA synthetases, these observations suggest that the two families are related, respectively, to the biochemical processes of amino acid activation and acyl transfer . Further evidence that the crystal polymorphism results from an underlying protein conformational polymorphism has now been obtained by quantitative analysis of how crystal growth depends on pH and the substrates tryptophan and ATP . The analysis consists first in showing that crystallization conditions for the unliganded protein are very favorable, suggesting that variation in crystal growth induced by pH and substrates under otherwise identical conditions is due to their effects on the protein conformation and not on incidental perturbations of crystal growth, per se . Next, crystal growth experiments are shown to be reproducible enough to support statistical analysis of quantitative scores assigned to the results . Finally, the observed variation in scores can be attributed at high confidence levels chiefly to three effects: that of pH alone, the synergistic effects of pH plus tryptophan, and of tryptophan plus ATP . These statistical inferences are consistent with other biochemical data, and support the conclusions based on crystal packing that representative stages of the enzyme mechanism have been trapped in the different crystal forms . The pH-tryptophan interaction implies that there is a pH-dependent conformational change favoring high affinity substrate binding at high pH . The pH-ATP interaction implies that a subsequent conformational change, not previously considered, occurs between tryptophan activation and acyl transfer.

Biochemistry, 1994 May 3, 33(17), 5000 - 10
Micellar bolaform and omega-carboxylate phosphatidylcholines as substrates for phospholipases; Lewis KA et al.; A series of mixed-chain diacyl-PCs which contain an omega-COOH on the sn-2 chain {1-Cx-2-Cy-(COOH)-PC} and bolaform (1-Cx-2,2'-Cy-1'-Cx-PC) phosphatidylcholines were synthesized and examined as substrates for phospholipase A2 (Naja naja naja) and C (Bacillus cereus) . There is very little detectable phospholipase A2 activity toward pure micellar 1-acyl-2-acyl-(omega-COOH) species . In addition, when these same omega-COOH species are present at concentrations above their CMCs, they are potent inhibitors of phospholipase A2 hydrolysis of other micellar lipids . In contrast, phospholipase C hydrolysis of the same 1-acyl-2-acyl-omega-COOH)-PC species proceeds with rates comparable to that of diheptanoyl-PC . The bolaform lipids, which are tethered through a common sn-2 acyl chain, (e.g., 1-C8-2,2'-C12-1'-C8-PC) display quite different kinetic results . Under limiting Ca2+ conditions (100 microM) all the available sn-2 acyl bonds of the dimer are hydrolyzed . However, at high Ca2+ concentrations (1-10 mM) the reaction curves have a biphasic nature, characterized by an initial burst of activity followed by much slower rate . This is consistent with only the micellar 1-acyl-2-acyl-(omega-COOH)-PC produced in situ from phospholipase A2 hydrolysis of the dimer acting as an inhibitor of subsequent phospholipase A2 activity . Phospholipase C hydrolysis of the PC dimer and the sn-2 omega-COOH PC is rapid, with both available glycerophosphate groups cleaved at presumably the same rate . These results are discussed in terms of the unique physical properties (as measured by NMR and fluorescence experiments) of these phospholipids.

Ann N Y Acad Sci, 1994 May 2, 721, 310 - 25
High-density cultivation of sporeformers; Liu WM et al.; Sporeformers are sources of a large number of industrially important biological products including enzymes, antibiotics, and bioinsecticides . Cultivation of these microorganisms to high cell densities offers potential for enhancing the rates of formation as well as the concentration of the desired products in the fermentation broths in bioreactors . With this objective, investigations have been carried out involving fed-batch cultivation of Bacillus thuringiensis, which is known to produce an insecticidal crystal protein during sporulation . With appropriate management of aeration and nutrient supply, it was possible to grow the cells to > 50 g DW/l density . Nevertheless, the achievement of high cell density did not enhance the formation of crystal protein in the same proportion as the cell concentration . Further examination of this system suggested a complex interplay of energetic requirements for protein turnover during sporulation . Energy reserve material, poly-beta-hydroxybutyric acid, appeared to be linked to formation of spores and crystal protein during the sporulation phase.

