Bacillus

Biosci Biotechnol Biochem, 1994 May, 58(5), 830 - 5
Cloning of a new cryIA(a) gene from Bacillus thuringiensis strain FU-2-7 and analysis of chimaeric CryIA(a) proteins for toxicity; Udayasuriyan V et al.; We cloned the cryIA(a) gene from Bacillus thuringiensis strain FU-2-7, one of the toxin genes encoding lepidopteran-specific protoxins . Sequence analysis of the gene showed two amino acid differences (Pro77 to Leu and Phe965 to Ser) from the CryIA(a) of B . thuringiensis strain HD-1 . We constructed chimaeric cryIA(a) genes using FU-2-7 and HD-1 cryIA(a) genes and isolated the chimaeric protoxins, as well as the parental ones, from Escherichia coli cells harboring the recombinant plasmids to examine the effects of the two amino acid variations on the toxicity . FU-2-7 CryIA(a) protein was about half as toxic against the smaller tea tortrix, Adoxophyes sp., and one-third as toxic against the silkworm, Bombyx mori, as that of HD-1 . On the other hand, a chimaeric CryIA(a) protein with a single replacement of Phe965 to Ser had nearly the same toxicity as the HD-1 CryIA(a) against the smaller tea tortrix and one-third the toxicity against silkworm as that of HD-1 . This improved property of the chimaeric CryIA(a) protoxin may be useful for widening its application to crop protection in sericultural countries.

Appl Microbiol Biotechnol, 1994 May, 41(3), 344 - 51
Cloning and sequencing of the leu C and npr M genes and a putative spo IV gene from Bacillus megaterium DSM319; Meinhardt F et al.; The leuC gene, encoding 3-isopropylmalate dehydrogenase, the nprM gene (neutral protease) and a sporulation gene coding for a putative spoIV protein (spoIV) from Bacillus megaterium DSM 319 were cloned and the nucleotide sequences were determined . The leuC gene is 1101 bp in length, preceded by a ribosome binding site; no promoter consensus sequence could be found . The nucleotide sequence from nprM when compared to the recently published gene from B . megaterium ATCC 14581 exhibited only a 17-base pair deviation . From a sporulation mutant isolated after transposon-mutagenesis with transposon Tn917 the insertion site of the transposon was cloned and adjacent chromosomal fragments were characterized . An open reading frame that encodes for a putative spo protein of 247 amino-acid residues was identified.

Trends Biotechnol, 1994 May, 12(5), 207 - 11
Engineering surface loops of proteins--a preferred strategy for obtaining new enzyme function; el Hawrani AS et al.; A prerequisite for the rational redesign of enzymes is that altering amino acids in an attempt to obtain new biological function does not unexpectedly alter the globular, natural framework of the native protein on which the design is being executed . The results of combinatorial-mutagenesis strategies suggest that random variation of amino acid sequence is most easily tolerated at the solvent-exposed surfaces of a protein . This review analyses effective redesigns of Bacillus stearothermophilus lactate dehydrogenase (bsLDH), in which all residue variations are at solvent-exposed surfaces . The majority of these variations were located within surface loops, which interconnect stable secondary structures traversing the globular core of the protein.

Lett Appl Microbiol, 1994 May, 18(5), 260 - 3
Characterization of Bacillus licheniformis with the RAPD technique (randomly amplified polymorphic DNA); Stephan R et al.; RAPD technique (randomly amplified polymorphic DNA) was used for epidemiological subtyping of Bacillus licheniformis and other Bacillus spp . Within 46 isolates of B . licheniformis, up to 10 strain types could be determined when two 10-mer primers were used . RAPD patterns, which were found in eight further strains of Bacillus spp., clearly differed from those of B . licheniformis . Thus RAPD technique proved to be a promising tool for characterization of Bacillus spp.

Anticancer Res, 1994 May-Jun, 14(3A), 837 - 40
Enhanced cytotoxicity caused by increased DNA strand breakage resulting from synergistic potentiation of bleomycin with Bacillus thuringiensis subsp . israelensis delta-endotoxin; Yokoyama Y et al.; We previously reported that a 25 kDa Bacillus thuringiensis subsp . israelensis (BTI) delta-endotoxin potentiates the cytotoxicity of bleomycin (BLM) in cultured cells . In order to clarify the mechanism involved, we examined the induction of DNA strand breakage by BLM in the presence of BTI toxin . Results showed that the increase in strand breakage resulted in enhanced cytotoxicity through the synergistic potentiation of BLM with BTI toxin.

J Clin Microbiol, 1994 May, 32(5), 1166 - 71
Isolation and characterization by immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis; Maurin M et al.; Rochalimaea quintana was isolated from the blood of a French human immunodeficiency virus-infected patient with bacillary angiomatosis . The isolate showed the typical growth characteristics of Rochalimaea species and was inert when typical biochemical testing was used . The purpose of the present work was to characterize and compare this new isolate with reference strains of R . quintana, Rochalimaea vinsonii, and Rochalimaea henselae by using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot), restriction fragment length polymorphism-PCR of the citrate synthase gene, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis . SDS-PAGE, Western blot, restriction fragment length polymorphism-PCR with TaqI enzyme, and 16S rRNA gene sequencing could differentiate the three Rochalimaea species and allowed characterization of the French isolate as R . quintana . However, identification of the Rochalimaea isolate to the species level was more easily obtained by immunofluorescence with specific murine antisera . Pulsed-field gel electrophoresis allowed differentiation of the French R . quintana isolate from R . quintana Fuller and may serve as an epidemiological tool.

J Bacteriol, 1994 May, 176(10), 2835 - 45
Tn5401, a new class II transposable element from Bacillus thuringiensis; Baum JA; A new class II (Tn3-like) transposable element, designated Tn5401, was recovered from a sporulation-deficient variant of Bacillus thuringiensis subsp . morrisoni EG2158 following its insertion into a recombinant plasmid . Sequence analysis of the insert revealed a 4,837-bp transposon with two large open reading frames, in the same orientation, encoding proteins of 36 kDa (306 residues) and 116 kDa (1,005 residues) and 53-bp terminal inverted repeats . The deduced amino acid sequence for the 36-kDa protein shows 24% sequence identity with the TnpI recombinase of the B . thuringiensis transposon Tn4430, a member of the phage integrase family of site-specific recombinases . The deduced amino acid sequence for the 116-kDa protein shows 42% sequence identity with the transposase of Tn3 but only 28% identity with the TnpA transposase of Tn4430 . Two small open reading frames of unknown function, designated orf1 (85 residues) and orf2 (74 residues), were also identified . Southern blot analysis indicated that Tn5401, in contrast to Tn4430, is not commonly found among different subspecies of B . thuringiensis and is not typically associated with known insecticidal crystal protein genes . Transposition was studied with B . thuringiensis by using plasmid pEG922, a temperature-sensitive shuttle vector containing Tn5401 . Tn5401 transposed to both chromosomal and plasmid target sites but displayed an apparent preference for plasmid sites . Transposition was replicative and resulted in the generation of a 5-bp duplication at the target site . Transcriptional start sites within Tn5401 were mapped by primer extension analysis . Two promoters, designated PL and PR, direct the transcription of orf1-orf2 and tnpI-tnpA, respectively, and are negatively regulated by TnpI . Sequence comparison of the promoter regions of Tn5401 and Tn4430 suggests that the conserved sequence element ATGTCCRCTAAY mediates TnpI binding and cointegrate resolution . The same element is contained within the 53-bp terminal inverted repeats, thus accounting for their unusual lengths and suggesting an additional role for TnpI in regulating Tn5401 transposition.

FEBS Lett, 1994 Apr 25, 343(2), 177 - 80
Stereochemical course of the hydrolysis reaction catalyzed by chitinases A1 and D from Bacillus circulans WL-12; Armand S et al.; Chitinases A1 and D were purified from the periplasmic proteins produced by Escherichia coli HB101 harbouring recombinant plasmids carrying respectively the chiA and chiD genes of Bacillus circulans WL-12 . HPLC analysis indicated that during the hydrolysis of chitotriose, both chitinases initially produce N-acetylglucosamine and only one anomer of chitobiose . 1H NMR spectroscopy of the hydrolysis of chitotetraitol showed that this anomer corresponds to beta-chitobiose, demonstrating that chitinases A1 and D act by a molecular mechanism that retains the anomeric configuration . This mechanism is similar to that of lysozymes although both chitinases belong to a family of proteins sharing no demonstrable amino acid sequence similarity with lysozymes.

Carbohydr Res, 1994 Apr 16, 257(1), 155 - 61
Effect of modifying histidine residues on the action of Bacillus amyloliquefaciens and barley-malt alpha-amylases; Nakatani H et al.; Modification of porcine pancreatic alpha-amylase (PPA) and Taka-amylase A(TAA) with diethyl pyrocarbonate (DEP) causes activation of the release of p-nitrophenol from p-nitrophenol alpha-maltoside (G2PNP), and a decrease in amylase activity (hydrolysis of alpha-1,4 glucosidic bonds in starch) . Among the possible sites of modification, attention focuses on three histidine residues present around the active site of alpha-amylases of many different origins . In PPA these are His 101, His 201, and His 299, with His 101 and His 299 being very close to the site of catalysis and thus perhaps directly or indirectly involved in the catalysis . On the other hand, His 201 is located on the aglycon side of the catalytic site, and we have suggested that it is involved in the increase of PNP release after chemical modification . Investigations of site-directed mutagenesis of the histidine residues of human pancreatic alpha-amylase support this identification . Although the degree of sequence similarity among alpha-amylases of different origins is low, there are several conserved short regions . Most belong to the structural components of the active site in PPA and TAA . Furthermore, there is a close similarity in the three-dimensional structures of PPA and TAA . The conserved residues around the active site in all alpha-amylases suggest some universal structural similarities in these active sites . Therefore, we examined the effects of the chemical modification of histidine residues in Bacillus amyloliquefaciens alpha-amylase(BLA) with DEP and made a comparison with modification of barley alpha-amylase isoenzyme II(BAII), using identical substrate systems . These two alpha-amylases have more substrate binding subsites than PPA and TAA, and have similar action patterns with malto-oligosaccharides.

Biochem Biophys Res Commun, 1994 Apr 15, 200(1), 359 - 64
A specific binding protein from Tenebrio molitor for the insecticidal toxin of Bacillus thuringiensis subsp . tenebrionis; Belfiore CJ et al.; Biopesticides based on the bacterium Bacillus thuringiensis have attracted wide attention as safe alternatives to chemical insecticides . In this paper, we report, for the first time, the identification of a single binding protein from a coleopteran insect, Tenebrio molitor, that is specific for the cryIII toxin of B . thuringiensis . The protein appeared as a single band of 144 kDa on radioligand and immunoblots of total proteins extracted from brush border membrane vesicles of the midgut of T . molitor . Radiolabelled cryIIIA toxin bound to the protein with a Kd value of 17.5 nM and could be specifically blocked by unlabelled toxin but not by toxins from other subspecies of B . thuringiensis . This study lays the groundwork to clone the cryIIIA toxin binding protein and to determine the molecular mechanism(s) of toxin action.