Br J Dermatol, 1994 May, 130(5), 665 - 8
Bacillary angiomatosis in a patient with lymphocytic leukaemia; Torok L et al.; A 78-year-old man, who suffered from chronic lymphocytic leukaemia and diabetes mellitus, but was human immunodeficiency virus (HIV)-negative, developed disseminated angiomatous papules following a cat scratch . Bacillary angiomatosis was diagnosed by light and electron microscopic demonstration of the causative bacteria in the vascular lesions . The lesions resolved completely when he was treated with erythromycin . This case demonstrates that bacillary angiomatosis can be an important cutaneous manifestation of immunodeficiency in individuals who are not infected with the human immunodeficiency virus.

Clin Exp Immunol, 1994 May, 96(2), 225 - 9
Antigen presentation by macrophages from bacille Calmette-Guérin (BCG)-resistant and -susceptible mice; Hilburger ME et al.; We have compared the antigen-presenting capacity of macrophages from congenic BALB/c.Bcgr and BALB/c.Bcgs mice that differentially express MHC class II glycoproteins . Several different criteria were used to evaluate the presentation of a protein antigen, ovalbumin (OVA), including limiting the concentration of antigen or the numbers of macrophages, and using both native OVA and OVA peptide 323-339 . No differences in the capacity of macrophages from Bcgr and Bcgs mice to present antigen to a OVA-specific T cell hybridoma were found . Splenic macrophages from BCG-infected congenic mice also induced an equivalent amount of IL-2 production by the T cell hybridoma . The relationship of these findings to other differences that have been attributed to Bcg are discussed.

J Bacteriol, 1994 May, 176(9), 2654 - 62
Analysis of core sequences in the D-Phe activating domain of the multifunctional peptide synthetase TycA by site-directed mutagenesis; Gocht M et al.; The D-phenylalanine-activating enzyme tyrocidine synthetase I (TycA) from Bacillus brevis ATCC 8185 was overexpressed in Escherichia coli, purified to homogeneity, and assayed for ATP-PPi exchange and covalent binding of phenylalanine by the thiotemplate mechanism . Amino acid exchanges in four different cores of TycA created by site-directed mutagenesis revealed the amino acid residues involved in aminoacyladenylate formation and in covalent thioester formation . Mutations in the putative ATP-binding site SGTTGKPKG caused a decreased phenylalanine-dependent ATP-PPi exchange activity to 10% of the wild-type level for a Lys-186-to-Arg substitution and an almost complete loss of activity (< 1%) for a Lys-186-to-Thr exchange . Alteration of Asp-401 to Asn in the ATPase motif TGDL of TycA decreased the phenylalanine-dependent ATP-PPi exchange activity to 75% of wild type, while an Asp-401-to-Ser mutation decreased the activity to 10% of the wild-type level . Replacement of Ser-562 in the putative thioester-binding motif LGGDSI to Ala or Gly caused a reduction in trichloroacetic acid-precipitable TycA-{14C}phenylalanine complex to one-third of the wild-type level . However, no cleavable {14C}phenylalanine could be detected after treatment with performic acid, indicating that the resulting mutant was unable to form thioester with phenylalanine . In E . coli, TycA was labeled with beta-{3H}alanine, a precursor of 4'-phosphopantetheine, indicating that TycA is modified with a beta-alanine-containing cofactor.

J Clin Oncol, 1994 May, 12(5), 1036 - 44
Improved survival in stage III melanoma patients with GM2 antibodies: a randomized trial of adjuvant vaccination with GM2 ganglioside; Livingston PO et al.; PURPOSE: To perform a double-blind randomized trial with American Joint Commission on Cancer (AJCC) stage III melanoma patients for the following reasons: (1) to confirm our previous finding that patients with antibodies against the melanoma differentiation antigen GM2 have an improved prognosis, and (2) to demonstrate clinical benefit from GM2 antibody induction . PATIENTS AND METHODS: One hundred twenty-two patients with AJCC stage III melanoma who were free of disease after surgery were randomized: 58 to receive treatment with the GM2/BCG vaccine, and 64 to receive treatment with bacille Calmette-Guerin (BCG) alone . All patients were pretreated with low-dose cyclophosphamide (Cy) . RESULTS: GM2 antibody was detected in 50 of 58 patients treated with GM2/BCG and seven of 64 patients treated with BCG alone . With a minimum follow-up period of 51 months, there was a highly significant increase in the disease-free interval (P = .004) and a 17% increase in overall survival (P = .02) in these 57 antibody-positive patients, confirming our earlier experience . Exclusion of all patients with preexisting GM2 antibodies (one in the GM2/BCG group and five in the BCG group) from statistical analysis resulted in a 23% increase in disease-free interval (P = .02) and a 14% increase in overall survival (P = .15) at 51 months for patients treated with the GM2/BCG vaccine . However, when all patients in the two treatment groups were compared as randomized, these increases were 18% for disease-free interval and 11% for survival in the GM2/BCG treatment group, with neither result showing statistical significance . CONCLUSION: (1) Vaccination with GM2/BCG induced immunoglobulin M (IgM) antibodies in most patients . (2) GM2 antibody production was associated with a prolonged disease-free interval and survival . (3) Comparison of the two arms of this trial as randomized fails to show a statistically significant improvement in disease-free interval or survival for patients treated with GM2/BCG vaccines.

Toxicon, 1994 May, 32(5), 629 - 33
Inhibition of NGF-induced neurite outgrowth of PC12 cells by Bacillus cereus sphingomyelinase, a bacterial hemolysin; Tamura H et al.; Sphingomyelinase of Bacillus cereus, a bacterial hemolysin, reduced nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells in a dose-dependent manner . At 200 mU/ml, sphingomyelinase repressed half the neurite outgrowth of the cells at 250 ng/ml NGF . The c-fos superinduction, one of the early responses induced by NGF, was not influenced by this treatment, suggesting that the repression by sphingomyelinase occurred via a protein kinase C-independent pathway.

Infect Control Hosp Epidemiol, 1994 May, 15(5), 319 - 20
Prevalence of tuberculin reactivity among healthcare workers from a Mexican hospital; Molina-Gamboa J et al.; The prevalence of tuberculosis (TB) infection in healthcare workers (HCWs) is completely unknown in Mexico . To evaluate the frequency of TB infection in our hospital, we performed a prevalence study of tuberculin reactivity among a random sample of asymptomatic HCWs . Our results showed an extremely high prevalence of PPD reactivity (70%) among 175 HCWs, probably related to the high incidence of active TB in the general population and to the absence of preventive programsPIP: Tuberculosis (TB) is again a fast growing health problem in the world . Recent data from the World Health Organization revealed 1.7 billion persons are infected with TB in the world, with at least 8 million new cases of active pulmonary TB and 2.9 million deaths every year . A prevalence study of tuberculin reactivity was performed among a random sample of 200 asymptomatic National Institute of Nutrition (NIN) health care workers (HCWs) in a Mexican hospital in order to evaluate the frequency of TB infection . Trained nurses inoculated purified protein derivative (PPD) using the Mantoux technique . All HCWs tested completed a questionnaire about demographic data, previous TB infection, previous contact with active tuberculous patients, Bacillus Calmette-Guerin (BCG) vaccine scar, previous PPD testing, and immunosuppressive conditions . 175 (87.5%) returned for test reading, including 51 nurses, 49 physicians, 36 office workers, 28 lab workers, and 11 janitors . There were 52 males and 123 females, with a median age of 25 years (range, 18-60) . Overall, 123 (70%) of 175 HCWs were PPD reactive . The rate of reactivity was higher among persons with a history of BCG vaccination than those without such history (105/139 {75.5%} versus 18/36 {50%}; P 0.01) and was higher among HCWs at least 30 years old than among those younger than 30 years (45/64 {70%} versus 78/120 {65%}; P 0.05) . The rate of PPD positivity was not influenced by sex, area of work, or reported contact with TB patients . Median induration among reactors was 17.5 mm, whereas it was 0 mm among nonreactors . Results showed an extremely high prevalence of PPD reactivity among our HCWs, which probably is explained by the high incidence of active TB in the general population and to the absence of preventive programs . It is useful to establish the PPD reactivity of HCWs for early recognition of active disease among symptomatic reactors and for follow up of nonreactors to detect subsequent conversions among them in order to prevent the nosocomial dissemination of tuberculosis .

Anticancer Res, 1994 May-Jun, 14(3A), 901 - 3
The antimetastatic effect of IV-inoculated BCG on adenocarcinomas in the prostate-seminal vesicle complex of L-W rats; Pollard M et al.; Adenocarcinomas were induced in the prostate-seminal vesicle complex of Lobund-Wister (L-W) rats by a single IV inoculation of N-methyl-N-nitrosourea . This was followed by three slow-release S.C . implants of testosterone propionate, each at intervals of 2 months . Small (0.5 cm diameter) palpable tumors developed which enlarged during the following month to 3-4 cm diameter . At the latter stage, tumor cells spread via lymphatics to the lungs and/or by direct extension into the peritoneal cavity . Rats with small palpable tumors were inoculated IV with viable Bacillus Calmette-Guerin (BCG) . One month later, the rats were killed and examined . Untreated rats with large tumors served as controls . Comparison of the two groups revealed that body weights and tumor sizes were similar and most of them had developed metastatic tumors in the peritoneal cavity . However, lung metastases were rare in the BCG-inoculated rats compared to controls . Spleen and liver weights were significantly heavier in the BCG-treated rats . It is speculated that an intra-vascular mechanism(s), engendered by BCG, immobilized the circulating tumor cells, but not those tumor cells that spread by direct extension from the primary tumor into the peritoneal cavity.