Ann Intern Med, 1994 Apr 15, 120(8), 653 - 62
Nosocomial pneumonia in mechanically ventilated patients receiving antacid, ranitidine, or sucralfate as prophylaxis for stress ulcer . A randomized controlled trial; Prod'hom G et al.; OBJECTIVE: To assess three anti-stress ulcer prophylaxis regimens in mechanically ventilated patients for bacterial colonization, early- and late-onset nosocomial pneumonia, and gastrointestinal bleeding . DESIGN: Randomized controlled trial . PATIENTS: Consecutive eligible patients with mechanical ventilation and a nasogastric tube . Of 258 eligible patients, 244 were assessable . SETTING: Medical and surgical intensive care units . INTERVENTION: At intubation, patients were randomly assigned to receive one of the following: antacid (a suspension of aluminum hydroxide and magnesium hydroxide), 20 mL every 2 hours; ranitidine, 150 mg as a continuous intravenous infusion; or sucralfate, 1 g every 4 hours . MEASUREMENTS: Using predetermined criteria, the incidence of gastric bleeding, gastric colonization, early-onset pneumonia, and late-onset pneumonia was assessed in patients intubated for more than 24 hours . RESULTS: Of 244 assessable patients, macroscopic gastric bleeding was observed in 10%, 4%, and 6% of patients assigned to receive sucralfate, antacid, and ranitidine, respectively (P > 0.2) . The incidence of early-onset pneumonia was not statistically different among the three treatment groups (P > 0.2) . Among the 213 patients observed for more than 4 days, late-onset pneumonia was observed in 5% of the patients who received sucralfate compared with 16% and 21% of the patients who received antacid or ranitidine, respectively (P = 0.022) . Mortality was not statistically different among the three treatment groups . Patients who received sucralfate had a lower median gastric pH (P < 0.001) and less frequent gastric colonization compared with the other groups (P = 0.015) . Using molecular typing, 84% of the patients with late-onset gram-negative bacillary pneumonia were found to have gastric colonization with the same bacteria before pneumonia developed . CONCLUSION: Stress ulcer prophylaxis with sucralfate reduces the risk for late-onset pneumonia in ventilated patients compared with antacid or ranitidine.

Biochim Biophys Acta, 1994 Apr 13, 1205(2), 183 - 8
Specificity of chitosanase from Bacillus pumilus; Fukamizo T et al.; Partially (25-35%) N-acetylated chitosan was digested by chitosanase from Bacillus pumilus BN-262, and structures of the products, partially N-acetylated chitooligosaccharides, were analyzed in order to investigate the specificity of the chitosanase . The chitosanase produced glucosamine (GlcN) oligosaccharides abundantly, indicating that the chitosanase splits the beta-1,4-glycosidic linkage of GlcN-GlcN . The chitosanase also produced hetero-oligosaccharides consisting of glucosamine and N-acetyl-D-glucosamine (GlcNAc) . Three types of the hetero-oligosaccharides purified by cation-exchange chromatography and HPLC were found to have GlcNAc residue at their reducing end and GlcN residue at their non-reducing end, indicating that the chitosanase can also split the linkage of GlcNAc-GlcN . The determination of the mode of action toward partially N-acetylated chitosan enables a classification of chitosanases according to their specificities and a more precise definition of chitosanases.

J Biol Chem, 1994 Apr 8, 269(14), 10378 - 83
Expression in Escherichia coli of genes encoding the E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex of Bacillus stearothermophilus and assembly of a functional E1 component (alpha 2 beta 2) in vitro; Lessard IA et al.; The E1 alpha and E1 beta subunits of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus were produced from two genes overexpressed separately in Escherichia coli . A functional E1 enzyme was generated from disrupted mixtures of cells containing the separately overexpressed E1 alpha and E1 beta genes . The purified E1 enzyme exhibited an apparent molecular mass of 150,000 Da, consistent with an alpha 2 beta 2 structure . The Km for pyruvate and kcat (30 degrees C) were found to be 0.9 +/- 0.2 microM and 0.47 +/- 0.03 s-1, respectively . The purified E1 alpha subunit existed as a monomer (42,000 Da), whereas the E1 beta subunit existed mainly (95%) in a tetrameric form (145,000 Da) . Mixing equimolar amounts of the pure recombinant E1 alpha and E1 beta subunits in vitro generated a functional E1 enzyme with a molecular mass and an E1 activity similar to those of the E1(alpha 2 beta 2) enzyme purified from disrupted mixtures of cells containing individually expressed subunits . Mixing individual subunits in vitro with one of the subunits in excess resulted in complete assembly of the lesser subunit into the intact E1 (alpha 2 beta 2) enzyme . Thus, no chaperonin is needed in vitro to promote the assembly of the separate subunits to form the E1 component of the pyruvate dehydrogenase multienzyme complex of B . stearothermophilus.

Biochemistry, 1994 Apr 5, 33(13), 3919 - 26
A calorimetric study of the thermal stability of barnase and its interaction with 3'GMP; Martinez JC et al.; We have used high-sensitivity differential scanning calorimetry to characterize the thermal stability of barnase from Bacillus amyloliquefaciens in the pH range 2.0-5.0 . The energetics of the interaction between barnase and its inhibitor 3'GMP have been studied by isothermal titration calorimetry in the temperature range 15-30 degrees C . Scanning calorimetry experiments were also made with the protein in the presence of various concentrations of 3'GMP at pH 4.5 . A novel, simple procedure is proposed to obtain binding parameters from scanning calorimetry data . This method is based on the calculation of the partition functions of the free and the ligand-bound protein . Isothermal calorimetry shows that at 25 degrees C 3'GMP binds to a single site in barnase with a delta Cp of -250 +/- 50 J/(K.mol) . Both free barnase and ligand-bound barnase undergo a highly reversible, two-state thermal unfolding process under our experimental conditions . delta G and delta Cp unfolding values are similar to others found for globular proteins, whereas delta H and delta S unfolding values are unusually high at the denaturation temperature of barnase . We have also found unexpectedly that the thermodynamic unfolding parameters of barnase fit neither the trend of values described in the literature for the correlation between delta Cp and delta H nor the limiting specific enthalpy value in the correlation between delta H and Tm for globular proteins . These discrepancies might be related to particular features of the folded and/or unfolded states of the protein.

J Appl Bacteriol, 1994 Apr, 76(4), 361 - 7
Chitinolytic properties of Bacillus pabuli K1; Frandberg E et al.; The chitinolytic properties of Bacillus pabuli K1 isolated from mouldy grain was studied . Chitinase activity was measured as the release of p-nitrophenol from p-nitrophenyl-N,N'-diacetylchitobiose . Influences of substrate concentration and different environmental variables on growth and chitinase activity were determined . The optimum environmental conditions for chitinase production were: 30 degrees C, initial pH 8, initial oxygen 10% and aw > 0.99 . Chitinase production was induced when B . pabuli K1 was grown on colloidal chitin . The smallest chito-oligosaccharide able to induce chitinase production was N,N'-diacetylchitobiose, (GlcNAc)2 . Production was also induced by (GlcNAc)3 and (GlcNAc)4 . When the bacterium was grown on glucose or N-acetylglucosamine, no chitinases were formed . The highest chitinase production observed was obtained with colloidal chitin as substrate . The production of chitinases by B . pabuli K1 growing on chitin was repressed by high levels (0.6%) of glucose . The production was also repressed by 0.6% starch, laminarin and beta-glucan from barley and by glycerol . The addition of pectin and carboxymethyl cellulose increased chitinase production.

J Appl Bacteriol, 1994 Apr, 76(4), 327 - 35
Multilocus enzyme electrophoresis on agarose gel as an aid to the identification of entomopathogenic Bacillus sphaericus strains; Zahner V et al.; Sixty strains of Bacillus sphaericus, including 31 insect pathogens were studied by multilocus enzyme electrophoresis and were classified into 44 zymovars (electrophoretic types) . Among the entomopathogenic strains, 11 belong to the same zymovar (Z59) indicating a widespread frequent genotype . Bands of enzyme activity were not detected among the strains for the loci GPI (E.C.5.3.1.9), G6P (E.C.1.1.1.49), 6PG (E.C.1.1.1.44) and ME (E.C.1.1.1.40) . The enzymatic loci NP (E.C.2.4.2.1) and ACON (E.C.4.2.1.3) were monomorphic while the other enzymes, MDH (E.C.1.1.1.37), LeDH (E.C.1.4.1.9), ADH (E.C.1.4.1.1), EST (E.C.3.1.1.1), PEP-2 (E.C.3.4.11.1), PEP-3 (E.C.3.4.11) and PEP-D (E.C . 3.4.13.9) were polymorphic . The genetic variation in the non-insect pathogenic group seemed to be greater than in the entomopathogenic group . This latter group appears to be distinct from other strains of these species . All insect pathogens were recovered in the same phenetic cluster and a diagnostic allele is reported for the identification of entomopathogenic strains.

J Appl Bacteriol, 1994 Apr, 76(4), 307 - 13
Characterization of larvicidal toxin protein from Bacillus thuringiensis serovar japonensis strain Buibui specific for scarabaeid beetles; Hori H et al.; The delta-endo toxin proteins from Bacillus thuringiensis which kill the larvae of various scarabaeid beetles such as Anomala cuprea, A . rufocuprea and Popillia japonica were purified by DEAE ion exchange chromatography . A protein with a molecular size of 130 kDa was purified . During the purification a minor peak was also detected which was estimated to be 67 kDa by SDS-PAGE . Both 130 and 67 kDa proteins showed larvicidal activity against A . cuprea . The lethal concentration of the 130 kDa protein which killed 50% of the larvae tested (LC50) against A . cuprea was 2 micrograms g-1 compost . A comparison by SDS-PAGE of the V8 protease digestion pattern of the 130 and 67 kDa larvicidal proteins showed that proteolytic resistant core peptides of approximately 60 kDa molecular size were resulted . The N-terminus amino acid sequence of the 130 and 67 kDa proteins was determined to be NH2-XXPNNQNEYEIIDAL and NH2-XSRNPGTFI, respectively, which is not identical to the sequence of CryIA, CryIB, CryIC and CryIII proteins.

Immunol Cell Biol, 1994 Apr, 72(2), 169 - 75
Assays for adjuvanticity of new formulations and of carrier proteins for inducing antibody responses to selected immunogens in the squirrel monkey Saimiri sciureus; Perraut R et al.; Different ways to improve antibody (Ab) responses following immunizations with selected antigens (TT and HSVgD) were investigated, and thus new adjuvant formulations and carrier molecules in a non-human primate experimental host, the squirrel monkey Saimiri sciureus, were assayed . Both quantitative and qualitative humoral responses were determined by means of radio-immunoassays using monoclonal Ab directed at Saimiri IgG . First, the adjuvanticity of the Syntex (SAF-1) adjuvant and of five new adjuvant formulations were assessed towards the selected Ag . This indicated that all the adjuvants induced similar antigen-specific Ab responses, although the adjuvants could modify to some extent the pattern of the qualitative Ab response . Second, we evaluated an adjuvant-free vaccine approach using a synthetic Ag from the circumsporozoite protein of Plasmodium falciparum as immunogen, this Ag being coupled to purified protein derivative (PPD) or to a recombinant heat shock protein from Mycobacterium tuberculosis . These constructs led to good antibody responses as well as an excellent memory effect . Bacille Calmette-Guerin (BCG) priming was required in conjunction with PPD as a carrier molecule to allow homogeneous Ab responses, whereas the heat shock protein construct gave a less homogeneous Ab response regardless of whether a BCG priming was done . We, in addition, discuss the relevance of Saimiri monkeys as experimental models for studies directed at evaluating the immunogenicity of further vaccine candidates.

Arch Biochem Biophys, 1994 Apr, 310(1), 126 - 33
Affinity isolation and characterization of cytochrome P450 102 (BM-3) from barbiturate-induced Bacillus megaterium; Black SD et al.; Cytochrome P450 102 (BM-3) is a catalytically self-sufficient enzyme from Bacillus megaterium that is presently accepted as an important model of the mammalian microsomal P450 monooxygenase system . We have developed a novel affinity approach to purify P450 102 in a single chromatographic step and have studied the spectroscopic, catalytic, nucleotide binding, and crystallization properties of the highly purified enzyme . B . megaterium ATCC 14581 was grown to high cell density, and P450 102 was purified rapidly and in high yield by chromatography on adenosine-2',5'-diphosphate agarose from crude cell-free extract . The cytochrome bound to the column with remarkable avidity, in contrast to the significantly weaker binding observed for NADPH-cytochrome P450 reductase . Chromatographic behavior also showed that the cytochrome bound NADP(+)-type nucleotides more tightly than any other cellular polypeptide . The purified protein was electrophoretically homogeneous and had essentially theoretical contents of FAD, FMN, and heme . Optical spectra showed the expected heme and flavin absorption bands, and three previously undescribed charge-transfer-type absorptions were characterized . Molar extinction coefficients in the oxidized, fully reduced, and ferrous carbonyl states have been determined; notable is the large soret extinction in the ferrous carbonyl state (epsilon 449 nm = 143,500 M-1 cm-1) . Final preparations were active in the oxidation of a wide variety of substrates . Of the C14 alkyl compounds studied, tetradecyltrimethylammonium bromide showed the highest substrate-dependent oxidation of NADPH, followed by myristate and myristyl alcohol; however, myristate exhibited the lowest Km value . Activities were tightly coupled to NADPH oxidation (> 97%) . Phenobarbital, benzphetamine, cocaine, cyclohexane, methanol, ethanol, retinoic acid, benzoate, heptaflourobutyrate, and 7-ethoxycoumarin were not substrates . NADP+ titrations showed, as expected, that the coenzyme was bound very tightly, with an average Kd of 580 nM . Our preparations of P450 102 are of sufficient purity and stability that crystals of the native holoenzyme have been grown.

J Bacteriol, 1994 Apr, 176(8), 2252 - 8
The internal pH of the forespore compartment of Bacillus megaterium decreases by about 1 pH unit during sporulation; Magill NG et al.; Previous work has shown that the internal pH of dormant spores of Bacillus species is more than 1 pH U below that of growing cells but rises to that of growing cells in the first minutes of spore germination . In the present work the internal pH of the whole Bacillus megaterium sporangium was measured by the distribution of the weak base methylamine and was found to decrease by approximately 0.4 during sporulation . By using fluorescence ratio image analysis with a fluorescein derivative, 2',7'-bis(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), whose fluorescence is pH sensitive, the internal pH of the mother cell was found to remain constant during sporulation at a value of 8.1, similar to that in the vegetative cell . Whereas the internal pH of the forespore was initially approximately 8.1, this value fell to approximately 7.0 approximately 90 min before synthesis of dipicolinic acid and well before accumulation of the depot of 3-phosphoglyceric acid . The pH in the forespore compartment was brought to that of the mother cell by suspending sporulating cells in a pH 8 potassium phosphate buffer plus the ionophore nigericin to clamp the internal pH of the cells to that of the external medium . We suggest that at a minimum, acidification of the forespore may regulate the activity of phosphoglycerate mutase, which is the enzyme known to be regulated to allow 3-phosphoglyceric acid accumulation during sporulation.

Clin Exp Immunol, 1994 Apr, 96(1), 86 - 90
T cell reactivity against antigen 85 but not against the 18- and 65-kD heat shock proteins in the early stages of acquired immunity against Mycobacterium leprae; Launois P et al.; T cell proliferation and interferon-gamma (IFN-gamma) production of peripheral blood mononuclear cells (PBMC) from 20 household contacts were tested against the 18- and 65-kD heat shock proteins from Mycobacterium leprae (ML18 and ML65 respectively) and antigen 85 from Myco . bovis bacille Calmette-Guerin (BCG) (Ag 85) during a 12-months follow-up study . Among the eight contacts that became positive, eight showed positive reactivity against Ag 85, 5/8 against ML65 and 4/8 against ML18 at the end of the study . Of the 16 contacts who were lepromin-positive either at first or second testing, all responded to Ag 85, 11 to ML 65, but only eight reacted to ML18 antigen . Contacts who were lepromin-positive at first testing developed responses to ML18 only at second testing . In contrast, among the four contacts that remained lepromin-negative during the follow up, three proliferated to Ag 85 either at first or second testing, but only one produced IFN-gamma against Ag 85 at the end of the study . These results demonstrated that T cell reactivity and particularly IFN-gamma secretion against Ag 85, but not against ML18 and ML65, might be a predominant mechanism in the early stages of acquired protective immunity against Myco . leprae.

Clin Exp Immunol, 1994 Apr, 96(1), 79 - 85
Cellular and humoral immune responses to recombinant 65-kD antigen of Mycobacterium leprae in leprosy patients and healthy controls; Ilangumaran S et al.; Cellular and humoral immune responses to recombinant 65-kD antigen of Mycobacterium leprae (rML65) were studied in leprosy patients and healthy contacts from a leprosy-endemic population . Peripheral blood mononuclear cells from a considerable proportion of tuberculoid leprosy patients, healthy contacts and non-contacts showed proliferative response to rML65 in vitro . A strong positive correlation was observed between the responses to rML65 and bacille Calmette-Guerin (BCG) or leprosin A . Addition of recombinant IL-2 (rIL-2) enhanced the proportion of responders to rML65 considerably in all groups of leprosy patients, healthy contacts and non-contacts . Among lepromatous patients this enhancement was more pronounced in the bacterial index (BI)-negative group . These results indicate that the 65-kD antigen of Myco . leprae is a dominant T cell immunogen in our study population . Though lepromatous patients showed poor lymphoproliferative response to rML65, their IgG antibody levels to the same antigen were markedly high . Most of the BI-positive lepromatous patients with elevated anti-rML65 IgG levels did not show T cell reactivity even with the addition of rIL-2 . On the other hand, tuberculoid leprosy patients, healthy contacts and non-contacts showed good T cell reactivity but low levels of IgG antibodies to rML65, thus indicating the presence of an inverse relationship between cell-mediated and humoral immune responses to a defined protein antigen of Myco . leprae in humans . A significant proportion of individuals among tuberculoid leprosy patients, healthy contacts and non-contacts showed neither T cell reactivity nor elevated levels of IgG antibody to rML65 . However, in most of these subjects, a T cell response to rML65 was demonstrable with the addition of rIL-2 . These results are discussed with reference to the immunoregulatory mechanisms occurring during Myco . leprae infection on the basis of differential activation of Th1 and Th2 subsets.

J Biol Chem, 1994 Apr 1, 269(13), 10088 - 92
A mixture of Manduca sexta aminopeptidase and phosphatase enhances Bacillus thuringiensis insecticidal CryIA(c) toxin binding and 86Rb(+)-K+ efflux in vitro; Sangadala S et al.; CryIA(c) delta-endotoxin, a member of the CryI family of Bacillus thuringiensis insecticidal proteins, specifically recognizes and binds with high affinity to target proteins in the midgut of susceptible insects . Protein blots of Manduca sexta brush-border membranes probed with 125I-CryIA(c) identify a major binding protein of 120 kDa and a minor binding protein of 65 kDa . Monoclonal antibodies were raised against the 120-kDa toxin binding protein . Using isoelectric focusing and monoclonal antibodies (2B3, 8G1, and 12B8) 120- and 65-kDa brush-border proteins were isolated . Labeled CryIA(c) and monoclonal antibodies probed to blots of the affinity-selected proteins recognized the 120- and 65-kDa proteins . When reconstituted into phospholipid vesicles, antibody-selected proteins increased toxin binding (35%) and enhanced toxin-induced 86Rb+ release up to 1000-fold . The 120-kDa protein was identified as aminopeptidase N (EC 3.4.11.2) . A CryIA(c)-sensitive phosphatase was also present in the 120/65-kDa protein mixture . These findings provide the first identification of B . thuringiensis toxin binding proteins, although confirmation is needed in vivo.

J Clin Pharm Ther, 1994 Apr, 19(2), 101 - 4
Intravesical BCG therapy of superficial bladder cancer: study of adverse effects; Galvan L et al.; Immunotherapy with intravesical bacillus Calmette-Guerin (BCG) has proved to be more effective than most chemotherapeutic agents in the prophylaxis and treatment of superficial bladder tumours . Side-effects, both local and systemic, are the main limitations against its use . With the aim of lowering the incidence and severity of side-effects, we started to use two vials of BCG, Connaught type, per instillation, instead of three vials, as recommended by the manufacturer . We prospectively reviewed adverse effects of BCG treatment at the lower dosage in 92 patients . Compared with other series, we found a similar incidence of adverse effects except for some local effects as haematuria which showed a higher incidence, but we also found a lower rate of tumour relapse . Four primary tumours were recorded during the study period . In our open study, a lower BCG intravesical dosage is not followed by a reduction in side-effects.

Eur J Biochem, 1994 Apr 1, 221(1), 87 - 100
Sequential 1H and 15N nuclear magnetic resonance assignments and secondary structure of the N-terminal lipoyl domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii; Berg A et al.; The N-terminal lipoyl domain (79 residues) of the transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been sub-cloned and produced in Escherichia coli . Over-expression exceeds the capacity of E . coli cells to lipoylate all expressed lipoyl domain, but addition of lipoic acid to the growth medium results in expression of fully lipoylated domain . A two-dimensional homo- and heteronuclear NMR study of the lipoyl domain has resulted in sequential 1H and 15N resonance assignments of the unlipoylated form of the protein . Small differences in chemical shift values for protons of residues in the vicinity of the lipoyl-lysine residue are observed for the lipoylated form of the domain, suggesting that the conformation of the lipoyl domain is not altered significantly by the coupled cofactor . From nuclear Overhauser effects, backbone coupling constants and slowly exchanging amide protons, two antiparallel beta-sheets, each containing four strands, were identified . The lipoyl-lysine residue is exposed to the solvent and located in a type-I turn between two strands . The N- and C-terminal residues of the folded chain are close together in the other sheet . Preliminary data on the relative three-dimensional orientation of the two beta-sheets are presented . Comparison with the solution structure of the lipoyl domain of the Bacillus stearothermophilous pyruvate dehydrogenase complex shows resemblance to a large extent, despite the sequence identity of 31%.