Anticancer Res, 1994 May-Jun, 14(3A), 1083 - 7
Induction of macrophage antitumor activity by a mycobacterial fraction . In vivo and in vitro studies; Rashid G et al.; A Methanol Extraction Residue (MER) of BCG tubercle bacillus induced cytostatic activity in vitro against the murine tumor-cell lines YAC-1 and MOPC-315 in resident murine peritoneal macrophages isolated from BALB/c mice . The induction of antitumor activity was not associated with increase in release of TNF-alpha . Macrophages from mice hyperimmunized with MER (MER 3x) or from mice injected once with MER in aqueous suspension released more PGE2 following stimulation in vitro with LPS . Macrophages cultured with either MER or MER+LPS prolonged the survival time of mice bearing palpable RPC5 plasmacytoma s.c . tumors.

Anal Biochem, 1994 May 1, 218(2), 405 - 12
Stable immobilization of lipid vesicles for kinetic studies using surface plasmon resonance; Masson L et al.; In order to study the kinetics of binding between membrane vesicle surface receptors to the lepidopteran insecticidal toxins from Bacillus thuringiensis using surface plasmon resonance, we have developed a technique to immobilize membrane vesicles purified from the brush border of dissected guts from the lepidopteran insect pest Choristoneura fumiferana . Two methods using immobilized immunoglobulins against either avidin or biotin were successful in achieving stable immobilization of the vesicles (> 1.5 h) . Specificity of the immobilized receptors exposed on the vesicle surface was demonstrated, in part, by the inability of bovine serum albumin to bind to the immobilized brush border membrane vesicles . Homologous and heterologous competition experiments further demonstrated specific binding of trypsin-activated CryIA(c) toxin to the cell-surface receptors on the vesicles . Kinetic rate constants for activated cryIA(b) toxin binding to brush border vesicles were determined, revealing the presence of a high-affinity receptor on the surface of the immobilized brush border membrane vesicles.

J Pharm Sci Technol, 1994 May-Jun, 48(3), 140 - 7
Parameters governing steam sterilization of deadlegs; Young JH et al.; Use of saturated steam for sterilization-in-place (SIP) is limited by factors effecting displacement of air from deadlegs . Effects of tube diameter, length, orientation and position within a deadleg were quantitatively studied by examining temperature profiles and rates of kill of Bacillus stearothermophilus spores . Tube diameter had the greatest effect on sterilization . For small diameter tubes, 0.4 cm inside diameter (ID), air displacement was minimal and due mainly to diffusion . 8.8 cm long tubes with 0.4 cm IDs could not be sterilized at 121 degrees C . As tube diameter was increased and buoyant driven convective flow became dominant over viscous forces, sterilization was achieved and tube orientation became critical . Sterilization time, as defined by a twelve log reduction in spore population, was 75 minutes in a 19.0 cm long vertical tube with 1.7 cm ID, whereas 167 minutes were required for an 8.8 cm long tube with 1.0 cm ID . For 8.8 cm long tubes, only the 1.7 cm ID tube could be sterilized when orientated 5 degrees above horizontal . Data show that length to diameter ratios, L/Ds, do not provide a general guideline which can be used to predict sterilization . In the absence of steam bleeders, equipment should be designed to assure strong buoyancy driven convective flow to assure adequate air removal . This requires elimination of small diameter deadlegs (0.4 cm ID and less) and vertical positioning of deadlegs.

Mol Biol (Mosk), 1994 May-Jun, 28(3), 602 - 9
{Barnase mutant Ser57Ala: preparation and properties}; Kolbanovskaia EIu et al.; Barnase, an extracellular ribonuclease produced by Bacillus amyloliquefaciens, belongs to a family of small microbial ribonucleases with similar structure and properties . These enzymes hydrolyze phosphodiester bonds on the 3' side of guanosine nucleotides in RNA . The guanylic specificity of barnase is more pronounced in the hydrolysis of dinucleotides or cyclonucleotide phosphates as substrates than in the hydrolysis of RNA or polynucleotides . To have an insight into the molecular basis of this phenomenon, we mutated amino acid residue Ser-57 in the "base recognition loop" of RNase Ba . The mutant protein was expressed in Escherichia coli producing system and purified for the study of the kinetic properties in the cleavage polynucleotide reactions . It was shown that the mutation of amino acid residue Ser-57 for Ala in the "recognition loop" of RNase Ba does not significantly influence the kinetic parametres of hydrolysis of polynucleotide substrates.

Mol Biol (Mosk), 1994 May-Jun, 28(3), 586 - 94
{Multiple genes of delta-endotoxins from Bacillus thuringiensis subspecies galleriae}; Shevelev AB et al.; Cloning and expression in E . coli delta-endotoxin genes cryIAb7, cryIG and, cryIX from Bacillus thuringiensis ssp galleriae has been performed . Restriction mapping and partial sequencing have shown the identity of the 3'-terminal parts of cryIG and cryIX, although their 5'-terminal halves corresponding to the toxin are unique . Sequencing 5'-flanking region of cryIX revealed no translation initiation site, which indicates a nonfunctional state of the gene . The clusterized locating of the cryIG and cryIG genes has been shown . The absence of a promoter-like structure in the 5'-flanking region of cryIG suggests transcription of the gene as a bicistronic mRNA with cryIX . The extremely high homology of the cryIG and cryIX 3'-terminal parts (2 kB long) suggests a recombination act in the gene origin.

J Gen Intern Med, 1994 May, 9(5), 286 - 94
New developments in tuberculosis and HIV infection: an opportunity for prevention; Curtis JR et al.; As we approach 2010, the year by which we were to have eliminated TB, we find this ancient disease is making a comeback . This comeback is due to many factors, but the role of HIV infection is clearly important . HIV infection can result in changes in the pathogenesis and presentation of infection with the tubercle bacillus . Consequently, as health care providers, we must respond with changes in our usual methods of prevention, treatment, and infection control . Whereas the increase in TB is currently limited to certain geographic areas, it is likely to spread more widely . All health care providers should be aware of the changing face of TB and have a high clinical index of suspicion for this disease.

Arch Bronconeumol, 1994 May, 30(5), 236 - 9
{The efficacy of bronchoalveolar lavage in the diagnosis of pulmonary tuberculosis}; Caminero Luna JA et al.; In order to analyze the usefulness of bronchoalveolar lavage (BAL) for conventional microbiological diagnosis of tuberculosis (TB) and other mycobacteria, and to assess the need to use it or not as a routine diagnostic technique in these diseases, we studied 30 patients with mycobacteria (26 TB and 4 Mycobacterium avium-intracellulare infections) by bronchoscopy, with BAL and bronchoaspirate (BAS) bacteriological analyses also available . The results were compared with those obtained for sputum taken before and after bronchoscopy when these specimens were available . The overall yield for BAL and BAS cultures was 90%, with BAL (83.3%) specimens being more productive than BAS (73.3%) specimens . Both performed far better than the 53.8% recorded for cultures of pre-bronchoscopy sputum and 60% for post-bronchoscopy sputum . BAL was the only diagnostic specimen from 7 patients, while BAS the only one from 4 . Sensitivity was similar for the two mycobacteria studied . The results for direct bacilloscopy, however, at 30% for the two specimens, rose to 36.6% when they were analyzed together with BAS and BAL . We conclude that bronchoscopy should be performed on all patients suspected of mycobacterial infection when sputum bacilloscopy is negative and patients have no expectoration . Performance of BAL should be routine since this simple and usually uncomplicated technique produces the most productive specimens.

Microbiology, 1994 May, 140 ( Pt 5), 1001 - 13
Prime time for Bacillus megaterium; Vary PS; It is evident that B . megaterium is an intriguing organism because of its biochemical versatility, its wide distribution ecologically, its ability to undergo sporulation, and its usefulness as an industrial production strain and expression host . With the progress in genetics and the availability of molecular tools such as new transposons, vectors and efficient transformation, an understanding of some of the organization and regulation of many genes is increasing rapidly . Such recent discoveries as the ability of oxetanocin to combat some medically significant, recalcitrant viruses further demonstrates that there is much to be learned and much to benefit from continued study of B . megaterium.