Biochem Mol Biol Int, 1994 Apr, 32(6), 1049 - 57
Heat and osmotic stress enhance the development of cytoplasmic serine proteinase activity in sporulating Bacillus megaterium; Vachova L et al.; The intracellular Ca(2+)-dependent serine proteinase (ISP1) activity in the cytoplasm of nongrowing Bacillus megaterium incubated in a sporulation medium was determined at 35 degrees C and at temperatures decreasing the sporulation frequency (42 degrees C) or suppressing sporulation (43.5 degrees C) . The enzyme in the crude cytoplasmic fraction was partially inhibited by a loosely bound inhibitor(s) because the ISP1 activity rose after protein fractionation by HPLC . Temperature shift-up or osmotic stress applied at 35 degrees C increased the development of the ISP1 activity several times . The increase was caused at least partially by the synthesis of the enzyme protein, as proved by SDS-PAGE and immunoblotting of the cytoplasm . This enzyme thus probably belongs among heat-shock proteins.

Int J Food Microbiol, 1994 Apr, 22(1), 11 - 22
Mechanism of inhibited growth of Bacillus pumilus by Propionibacterium freudenreichii subsp . shermanii; Marshall DL et al.; Physiological studies were conducted in an attempt to elucidate the mechanism of inhibition of Bacillus pumilus by Propionibacterium freudenreichii subsp . shermanii . Inhibition of B . pumilus by P . shermanii occurred in media supplemented with 1% glucose, indicating that glucose utilization by the latter bacterium was not responsible for growth inhibition of the former bacterium . The medium pH in which P . shermanii inhibited the growth of B . pumilus was 4.3 . Propionic acid was positively identified in the culture medium in which B . pumilus was inhibited by P . shermanii . The presence of propionic acid and a low medium pH may account for the inhibition of B . pumilus by P . shermanii . Sodium lactate concentrations of 0.8-1.0% were essential for the continuous growth of and propionic acid production by P . shermanii . Thus, use of P . shermanii to inhibit B . pumilus in foods would likely require a lactate source.

Protein Expr Purif, 1994 Apr, 5(2), 118 - 24
Purification procedure for bacterial translational initiation factors IF2 and IF3; Soffientini A et al.; In bacteria the initiation of protein synthesis is a complex phenomenon in which specific proteins, termed initiation factors (IFs) IF1, IF2, and IF3, are involved . Notwithstanding the progress made in understanding their functions, the precise molecular mechanisms of action of these factors remain somewhat obscure . One reason for this lack of knowledge is the difficulty involved in purifying sufficient quantities of these proteins . We have developed a new procedure for purification of IFs from recombinant Escherichia coli strains producing high levels of E . coli IF3 and Bacillus stearothermophilus IF2 . This new procedure is quicker than previous methods, easily scaled up to large volumes, and can be used, with only minor modifications, for different IFs . This new purification method consists essentially of one chromatographic (FPLC) separation on an ion-exchange resin (S-Sepharose fast-flow or Mono-S HR) . Using this procedure we have been able to obtain chromatographically pure and biologically active preparations of both IF2 and IF3.

Am J Dent, 1994 Apr, 7(2), 98 - 102
Effectiveness of two types of sterilizers on the contents of sharps containers; Palenik CJ et al.; The purpose of this study was to evaluate the killing effect that a gravity steam autoclave (GSA), a high-vacuum steam sterilizer (HVA) or an unsaturated chemical vapor sterilizer (UCV) had on endospores present on strips or applied to dental needles within three sizes of sharps containers . Commercial spore strips containing 1.7 x 10(5) Bacillus stearothermophilus endospores were used, while the needles were soiled with an equal number of spores or with spores mixed with blood . Needles were tested capped and uncapped . Initial operating conditions were for the: (1) GSA were 129 kPa at 124 degrees C for 30 minutes; (2) HVA were 214 kPa at 131 degrees C for 10 minutes and (3) UCV, 172 kPa at 134 degrees C for 20 minutes . Strips and needles were placed within 3/4 filled containers . Fill materials consisted of needles, syringes, plastic sheaths and scalpel blades . Containers were processed upward or on their sides . Twenty-five strips or needles comprised a test group for each operational parameter . Processed strips and needles were aerobically cultured at 56 degrees C for 7 days . If sterilization was not accomplished after the initial period, additional exposure time was added . Results included: (1) soiled needles are more difficult than strips to sterilize; (2) capping or the presence of blood did not affect sterilization efficiency; (3) container positioning was important only for the smallest container; (4) additional exposure time was usually required when sterilizing soiled needles; (5) the HVA killed all spore challenges in a single sterilization cycle and (6) GSA and UCV were approximately equal in sterilization efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Hawaii Med J, 1994 Apr, 53(4), 116 - 8
Helicobacter pylori infection and chronic active gastritis; Ro MS et al.; Helicobacter pylori (H . pylori) is an S-Shaped, gram-negative bacillus that recently has been implicated in the pathogenesis of chronic active gastritis and other peptic ulcer disease . These findings have encouraged gastroenterologists to provide a new rationale for patient management, with hope of providing more successful treatment of peptic ulcer disease, particularly gastritis . Therefore, a cooperative diagnostic effort was made at the pathology laboratory of St Francis Medical Center to adopt a simple and reliable method for the identification of H . pylori in tissue sections of endoscopic biopsies of stomach and duodenum . We attempted to estimate the prevalence of H . pylori infection in patients biopsied for upper Gl disorders who were refractory to medication . A prevalence of H . pylori infection among different ethnic groups also was studied.

Vet Immunol Immunopathol, 1994 Apr, 40(4), 325 - 39
Further characterisation of the immune response of the koala; Wilkinson R et al.; Sensitive enzyme immunoassays (EIA) were developed to monitor antibody (Ab) production in the koala, in response to both soluble and particulate antigens (Ag) . When compared with a eutherian mammal, the rabbit, both the dynamics and kinetics of Ab production in the koala were found to be severely retarded . In vitro, Ag specific lymphocyte proliferative responses were demonstrated for the first time in this animal by sensitising koalas in vivo with Bacillus Calmet-Guerin (BCG), with the level and timing of this cell mediated immune (CMI) response comparable with those seen in non-metatherian mammals . Levels of circulating B lymphocytes were examined in an attempt to clarify the retarded humoral responses to foreign Ags . In addition, peripheral mononuclear cells (PMC) from koalas, were examined for their reactivity to a range of monoclonal Abs and lectins in an attempt to characterise these cells further . The lectins examined, demonstrated an all or none reactivity with koala lymphocytes and were therefore considered unsuitable as markers for identifying lymphoid subsets in this animal . A monoclonal Ab directed at class II MHC Ags in the mouse, demonstrated cross reactivity with a high percentage of all koala monocytes tested . Using this Ab to probe CMI responses in vitro, it is concluded that immune interactions required for such responses in the koala parallel those seen in other mammals.

J Natl Med Assoc, 1994 Apr, 86(4), 313 - 5
Eikenella corrodens endocarditis in an intravenous drug user: case report and literature review; Olopoenia LA et al.; A rare case of Eikenella corrodens endocarditis in an intravenous drug user is reported . Repeated blood cultures from the patient established the diagnosis of this infection . However, evaluation of the cardiac function using two-dimensional echocardiography with Doppler flow demonstrated a large pedunculated tricuspid vegetation . Also evident on this study was a dilated right ventricle with diminished contractility and regurgitation . Complete sterilization of the blood was achieved after a 2-week course of intravenous penicillin and gentamicin followed by an additional 4-week course of intravenous penicillin alone . Clinicians treating suspected IV drug users should be aware of the potential pathogenicity of this rare, facultative, anaerobic gram-negative bacillus (E corrodens) . A combination of intravenous penicillin and aminoglycoside should be considered as the initial treatment followed by an additional course of intravenous penicillin for such patients with valvular vegetation.

Immunology, 1994 Apr, 81(4), 618 - 25
T-helper 1-like subset selection in Mycobacterium bovis bacillus Calmette-Guérin-infected resistant and susceptible mice; Kramnik I et al.; The Bcg gene has been shown to control natural resistance of mice to intravenous infection with low doses of Mycobacterium bovis (bacillus Calmette-Guerin; BCG) . In the present study, we evaluated the impact of the Bcg gene on the development of T-cell reactivity during the early stages of infection . Congenic strains of mice, bearing 'r' and 's' alleles of the Bcg gene on B10.A and BALB/c backgrounds, were studied at different time-points for 2 weeks after infection . The in vitro proliferative response of spleen cells, induced by mycobacteria or concanavalin A, was depressed in the Bcgs mice compared to the Bcgr congenic mice 14 days after infection with 10(5) colony-forming units (CFU) of BCG . Polymerase chain reaction (PCR)-based methodology was used to compare the level of lymphokine gene expression in the spleens of infected congenic mice both ex vivo and after in vitro stimulation . In both cases, preferential expression of interferon-gamma (IFN-gamma), lymphotoxin, interleukin-2 (IL-2) and IL-2 receptor genes was observed . The lymphokine gene expression profiles indicated that T lymphocytes activated in the course of the BCG infection preferentially expressed the T-helper 1-specific pattern, irrespective of the allele of the Bcg gene . We showed that this bias in T-cell differentiation could not be attributed to either down-regulation of IL-4 gene expression or modulation of the macrophage co-stimulatory activity by live M . bovis BCG . We conclude that the mechanism of phenotypic expression of the Bcg gene resides in the differential ability of macrophages to be activated by lymphokines produced by protective T cells, rather than in the lack of these lymphokines in susceptible animals.

Protein Eng, 1994 Apr, 7(4), 571 - 7
A proposal for the catalytic mechanism in phospholipase C based on interaction energy and distance geometry calculations; Sundell S et al.; The non-specific phospholipase C from Bacillus cereus preferentially hydrolyses phosphatidylcholine but is also active against phosphatidylserine, phosphatidylethanolamine and at a much lower level, sphingomyelin . A minimal substrate model containing all required structural and configurational elements of a high affinity substrate was docked into the active site . The enzyme-substrate attachment points were from molecular interaction energy calculations using the program GRID and from a previous phosphate inhibitor complex structure . Available conformational space for the substrate was sampled by distance geometry calculations using the program DGEOM . This investigation clearly identifies the attacking nucleophile, a catalytically favourable orientation of the phosphate group in its tetra-, as well as its penta-, coordinated state and a crucial stabilizing environment for the alkoxide intermediate . Based on this information a complete catalytic cycle is proposed.