J Invertebr Pathol, 1994 May, 63(3), 244 - 8
Protozoan-enhanced toxicity of Bacillus thuringiensis var . israelensis delta-endotoxin against Aedes aegypti larvae; Manasherob R et al.; The toxicity of Bacillus thuringiensis var . israelensis (Bti) in mosquito larvae was enhanced by encapsulation in the protozoan Tetrahymena pyriformis . Aedes aegypti larvae which fed on T . pyriformis loaded with Bti died about three times faster than when fed on the same concentrations of Bti alone due to ingestion of higher toxin concentrations, reflected by shorter death times of exposed populations . The best larvicidal activities were achieved at ratios of cell/spore numbers in the range of 1:200 to 1:500 . This enhancement of mortality by preincubation with T . pyriformis was higher at low Bti concentrations or in late third-instar larvae . Ninety minutes of preincubation yielded the best enhancement effect . Toxicity enhancement is very likely a consequence of concentrating large quantities of Bti spores and crystals (containing delta-endotoxin) by T . pyriformis cells and delivering them to the larvae . Shortening larval mortality time by encapsulation in T . pyriformis should reduce the exposure time of Bti to unfavorable field conditions that inactivate its larvicidal activity . Whether this method will indeed improve Bti efficacy is still to be determined.

Plant Mol Biol, 1994 May, 25(2), 141 - 57
Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability; Svensson B; Most starch hydrolases and related enzymes belong to the alpha-amylase family which contains a characteristic catalytic (beta/alpha)8-barrel domain . Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme . Crystal structures have been reported in three of these enzyme classes: the alpha-amylases, the cyclodextrin glucanotransferases, and the oligo-1,6-glucosidases . Throughout the alpha-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn . However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the beta-->alpha loops . Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of alpha-amylases, changed the distribution of alpha-, beta- and gamma-cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative alpha-1,4:alpha-1,6 dual-bond specificity of neopullulanase . Barley alpha-amylase isozyme hybrids and Bacillus alpha-amylases demonstrate the impact of a small domain B protruding from the (beta/alpha)8-scaffold on the function and stability . Prospects for rational engineering in this family include important members of plant origin, such as alpha-amylase, starch branching and debranching enzymes, and amylomaltase.

Appl Environ Microbiol, 1994 May, 60(5), 1646 - 51
Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar; Beecher DJ et al.; Bacillus cereus causes distinct exotoxin-mediated diarrheal and emetic food poisoning syndromes and a variety of nongastrointestinal infections . Evidence is accumulating that hemolysin BL is a major B . cereus virulence factor . We describe two methods for detection of hemolysin BL in crude samples and on primary culture media . In the first method, the highly unusual discontinuous hemolysis pattern that is characteristic of pure hemolysin BL was produced in sheep and calf blood agar around wells filled with crude culture supernatant from hemolysin BL-producing strains . In the second method, the pattern was formed surrounding colonies of hemolysin BL-producing strains grown on media consisting of nutrient agar, 0.15 M NaCl, 2% calf serum, and sheep or calf blood . Hemolysin BL production was detected with these methods in 41 of 62 (66%) previously identified B . cereus isolates and in 46 of 136 (34%) presumptive B . cereus isolates from soil . All nine isolates tested that were associated with diarrhea or nongastrointestinal illness were positive for hemolysin BL . The methods presented here are specific, simple, inexpensive, and applicable to the screening of large numbers of samples or isolates.

FEMS Microbiol Lett, 1994 May 1, 118(1-2), 129 - 33
Analysis of non-active engineered Bacillus thuringiensis crystal proteins; Bosch D et al.; Crystal proteins of Bacillus thuringiensis are known for their insecticidal specificity . This specificity is, to a large extent, determined by the interaction of the proteins with high-affinity binding sites on the epithelial membrane of the midgut of sensitive insects . In particular, domain II of the three domains of the toxic moiety has been implicated in specificity . To determine which sequences of the protein are involved in binding, loops of domain II which terminate in the molecular apex of CryIA(b) were replaced by the corresponding regions of CryIE, a protein with different binding characteristics and insect specificity . In contrast to expression of the wild-type genes, expression of the mutant alleles in Escherichia coli resulted in the formation of biologically inactive, insoluble aggregates . Although these aggregates could be solubilized in vitro using urea, in contrast to the wild-type CryIA(b), the mutant proteins did not correctly refold as is shown by their increased protease sensitivity and lack of biological activity . The results indicate that engineering CryI proteins, based on the CryIIIA structure, is likely to prove difficult, particularly since the conformation of CryIIIA and CryI proteins might differ in domain II.