J Clin Microbiol, 1994 Apr, 32(4), 942 - 8
Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR; Anderson B et al.; A PCR assay was developed by using degenerate primers that allow amplification of a 414-bp fragment of DNA from the rickettsia-like organisms Rochalimaea henselae and R . quintana . Internal oligonucleotides were used as hybridization probes, permitting rapid differentiation of these two Rochalimaea species . DNAs from 12 different isolates of R . henselae were amplified with the PCR primers, and the resulting 414-bp PCR product hybridized only with the R . henselae-specific probe . DNAs from four different isolates of R . quintana were amplified and produced a PCR product of the same size that hybridized only with the R . quintana-specific probe . DNAs from isolates of R . elizabethae, R . vinsonii, Bartonella bacilliformis, and Afipia felis failed to amplify the 414-bp fragment in the PCR assay . This two-step assay was applied to DNAs extracted from 16 fresh (unfixed) lymph node biopsy specimens and nine aspirates from patients with clinical cat scratch disease (CSD) to assay for the presence of R . henselae or R . quintana DNA in these samples . Twenty-one (84%) of 25 lymph node samples from CSD patients were positive for R . henselae, while none were positive for R . quintana . The characteristic 414-bp fragment was not amplified from eight lymph node tissue samples from non-CSD cases . These results provide evidence that R . henselae, and not R . quintana, plays the central role in the etiology of CSD.

Cytometry, 1994 Apr 1, 15(4), 283 - 93
Neural network analysis of flow cytometric data for 40 marine phytoplankton species; Boddy L et al.; Flow cytometry data (time of flight, horizontal and vertical forward light scatter, 90 degrees light scatter, and "red" and "orange" integral fluorescence) were collected for laboratory cultures of 40 species of marine phytoplankton, from the following taxonomic classes, the Dinophyceae, Bacillariophyceae, Prymnesiophyceae, Cryptophyceae, and other flagellates . Single-hidden-layer "back-propagation" neural networks were trained to discriminate between species by recognising patterns in their flow cytometric signatures, and network performance was assessed using an independent test data set . Two approaches were adopted employing: (1) a hierarchy of small networks, the first identifying to which major taxonomic group a cell belonged, and then a network for that taxonomic group identified to species, and (2) a single large network . Discriminating some of the major taxonomic groups was successful but others less so . With networks for specific groups, cryptophyte species were all identified reliably (probability of correct classification always being > 0.75); in the other groups half of the species were identified reliably . With the large network, dinoflagellates, cryptomonads, and flagellates were identified almost as well as by networks specific for these groups . The application of neural computing techniques to identification of such a large number of species represents a significant advance from earlier studies, although further development is required.

Bioorg Khim, 1994 Apr, 20(4), 382 - 92
{Primary structure of an intracellular serine proteinase from Bacillus amyloliquefaciens . II . Amino acid sequence of peptides in a chymotrypsin hydrolysate}; Surova IA et al.; Chymotrypsin hydrolyzate of the intracellular serine protease was separated by ion-exchange chromatography on a sulphocationite resin followed by HPLC to yield fifty one individual peptides . Their sequences, corresponding in total to 381 amino acid residues, were determined by the manual Edman procedure.

Rev Prat, 1994 Apr 1, 44(7), 915 - 9
{New infectious risks}; Carbon C; New infectious risks have been identified or better elucidated in the last few years . The concept of infectious risks implies: a receptive host; or more or less pathogenic agent, inducing pathologic phenomena with variable facility, depending the receptivity of the host; and individual or community environmental factors (risk of accidental and massive contact with a pathogenic agent) . These new risks are linked to: 1 . the appearance of new or newly identified pathogenic agents, and the periodic changes in the pathogenic strength of some microorganisms, modifying the epidemiology of some infections; 2 . changes in host receptivity (immunodepression, either acquired or linked to aggressive therapy which leads to prolonged survival of receptive patients); 3 . environmental modifications (spread of resistant pathogenic bacteria, for example, presently the tuberculosis bacillus; declining socioeconomic conditions; reduced efficacy of community measures for prevention); 4 . exposure to new risks linked to progress in diagnostic and therapeutic techniques (invasiveness of medical treatments, new treatment techniques, transplantations, implantation of foreign materials, transfusions) . These phenomena illustrate the constant change in infectious risks and the need for a multidisciplinary approach to the problem and its effects.

Histochemistry, 1994 Apr, 101(4), 253 - 62
Distribution of endogenous and exogenous 5'-nucleotidase on bovine spermatozoa; Schiemann PJ et al.; A polyclonal rabbit antibody against 5'-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa . Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion . Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa . On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development . This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa . Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region . Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane . Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor . Our findings show that endogenous 5'-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction . Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound . It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.

Curr Biol, 1994 Apr 1, 4(4), 351 - 3
Tuberculosis . Beating the bacillus; Young DB; The identification of the target of isoniazid in the mycobacterial tuberculosis pathogen opens the way for the developoment of new drugs and diagnostic tests to combat drug-resistant pathogen strains.

Mol Membr Biol, 1994 Apr-Jun, 11(2), 87 - 92
Structural and functional studies of a synthetic peptide mimicking a proposed membrane inserting region of a Bacillus thuringiensis delta-endotoxin; Cummings CE et al.; In order to study the mechanism of action of Bacillus thuringiensis delta-endotoxins, a synthetic 31-mer peptide corresponding to the sequence of a putative pore-forming segment of the CrylA(c) toxin was characterized structurally and functionally . The peptide maps onto the central helix (alpha 5) of the six-helix bundle of domain I of the crystal structure of the CryIIIA toxin . CD and NMR spectroscopic studies indicated that the peptide exists as an alpha-helix in methanol and a random coil in water . The peptide associated with liposomes at pH 4.7 and formed discrete, characterizable channels in planar lipid bilayers at low pHs . These channels had a conductance value of 60 picosiemens (pS) . It is possible that this helix is a component of the transmembrane pore formed by B . thuringiensis delta-endotoxins in vivo.

J Gastroenterol, 1994 Apr, 29(2), 120 - 4
A clinico-epidemiological analysis of Helicobacter pylori (H . pylori) by Southern blotting with A urease gene probe; Tonokatsu Y et al.; Helicobacter pylori (H . pylori) is a gram-negative bacillus thought to be involved in such diseases of the upper gastrointestinal tract as gastritis, peptic ulcers, and gastric cancer . Urease is regarded as the factor responsible for the pathogenic nature of this bacterium . Therefore, in our examination of the genetic polymorphism of H . pylori, by means of Southern blotting, we used the urease gene as a probe . The Southern blot patterns of H . pylori isolated from different patients differed greatly, the inter-individual variation being so marked that it allowed approximate distinction between individual patients . The Southern blot patterns of individual strains of H . pylori did not change, even when they were stored and passed from generation to generation in our laboratory . These results suggest that DNA fingerprints with a urease gene probe will be useful in epidemiologically tracing H . pylori infection . Almost all strains of H . pylori isolated from different sites in the stomach of a patient on different occasions showed the same pattern, allowing us to confirm that only one strain of H . pylori was responsible for H . pylori infection in individual patients.

Rev Prat, 1994 Apr 1, 44(7), 865 - 9
{A new bacterium: Rochalimaea}; Maurin M et al.; Originally limited to trench fever, infections due to Rochalimaea now comprise manifestations particular to patients with human immunodeficiency virus (bacillary angiomatosis and hepatic peliosis), but also manifestations as diverse as isolated fever, septicaemia, endocarditis, lymphocytic meningitis, or central neurological disorders, in immunodepressed or immunocompetent subjects . The involvement of Rochalimaea in cat-scratch fever remains debated . Microbiological analysis used for diagnosis has been modified to allow isolation of these new bacteria, whose culture is slow and difficult, in the course of the above-cited clinical manifestations, which should further extend the range of Rochalimaea infections.

Lab Anim Sci, 1994 Apr, 44(2), 153 - 8
Development of a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay for detection of Bacillus piliformis isolate-specific antibodies in laboratory animals; Boivin GP et al.; A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect Bacillus piliformis isolate-specific antibodies in serum specimens from rats and gerbils experimentally infected with B . piliformis isolates R1, R2, or M . Detection was based on the ability of serum antibodies to block binding of B . piliformis isolate-specific monoclonal antibodies to purified B . piliformis flagella . Application of this assay to serum specimens collected from sham-infected or experimentally infected rats and gerbils demonstrated that the serum specimens were capable of specifically inhibiting the binding of B . piliformis isolate-specific monoclonal antibodies to homologous flagella preparations (> 70% inhibition) only when the serum specimens were from animals infected with the homologous B . piliformis isolate . Only one false-negative and false-positive result were obtained when 80 serum specimens were tested by this competitive inhibition ELISA . In addition, we demonstrated that little nonspecific inhibition of monoclonal antibody binding occurred (< 30% inhibition) in this immunoassay specific inhibition of monoclonal antibody binding by serum was due to serum antibody and a serum's ability to inhibit binding of monoclonal antibodies to purified B . piliformis flagella was correlated with antibody reactivity with B . piliformis flagella but not with serum antibody reactivity to whole B . piliformis organisms . These results suggest that this monoclonal antibody-based competitive inhibition assay could be successfully applied to the serologic identification of isolates involved in naturally occurring B . piliformis infections in laboratory animals.

Appl Environ Microbiol, 1994 Apr, 60(4), 1213 - 20
Cloning and DNA sequencing of bgaA, a gene encoding an endo-beta-1,3-1,4-glucanase, from an alkalophilic Bacillus strain (N137); Tabernero C et al.; The gene bgaA encoding an alkaline endo-beta-1,3-1,4-glucanase (lichenase) from an alkalophilic Bacillus sp . strain N137, isolated in our laboratory, was cloned and expressed from its own promoter in Escherichia coli . The nucleotide sequence of a 1,416-bp DNA fragment containing bgaA was determined and revealed an open reading frame of 828 nucleotides . The deduced protein sequence consists of 276 amino acids and has a 31-amino-acid putative signal peptide which is functional in E . coli, in which the BgaA protein is located mainly in the periplasmic space . The lichenase activity of BgaA is stable between pH 6 and 12, it shows optimal activity at a temperature between 60 and 70 degrees C, and it retains 65% of its activity after incubation at 70 degrees C for 1 h . This protein is similar to some other lichenases from Bacillus species such as B . amyloliquefaciens, B . brevis, B . licheniformis, B . macerans, B . polymyxa, and B . subtilis . However, it has a lysine-rich region at the carboxy terminus which is not found in any other published lichenase sequence and might be implicated in the unusual biochemical properties of this enzyme . The location of the mRNA 5' end was determined by primer extension and corresponds to nucleotide 235 . A typical Bacillus sigma A promoter precedes the transcription start site.

Biochem Biophys Res Commun, 1994 Mar 30, 199(3), 1194 - 9
Detection of Choristoneura fumiferana brush border membrane-binding molecules specific to Bacillus thuringiensis delta-endotoxin by crossed affinity immunoelectrophoresis; Pang AS; Brush border membrane vesicles (BBMVs) prepared from midguts of Choristoneura fumiferana larvae were pretreated separately with the three CryIA delta-endotoxins from Bacillus thuringiensis subsp . kurstaki HD-1 . Crossed affinity immunoelectrophoresis of the solubilized toxin-BBMV complexes, using antibodies raised against the toxins, revealed at least two peaks for each toxin . All complexes (BBMV bound toxin) migrated in the opposite direction to that of the free toxins . It is concluded that the toxins form stable complexes with membrane acceptor molecules, possibly receptors, and that there are at least two types of acceptor molecules present in the midgut epithelial cells of C . fumiferana that bind to all three toxins . The relative size of one of the BBMV-toxin peaks correlates with the in vivo potency of the toxins against C . fumiferana.