Trans R Soc Trop Med Hyg, 1994 May-Jun, 88(3), 349 - 53
Difficulties of interpreting PPD reactions of women living in Madang, Papua New Guinea; Brabin L et al.; Malaria surveys in Madang, Papua New Guinea, previously distinguished 2 populations of women with significantly different spleen rates and immune responses to malaria . Differences between the high (HS) and low (LS) spleen rate groups suggested a defect in cellular immunity in the HS group . This paper reports a survey of purified protein derivative (PPD) responses in a sample of HS and LS women . Eighty-eight of 162 women were PPD positive (reaction size > 5 mm) . There was a marked difference in the range and size of PPD reaction between the HS and LS groups . Mean size in the LS group was 20.7 mm and in the HS group it was 12.1 mm (P = 0.02) . Failure to show differences in other indicators of specific malaria immunity indicated that the difference in PPD response was not the result of malaria-specific cell-mediated immune suppression . Many women were PPD non-responders in spite of evidence of multiple bacillus Calmette-Guerin (BCG) vaccination scars . There was no difference between HS and LS groups in the level of non-response to PPD . The results confirm an early tuberculosis survey indicating that New Guineans rapidly lose PPD reactivity to BCG vaccination . Ability to maintain a PPD response, and the quality of response, may vary according to sex and genetic background.

Cell Signal, 1994 May, 6(4), 393 - 403
Diacylglycerol kinase activity in rat liver nuclei; Previati M et al.; Membrane-depleted rat liver nuclei contain diacylglycerol (DAG) kinase showing a specific activity which doubles that of the whole homogenate . In contrast, cytoplasmic and plasma membrane marker enzymes attain a specific activity of 0.4% at the most, when nuclear DAG kinase approaches 4.5% of the total tissue activity . The enzyme shows a Km of 161 and 200 microM for ATP in both nuclei and microsomes whereas the Km for DAG is 75 microM in nuclei and 658 microM in microsomes . Octylglucoside, CHAPS and Triton X-100 behave mainly as inhibitors, while deoxycholate stimulates the enzyme activity in both cellular fractions, increasing specific activity (3.2-fold in nuclei and 29.1-fold in microsomes) and decreasing Km for DAG (39 microM in nuclei and 237 microM in microsomes) . Phospholipids and ceramide stimulate the enzyme activity in isolated nuclei, while no effect occurs in the microsomal fraction . At variance, sphingosine behaves as an inhibitor in both cellular fractions . DAG kinase also utilizes endogenous substrates mobilized by Bacillus cereus phospholipase C, which hydrolyses nuclear phosphatidylcholine and phosphatidylethanolamine and by phosphatidylinositol-specific phospholipase C, which hydrolyses nuclear PI and PIP . These data indicate that nuclear DAG can be controlled by converting it into phosphatidic acid by the action of a nuclear enzyme and support the contention that protein kinase C activity can be modulated at the nuclear level by a discrete system involving phospholipase C and DAG kinase that could operate independently from the cytoplasm.

Sante, 1994 May-Jun, 4(3), 173 - 82
{Routine vaccinations in children and adults infected with HIV}; Dabis F et al.; Five questions were raised in 1986 regarding routine immunization of children infected by the HIV: do vaccines protect these children, both in terms of immunogenicity and clinical efficacy? is immunization, particularly with live attenuated vaccines associated with an increased risk of adverse events? could the stimulation by vaccine antigens precipitate the course of paediatric HIV infection and therefore be dangerous? what are the clinical and epidemiological features of vaccine preventable diseases among HIV-infected children? what is the risk of nosocomial transmission of HIV associated with immunization practices? Based on the best available information, the WHO formulated recommendations in 1987 and updated them in 1989 . These recommendations are in general agreement with those proposed during the same period in the USA and in France (table 1) . This paper provides an update on the scientific knowledge in this field, focusing on routine childhood immunization in the context of HIV infection, especially in developing countries . The cases of bacillus Calmette-Guerin (BCG), measles vaccine, diphtheria-tetanus-pertussis and poliomyelitis vaccines are reviewed . For each of these antigens, the experience of the authors in Kigali, the capital city of Rwanda, is used as an example . A brief overview of the issue of adult immunization in the context of HIV infection concludes this review . Paediatric HIV infection should not be considered as a limiting factor in the implementation and the progression of the EPI worldwide . Experience accumulated over the last seven years, particularly in Africa, indicates that the WHO recommendations should not be modified.