Proc Natl Acad Sci U S A, 1994 Mar 29, 91(7), 2428 - 9
Evolution of drug-resistant tuberculosis: a tale of two species; Iseman MD; The history of the disease tuberculosis is briefly discussed . Now human societal failures have potentiated the evolution of drug-resistant strains of the tubercle bacillus in the United States and around the world . Until recently, this evolutionary change largely posed a threat to the health and survival of the individual in whom inadequate therapy promoted the drug resistance . However, the human immunodeficiency virus epidemic threatens to promote wholesale transmission of multidrug-resistant tuberculosis with the potential for immense morbidity and mortality . Reinforced treatment and control programs for tuberculosis are vital.

Biochemistry, 1994 Mar 29, 33(12), 3464 - 74
Phosphatidylinositol-specific phospholipase C from Bacillus cereus at the lipid-water interface: interfacial binding, catalysis, and activation; Volwerk JJ et al.; Binding characteristics of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus binding to the phospholipid-water interface were determined by spectroscopic methods and correlated with PI-PLC's catalytic properties . Binding of the enzyme to micelles and bilayers of zwitterionic phosphocholines is accompanied by an increase in the fluorescence emission from tryptophan, whereas a decrease in the emission is observed with synthetic anionic lipids containing a phosphomethanol head group . A similar decrease in the tryptophan emission is observed with phosphatidylinositol (PI) analogues containing the phospho-D-1-myo-inositol head group, but not with the enantiomeric L-1-myo-inositol . In covesicles of PI and phosphatidylcholine (PC), the rate of cleavage of PI is reduced because, as a neutral diluent, PC effectively reduces the surface concentration of PI that the bound enzyme "sees" in the interface . This permits determination of the interfacial Michaelis constant (KM*) as 0.26 mol fraction for PI as substrate . On the other hand, ditetradecylglycerophosphomethanol (DTPM) acts as a kinetic competitive inhibitor in the covesicles . The spectroscopic and catalytic activity data taken together show that PI-PLC binds to the interface of aqueous dispersions of phospholipids with an apparent Kd (in terms of the lipid monomers) of about 10-50 microM . However, only lipids with an anionic head group, such as phosphomethanol and phospho-D-1-myo-inositol, are able to bind as single molecules into the active site of the enzyme at the interface . Enantiomeric phospho-L-1-myo-inositol or the zwitterionic phosphocholine head group has little affinity for the enzyme at the interface . Thus, PI-PLC appears to obey the two-stage, Michaelis-Menten adaptation of interfacial catalysis, according to which the binding of the enzyme to the interface precedes the steps of the catalytic turnover at the interface . Limit estimates suggest that on PI or PI/PC vesicles the catalysis occurs in the "scooting" mode with a moderate processivity . DTPM vesicles also inhibit the activity of PI-PLC toward the synthetic water-soluble substrate myo-inositol 1-(4-nitrophenyl phosphate), but the activity is enhanced severalfold in the presence of vesicles of zwitterionic phosphatidylcholine . Several possible explanations of this interfacial activation are considered within the general context of the kinetic scheme for interfacial catalysis . The kinetic results for the action of PI-PLC bound to vesicles are consistent with a model in which the interface acts as an "allosteric" effector of the catalytic rate constant, kcat, without affecting the substrate binding.

Proc Natl Acad Sci U S A, 1994 Mar 29, 91(7), 2654 - 8
Rice tungro bacilliform virus encodes reverse transcriptase, DNA polymerase, and ribonuclease H activities; Laco GS et al.; Rice tungro bacilliform virus (RTBV) is a newly described badnavirus and proposed member of the plant pararetrovirus group . RTBV open reading frame 3 is predicted to encode a capsid protein, protease (PR), and reverse transcriptase (RT) and has the capacity to encode other proteins of as yet unknown function . To study the possible enzymatic activities encoded by open reading frame 3, a DNA fragment containing the putative PR and RT domains was used to construct the recombinant baculovirus PR/RT-BBac . Trichoplusia ni insect cells infected with PR/RT-BBac were used in pulse-labeling experiments and demonstrated synthesis of an 87-kDa polyprotein that corresponds in molecular mass to that predicted from the PR/RT DNA coding sequence . The 87-kDa polyprotein was processed with concomitant accumulation of 62-kDa (p62) and 55-kDa (p55) proteins . Amino-terminal sequencing of p62 and p55 determined that they mapped to the PR/RT domain and shared common amino termini . p62 and p55 were purified and exhibited both RT and DNA polymerase activities using synthetic primer/template substrates . Only p55 had detectable ribonuclease H activity, an activity intrinsic to all reverse transcriptases studied to date . Characterization of the RTBV RT provides a biochemical basis for classifying RTBV as a pararetrovirus and will lead to further studies of these proteins and their role in virus replication.

Biochemistry, 1994 Mar 22, 33(11), 3424 - 31
Kinetic characteristics of phosphofructokinase from Bacillus stearothermophilus: MgATP nonallosterically inhibits the enzyme; Byrnes M et al.; The kinetic mechanism of phosphofructokinase from Bacillus sterothermophilus has been investigated using steady-state measurements . The double-reciprocal patterns observed for initial velocity, product inhibition, and mixed alternate substrate studies of the reverse reaction establish that the mechanism involves rapid-equilibrium random binding of substrates and the formation of an abortive complex composed of enzyme, MgADP, and fructose 6-phosphate (E-MgADP-Fru-6P) . Initial velocity patterns for the forward reaction show significant nonlinearity and resemble those seen for competitive substrate (MgATP) inhibition of an enzyme that obeys a random mechanism . A mutant BsPFK enzyme (GV212) was used to show that the inhibition is not due to MgATP binding in the effector site . Product and dead-end inhibition studies of the forward reaction are consistent with a random mechanism, after taking into account the effects of substrate inhibition by MgATP . Initial velocity measurements at low MgATP concentration show that the binding of MgATP is not a rapid-equilibrium process; i.e., the rate of catalysis is faster than the rate of substrate binding . It is concluded that the kinetic mechanism of the forward reaction is sequential random, with the rate of MgATP binding slower than the catalytic rate . A model is presented that incorporates these results and proposes that substrate binding proceeds through two alternative pathways, one of which is kinetically disfavored . The observed MgATP substrate inhibition arises from both reaction flux through the disfavored pathway and, to some extent, abortive binding of MgATP in the Fru-6P site.

Biochemistry, 1994 Mar 22, 33(11), 3260 - 5
Characterization of the two anion-recognition sites of glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis and chemical modification; Corbier C et al.; The active site of the glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) contains two anion recognition sites which have been attributed to the phosphate binding of the substrates, namely, glyceraldehyde 3-phosphate (Ps site) and inorganic phosphate (Pi site) {Moras et al . (1975) J . Biol . Chem . 250, 9137-9162} . In order to probe the role of both sites during the catalytic event, Arg 195 from the Pi site and Arg 231 from the Ps site of the Bacillus stearothermophilus enzyme have been changed to Leu and Gly, respectively, by site-directed mutagenesis . A comparative study of the chemical reactivity of the mutants and wild type toward 2,3-butanedione revealed a similarly high reactivity only for the R195L mutant and wild type, suggesting that only Arg 231 is chemically reactive toward 2,3-butanedione and that its reactivity is not influenced by the presence of the residue Arg 195, which is only 4 A distant . The kinetic consequences of the mutations were also analyzed for the consecutive steps in the forward catalytic reaction . The replacement of Arg 195 by Leu leads to a marked decrease of the rate of the first steps of the reaction which lead to the acylenzyme formation, in particular, the rate of enzyme-substrate association, while these steps occur at a similar or higher rate when Arg 231 is replaced by Gly . Furthermore, the mutations R195L and R231G also result in a 550-fold and 16,400-fold decrease in the second-order rate constant of phosphorolysis . This step becomes rate-determining for the R195L mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1994 Mar 18, 237(1), 163 - 4
Crystallization and preliminary X-ray studies of cyclodextrin glucanotransferase from alkalophilic Bacillus sp . 1011; Haga K et al.; Large crystals of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp . 1011, a typical alkalophilic enzyme, have been obtained at room temperature using polyethylene glycol 3000 and 2-propanol as precipitant . They belong to the triclinic space group P1 with the following unit cell constants: a = 64.93 A, b = 74.45 A, c = 79.12 A, alpha = 85.2 degrees, beta = 105.0 degrees and gamma = 101.0 degrees . The crystallographic asymmetric unit seems to contain two molecules of CGTase, with crystal volume per protein mass (Vm) of 2.41 A3/Da and solvent content of 49% by volume . The crystals diffract to at least 2.0 A resolution and they are suitable for X-ray analysis.

MMWR Morb Mortal Wkly Rep, 1994 Mar 18, 43(10), 177 - 8
Bacillus cereus food poisoning associated with fried rice at two child day care centers--Virginia, 1993; Flexibility in the protoxin composition of Bacillus thuringiensis; Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907In at least three Bacillus thuringiensis subspecies, multiple protoxin genes are confined to just a few of the many plasmids with two or more on one of > 100 mDa and a particular gene, cryIA(b), on a 40-50 mDa plasmid . The latter is unstable but can be maintained in the population by cell mating . Cells which had lost this plasmid compensated by increasing transcription of the remaining protoxin genes resulting in the formation of inclusions which differed from those in the parental strains in their toxicity profiles for selected insects as well as their solubility . Instability of a particular protoxin-encoding plasmid appears to be a mechanism for rapidly shifting the protoxin gene complement and thus the toxicity profiles of these bacteria.

Eur J Biochem, 1994 Mar 15, 220(3), 981 - 5
Protein stabilization by hydrophobic interactions at the surface; Van den Burg B et al.; The contribution of the solvent-exposed residue 63 to thermal stability of the thermolysin-like neutral protease of Bacillus stearothermophilus was studied by analyzing the effect of twelve different amino acid substitutions at this position . The thermal stability of the enzyme was increased considerably by introducing Arg, Lys or bulky hydrophobic amino acids . In general, the effects of the mutations showed that hydrophobic contacts in this surface-located region of the protein are a major determinant of thermal stability . This observation contrasts with general concepts concerning the contribution of surface-located residues and surface hydrophobicity to protein stability and indicates new ways for protein stabilization by site-directed mutagenesis.