Biol Pharm Bull, 1994 May, 17(5), 759 - 61
Nitric oxide production in mouse peritoneal macrophages enhanced with glycyrrhizin; Kondo Y et al.; The enhancement of nitric oxide (NO) production in glycyrrhizin (GL)-induced macrophages (M phi) in response to lipopolysaccharide (LPS) was investigated . No production in GL-induced macrophage culture supernatants was stimulated in response to LPS (10 micrograms/ml) for 24- or 48- h cultures, and these levels were compared three times with the levels in saline-induced peritoneal exudate cell cultures . Furthermore, M phi induced with proteose peptone (PP) containing GL could generate greater NO production than M phi induced with PP alone . However, no stimulation of NO production was observed by addition of GL in the cultures of M phi induced with thioglycollate or Bacillus Calmette Guerin . Moreover, GL-induced M phi showed cytostasis against such tumor target cells as L 1210 and P 388 lymphoma cell lines . These observations indicate that GL can activate the M phi in vivo system and stimulate NO production in response to LPS.

Immunol Lett, 1994 May, 40(2), 117 - 24
Autocrine regulation of ICAM-1 expression on bladder cancer cell lines: evidence for the role of IL-1 alpha; Alexandroff AB et al.; We have studied the autocrine regulation of essential expression of the intercellular adhesion molecule-1 (ICAM-1) on 8 transitional cell carcinoma (TCC) cell lines (histopathological grades 1-3) . The constitutive expression of ICAM-1 was regulated by soluble factors in an autocrine fashion . These factors were produced by all cell lines, with the exception of the MGH-U1 cell line . The effects observed could be largely attributed to IL-1 alpha . However, the residual ICAM-1 inducing activity (up to 30% of ICAM-1 induction) could not be associated with any known ICAM-1 inducers (IFN gamma, TNF alpha, TNF beta, IL-1 alpha, IL-1 beta, IL-4, retinoic acid, LPS) . In contrast to recombinant derived cytokines, the IL-1 alpha present in tissue culture supernatant was only able to induce ICAM-1 on high-grade tumours and not low-grade cells . This discriminative effect is similar to that noted following in vitro culture of tumour cells with bacillus Calmette-Guerin organisms . Whether the production of soluble factors (e.g., IL-1 alpha) by TCC cell lines plays an essential autocrine role for bladder tumours and/or affects the interaction with cells of the immune system needs to be investigated further.

Biochem J, 1994 May 1, 299 ( Pt 3), 671 - 8
Site-directed mutagenesis of beta-lactamase I: role of Glu-166; Leung YC et al.; Two Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A) . Their kinetic properties were studied and compared with those of the wild-type and the E166D mutant (with a shortened side chain) previously made by Gibson, Christensen and Waley (1990) (Biochem . J . 272, 613-619) . Surprisingly, with good penicillin substrates, Km, kcat . and kcat./Km of the two conservative mutants (E166Cmc and E166D) are similar to those of the non-conservative mutant E166A . Their kcat . values are 3000-fold lower than that of the wild-type enzyme, showing that Glu-166 is a very important residue . The acylenzyme intermediate of E166A and a good substrate, penicillin V, was trapped by acid-quench and observed by electrospray ionization mass spectrometry, suggesting that Glu-166 is more important in catalysing the deacylation step than the acylation step . The beta-lactamase I E166A mutant is about 200-fold more active than the Bacillus licheniformis E166A mutant with nitrocefin or 6 beta-furylacryloyl-amidopenicillanic acid as substrate . This suggested that other groups in the active site of the beta-lactamase I mutant may activate the catalytic water molecule for deacylation.

Biosci Biotechnol Biochem, 1994 May, 58(5), 947 - 9
Nucleotide sequence of the pectate lyase gene from alkali-tolerant Bacillus sp . YA-14; Kim JM et al.; The nucleotide sequence of the pectate lyase gene (pe lK) from alkali-tolerant Bacillus sp . was identified and analyzed . A 1,260-base pair open reading frame for the pe lK gene was observed and encoded for a protein of 420 amino acids . The signal peptide was composed of 21 amino acid residues . In the deduced primary structure of this enzyme, the three conserved regions of several pectate lyases were found and showed high homologies.

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