Biochem J, 1994 Mar 15, 298 Pt 3, 697 - 703
Cytodifferentiation in Tetrahymena vorax is linked to glycosyl-phosphatidylinositol-anchored protein assembly; Yang X et al.; The role of glycosyl-PtdIns (GPI)-anchored proteins in the cytodifferentiation of Tetrahymena vorax was examined . Labelling of cells with {3H}myristate or {3H}palmitate followed by electrophoresis showed an array of proteins carrying covalently bound lipids . Electrophoresis of protein from cells labelled with the GPI-anchor components {3H}Ins and {14C}ethanolamine revealed three polypeptides on fluorograms which have apparent molecular masses of approx . 28, 50 and 82 kDa . Labelled lipid associated with these polypeptides was susceptible to release by in vitro exposure to Bacillus thuringiensis PtdIns-specific phospholipase C (PI-PLC) . Using labelled fatty acids, cells induced to differentiate showed altered GPI-anchored protein-labelling patterns in comparison with undifferentiated control cells, with a heavily labelled 32 kDa band appearing upon differentiation . Pre-incubation of cells in 10 mM D-mannosamine, an inhibitor of GPI incorporation into protein, resulted in a reduction of the incorporation of label into the three GPI-anchored proteins, nearly complete inhibition of differentiation and a reduction in the rate of digestive vacuole formation . A 50% inhibition of differentiation was obtained using 500 microM mannosamine . The inhibitory impact of D-mannosamine on differentiation could be competitively and completely reversed by the inclusion of D-mannose, but not D-glucose . Neither glucosamine nor tunicamycin inhibited differentiation . Incubation of cells in PI-PLC (5 units/ml) plus the differentiation inducer resulted in an acceleration of differentiation and generally higher percentages of differentiated cells versus controls.

Proc Natl Acad Sci U S A, 1994 Mar 15, 91(6), 2225 - 9
Activation of the interleukin 6 gene by Mycobacterium tuberculosis or lipopolysaccharide is mediated by nuclear factors NF-IL6 and NF-kappa B; Zhang Y et al.; The host response to Mycobacterium tuberculosis includes granuloma formation at sites of infection and systemic symptoms . Cytokines have been identified by immunohistochemistry in granulomas in animal models of bacillus Calmette-Guerin (BCG) infection and are released by mononuclear phagocytes upon stimulation by mycobacterial proteins . In this regard, the cytokine interleukin 6 (IL-6) may play a role in the clinical manifestations and pathological events of tuberculosis infection . We have demonstrated that lipoarabinomannan (LAM) from the mycobacterial cell wall, which was virtually devoid of lipopolysaccharide (LPS), stimulated mononuclear phagocytes to release IL-6 in a dose-response manner . LAM and LPS were potent inducers of IL-6 gene expression in peripheral blood monocytes . Both LAM- and LPS-inducible IL-6 promoter activity was localized to a DNA fragment, positions -158 to -49, by deletion analysis and chloramphenicol acetyltransferase assay . Two nuclear factor NF-IL6 (positions -153 to -145 and -83 to -75) and one nuclear factor NF-kappa B (positions -72 to -63) motifs are present within this fragment . Site-directed mutagenesis of one or more of these motifs within the IL-6 promoter demonstrated that each has positive regulatory activity and that they could act in a function- and orientation-independent manner . Deletion of all three elements abolished inducibility of IL-6 promoter activity by both LAM and LPS . We conclude that the NF-IL6 and NF-kappa B sites mediate IL-6 induction in response to both LPS and LAM, acting as bacterial or mycobacterial response elements.

J Biotechnol, 1994 Mar 15, 33(1), 55 - 62
Production of human protein disulfide isomerase by Bacillus brevis; Tojo H et al.; Human protein disulfide isomerase (PDI; EC 5.3.4.1) was expressed and secreted into the culture medium using Bacillus brevis as host and pNU200 which codes the promoter and signal sequence of major cell wall protein of B . brevis as vector . The accumulation of recombinant human PDI (rhPDI) reached about 5 mg l-1 in the late exponential phase of the bacterial growth . The purified rhPDI was found to be exactly processed at the carboxyl terminus of the signal sequence . It was as active as natural PDI derived from human placenta as determined by its ability to reactivate scrambled ribonuclease that was a fully oxidized mixture containing randomly formed disulfide bonds . The activity was significantly accelerated in the presence of dithiothreitol or a mixture of reduced and oxidized glutathione . These indicate that the characteristics of rhPDI are similar to those reported for mammalian PDI and that it can be used for refolding inactive proteins having incorrect disulfide bonds.

Arch Intern Med, 1994 Mar 14, 154(5), 524 - 8
Bacillary angiomatosis . Clinical and histologic features, diagnosis, and treatment; Cotell SL et al.; Bacillary angiomatosis is a relatively new infection affecting primarily patients with human immunodeficiency virus or others with impaired host defenses . It presents most commonly with multiple red skin lesions, but visceral involvement may also occur, including involvement of the liver and spleen . Because of the dermatologic manifestations, bacillary angiomatosis may be mistaken for Kaposi's sarcoma . The diagnosis is made by identification of the characteristic histologic findings or genetic amplification by means of polymerase chain reaction . The causative agent was recently identified as Rochalimaea henselae, although Rochalimaea quintana may also play a role . Therapy with erythromycin or doxycycline is usually effective.

J Biol Chem, 1994 Mar 11, 269(10), 7262 - 6
Involvement of conserved lysine 68 of Bacillus stearothermophilus leucine dehydrogenase in substrate binding; Sekimoto T et al.; Lysine 68 of Bacillus stearothermophilus leucine dehydrogenase is highly conserved in the corresponding regions of NAD(P)+-dependent amino acid dehydrogenase sequences . To elucidate its functional role, the lysyl residue of the recombinant enzyme has been replaced with alanine or arginine by site-directed mutagenesis . Either mutation resulted in nearly complete loss of activity in the oxidative deamination, whereas only the mutation to alanine led to a marked increase in Michaelis constants for both amino and keto acid substrates . On the other hand, an ionizable group in the wild-type enzyme with a pKa value of 10.1-10.7, which must be protonated for binding of substrate and competitive inhibitor with an alpha-carboxyl group, was unobservable in both mutant enzymes . These results altogether led to the conclusion that Lys-68 is located at the active site of the enzyme and involved in binding of the alpha-carboxyl group of substrate through an ionic interaction . In addition, the alanine mutant enzyme that is almost inactive in the deamination but significantly active in the amination was greatly stimulated by exogenously added ammonia, suggesting that proper binding of the substrate alpha-carboxyl group at Lys-68 is essential for catalysis.

Gene, 1994 Mar 11, 140(1), 103 - 7
Primary sequence of the chitosanase from Streptomyces sp . strain N174 and comparison with other endoglycosidases; Masson JY et al.; A 1.6-kb DNA fragment from the soil actinomycete, Streptomyces sp . strain N174, containing the gene (csn) encoding an extracellular chitosanase (CSN), has been isolated and its complete nucleotide sequence determined . The gene was expressed in Escherichia coli and Streptomyces lividans using appropriate vectors . The sequence was found to contain one large ORF which encodes a protein of 238 amino acids (aa) . The deduced aa sequence begins with a signal peptide which has an unusual C-terminal segment, well recognized by the Streptomyces secretion system, but poorly in Escherichia coli . The strain N174 CSN aa sequence was compared with those of other proteins and significant homology was found only with the Bacillus circulans MH-K1 CSN but not with known chitinases or lysozymes . This suggests that CSN form a new enzyme family distinct from other endoglycosidases.

Genetika, 1994 Mar, 30(3), 367 - 72
{The effect of Culex family mosquito larva on the sensitivity of Anopheles mosquitos with various karyotypes to the entomopathogenic bacteria Bacillus thuringiensis subsp . Israelensis}; Gordeev MI et al.; Culex (C . modestus) and Anopheles (A . beklemishevi and A . messeae) larvae mosquito were treated with Bacillus thuringiensis subsp . israelensis (Bti) bacterium together and separately . It was determined that the presence of C . modestus reduced the mortality of Anopheles larvae and altered the results of the selection of genotypes resistant to Bti of the polymorphic species of A . messeae, C . modestus larvae consumed the pathogen more rapidly and protected the sensitive individuals of A . messeae with "northern" chromosomal inversions from destruction . The correlation of the selection of cohabiting Anopheles and Culex mosquitoes as well as the relation between the specific diversity and the stability of communities are discussed.

Mol Biol (Mosk), 1994 Mar-Apr, 28(2), 468 - 71
{Cloning of the gene for extracellular Bacillus circulans RNAase}; Fedorova ND et al.; The gene for extracellular low molecular weight ribonuclease of Bacillus circulans BCF 247 was cloned . The strain was isolated from permafrost deposits of the Kolyma lowland . The gene for the ribonuclease from Bacillus intermedius (binase) was used as a specific probe . The cloning succeeded only in the E . coli strain producing the inhibitor of ribonuclease form Bacillus amyloliquefaciens . Selected clones secreted the active ribonuclease into the growth media . Deletion derivatives of the parental recombinant plasmid were constructed . The smallest DNA fragment which enclosed a functional ribonuclease gene in E . coli was determined to be 0.6 kb in length.

Mol Biol (Mosk), 1994 Mar-Apr, 28(2), 453 - 63
{Superproduction of Bacillus intermedius 7P ribonuclease (binase) in Escherichia coli}; Shul'ga AA et al.; We have reported previously about the cloning of the binase gene in E . coli . In this work, using an original approach named "homolog gene recombination" method (HGR), vectors for binase expression in E . coli have been constructed . Transcription of the binase gene have been directed through either tac-promoter or PR-promoter of bacteriophage lambda under the control of temperature-sensitive CI857 repressor . The last promoter gave the maximum yield of binase, up to 100 mg of protein per litre of heat-induced bacterial culture . The location of the transcription terminator at the 3' terminus of the binase gene raised the expression approximately two times more . A chromatographic method have been developed and applied for the control of binase accumulation in growth medium without measuring the ribonuclease activity.

Protein Eng, 1994 Mar, 7(3), 393 - 400
Structure-function relationships in the catalytic and starch binding domains of glucoamylase; Coutinho PM et al.; Sixteen primary sequences from five sub-families of fungal, yeast and bacterial glucoamylases were related to structural information from the model of the catalytic domain of Aspergillus awamori var . X100 glucoamylase obtained by protein crystallography . This domain is composed of thirteen alpha-helices, with five conserved regions defining the active site . Interactions between methyl alpha-maltoside and active site residues were modelled, and the importance of these residues on the catalytic action of different glucoamylases was shown by their presence in each primary sequence . The overall structure of the starch binding domain of some fungal glucoamylases was determined based on homology to the C-terminal domains of Bacillus cyclodextrin glucosyl-transferases . Crystallography indicated that this domain contains 6-8 beta-strands and homology allowed the attribution of a disulfide bridge in the glucoamylase starch binding domain . Glucoamylase residues Thr525, Asn530 and Trp560, homologous to Bacillus stearothermophilus cyclodextrin glucosyltransferase residues binding to maltose in the C-terminal domain, could be involved in raw-starch binding . The structure and length of the linker region between the catalytic and starch binding domains in fungal glucoamylases can vary substantially, a further indication of the functional independence of the two domains.

J Invertebr Pathol, 1994 Mar, 63(2), 123 - 9
Cellular toxicities and membrane binding characteristics of insecticidal crystal proteins from Bacillus thuringiensis toward cultured insect cells; Johnson DE; The pattern of in vitro toxicity of activated toxins from several classes of entomocidal inclusion genes from Bacillus thuringiensis was measured using eight established cell lines from lepidopteran insects . Protoxins representing CryIA(b), CryIA(c), and a mixture of all three CryIA toxins (subtypes a, b, and c; from B . thuringiensis subsp . kurstaki HD-1) were compared with the protoxin representing CryIC in a bioassay which measured the viability of cultured insect cells upon exposure to entomocidal toxin proteins . The responses of the various cell lines were very specific toward the individual toxin proteins . CryIC activated protoxin was toxic for cells of Manduca sexta, Plodia interpunctella, and to a lesser extent Spodoptera frugiperda . CryIA(b) and CryIA(c) proteins were toxic toward M . sexta but relatively non-toxic for P . interpunctella or S . frugiperda . The toxicity of CryIA(b), CryIA(c), and the composite CryIA activated toxins toward cells of Choristoneura fumiferana varied substantially, with the CryIA mixture being slightly more toxic than CryIA(c) alone . CryIC toxin had no effect toward C . fumiferana cells . Probit regression analysis of dose-response relationships between insect species and crystal protein composition demonstrated specific patterns of toxicity which may be related to membrane-receptor site binding by specific toxins . Membrane binding analysis of 125I-labeled CryIA(b), CryIA(c), and CryIC toxins to insect cells from three of the cell lines yielded high specific binding only with M . sexta cells toward CryIA(c) toxin . Lower levels of binding were observed with CryIA(b) and CryIC toward cells of C . fumiferana and P . interpunctella . Although relatively low binding levels for CryIC were observed with P . interpunctella cells, toxicity was high for these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Crystallogr A, 1994 Mar 1, 50 ( Pt 2), 164 - 82
Overcoming non-isomorphism by phase permutation and likelihood scoring: solution of the TrpRS crystal structure; Doublie S et al.; Entropy maximization to maximum likelihood, constrained jointly by the best available experimental phases and by a sufficiently good envelope, can bring about substantial model-independent map improvement, even at medium (3.1 A) resolution {Xiang, Carter, Bricogne & Gilmore (1993) . Acta Cryst . D49, 193-212} . In the crystal structure determination of the Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS), however, the following had to be dealt with simultaneously: (1) a serious lack of isomorphism in the heavy-atom derivatives, resulting in large starting-phase errors; and (2) an initially poorly known molecular envelope . Because the constraints--both phases and envelope--were insufficiently well determined at the outset, maximum-entropy solvent flattening as previously applied was unsuccessful . Rather than improving the maps, it led to a deterioration of their quality, accompanied by a dramatic decrease of the log-likelihood gain as phases were extended from about 5 A resolution to the 2.9 A limit of the diffraction data . This deadlock was broken by the identification of strong reflections, which were initially unphased and which were inaccessible by maximum-entropy extrapolation from the phased ones, and by permutation of the phases of these reflections so as to sample the space of possible electron-density and envelope modifications they represented . Permutation was carried out by successive full and incomplete factorial designs {Carter & Carter (1979) . J . Biol . Chem . 254, 12219-12223} for 28 strong reflections selected in decreasing order of their 'renormalized' structure-factor amplitudes . The permuted reflections included one reflection for which the probability distribution from multiple isomorphous replacement with anomalous scattering (MIRAS) indicated an incorrect phase with a high figure of merit and which consequently had a large renormalized structure factor . A similar permutation was carried out for six different binary choices related to the calculation and description of the molecular envelope . Permutation experiments were scored using the log-likelihood gain and contrasts for each main effect were analyzed by multiple-regression least squares . Student t tests provided significant and reliable indications for a large majority of the permuted reflections and for all six hypotheses related to the molecular envelope . The resulting phase improvement made it possible to assign positions (hitherto unobtainable) for nine of the ten selenium atoms in an isomorphous difference Fourier map for selenomethionine-substituted TrpRS crystals and hence to solve the structure . Phase-permutation methods continued to be useful in producing improved maps from all the available isomorphous-replacement phase information and therefore played a critical role in solving the structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Appl Environ Microbiol, 1994 Mar, 60(3), 896 - 902
Insecticidal activity of the CryIIA protein from the NRD-12 isolate of Bacillus thuringiensis subsp . kurstaki expressed in Escherichia coli and Bacillus thuringiensis and in a leaf-colonizing strain of Bacillus cereus; Moar WJ et al.; A 4.0-kb BamHI-HindIII fragment encoding the cryIIA operon from the NRD-12 isolate of Bacillus thuringiensis subsp . kurstaki was cloned into Escherichia coli . The nucleotide sequence of the 2.2-kb AccI-HindIII fragment containing the NRD-12 cryIIA gene was identical to the HD-1 and HD-263 cryIIA gene sequences . Expression of cryIIA and subsequent purification of CryIIA inclusion bodies resulted in a protein with insecticidal activity against Heliothis virescens, Trichoplusia ni, and Culex quinquefasciatus but not Spodoptera exigua . The 4.0-kb BamII-HindIII fragment encoding the cryIIA operon was inserted into the B . thuringiensis-E . coli shuttle vector pHT3101 (pMAU1) . pMAU1 was used to transform an acrystalliferous HD-1 strain of B . thuringiensis subsp . kurstaki and a leaf-colonizing strain of B . cereus (BT-8) by using electroporation . Spore-crystal mixtures from both transformed strains were toxic to H . virescens and T . ni but not Helicoverpa zea or S . exigua.

J Appl Bacteriol, 1994 Mar, 76(3), 259 - 63
Evaluation of a chromogenic chito-oligosaccharide analogue, p-nitrophenyl-beta-D-N,N'-diacetylchitobiose, for the measurement of the chitinolytic activity of bacteria; Frandberg E et al.; Three methods of quantifying chitinase activity were compared . The activities of crude chitinases of 10 bacterial isolates from different environments were estimated in terms of (1) the release of p-nitrophenol from the chromogenic chito-oligosaccharide analogues, p-nitrophenyl-beta-D-N,N'-diacetylchitobiose, p-nitrophenyl-N-acetyl-beta-D-glucosamine and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, (2) the release of reducing sugars from chitin and (3) the formation of clearing zones on chitin agar . When crude chitinase from Bacillus pabuli was used the hydrolysis of p-nitrophenyl-beta-D-N,N'-diacetylchitobiose correlated well with the release of reducing sugars from chitin and the formation of clearing zones on chitin agar . However, when the activity of crude chitinases from the different bacterial isolates were compared no agreement was found between the hydrolysis of p-nitrophenyl-beta-D-N,N'-diacetylchitobiose and the release of reducing sugars from chitin or the formation of clearing zones on chitin agar . It was concluded that the assay with chromogenic p-nitrophenyl chito-oligosaccharide analogues is not well suited for studies that compare the chitinase activity of different bacteria.

Biol Mass Spectrom, 1994 Mar, 23(3), 159 - 64
beta-Lactamase ragged ends detected by electrospray mass spectrometry correlates poorly with multiple banding on isoelectric focusing; Payne DJ et al.; Purified preparations of TEM-2, P99, Bacillus cereus I and B . cereus II beta-lactamases were examined by electrospray (ES) mass spectrometry . The ES mass spectra of the B . cereus enzymes revealed the presence of four to five components of different mass, corresponding to the loss of different numbers of N-terminal amino acids (ragged ends) . The ES mass spectra of both TEM-2 and P99 consisted of a single component with no evidence of ragged ends . All four beta-lactamase preparations were visualized on isoelectric focusing (IEF) gels stained with nitrocefin to investigate a possible correlation between IEF patterns and ragged ends . Multiple banding patterns were seen with each beta-lactamase preparation . Although these may correlate with the presence of ragged ends in the two B . cereus preparations, the satellite bands seen with P99 and TEM-2 were not associated with differences detected by ES mass spectrometry . In this study we have shown for the first time that beta-lactamase satellite bands seen on IEF are not always associated with ragged ends . Furthermore, we have illustrated the use of ES mass spectrometry to characterize the extent of ragged end formation in protein samples . This is of particular significance if the sample is required for detailed biochemical or crystallography experiments.

Cell Tissue Res, 1994 Mar, 275(3), 577 - 85
A monoclonal antibody (ER-HR3) against murine macrophages . II . Biochemical and functional aspects of the ER-HR3 antigen; de Jong JP et al.; We describe the purification and intracellular distribution of an antigen present on a subpopulation of murine macrophages and recognized by monoclonal antibody ER-HR3 against bone marrow-derived haemopoietic reticulum cells . Using the ER-HR3 antibody as an immobilizing ligand, two proteins were isolated as determined by SDS polyacrylamide gel electrophoresis . Under non-reducing conditions, there was a major band with an apparent molecular mass of 69 kDa and a minor band of 55 kDa . Under reducing conditions, the apparent molecular mass of each band was estimated as 76 kDa and 67 kDa, respectively . Intracellularly, these proteins occurred in close association with membranous structures, as demonstrated with gold-labelled protein A in an electron-microscopic study of the ER-HR3-positive cell line AP284 . Some of the antigen was present in vesicles . To gain further insight into the possible function of the ER-HR3 antigen, its tissue distribution was investigated under distinct experimental conditions . In mice infected with Bacillus Calmette Gurerin, ER-HR3-positive cells were observed in many, but not all, granulomata of the spleen, the lung and the liver . The ER-HR3 reactivity in these mice clearly differed from that of other antimacrophage monoclonal antibodies, such as F4/80, M5/114 and M1/70 . Furthermore, phenylhydrazine-induced extramedullary erythropoiesis in the liver was accompanied by ER-HR3 expression on a subpopulation of macrophages . Finally, the addition of ER-HR3 to an antigen-specific T cell proliferation assay did not inhibit T cell proliferation.

Arch Biochem Biophys, 1994 Mar, 309(2), 273 - 80
Identification of the lipid moiety and further characterization of the novel lipophosphoglycan-like glycoconjugates of Trichomonas vaginalis and Trichomonas foetus; Singh BN et al.; The lipid moiety of the lipophosphoglycan (LPG)-like glycoconjugates of Trichomonas vaginalis and Trichomonas foetus, parasites of the urogenital tract of human and cattle, respectively, has been isolated and characterized by a combination of enzymatic and chemical degradation, chromatography, and mass spectrometry . The carbohydrate composition of the glycan inositol lipid core is also reported . The glycan inositol core of trichomonad glycoconjugates is unique in having more than one GlcN and is significantly larger than any other glycan core reported so far . T . vaginalis glycoconjugate binds strongly to the lectin RCA-I, which suggest that the macromolecule possesses terminal beta 1,4-linked galactosyl residues . The binding of T . foetus glycoconjugate to the lectin UEA-I suggests the presence of terminal alpha 1,2-linked fucose . Acid hydrolysis of deaminated and reduced LPG products yields a {3H}anhydromannitol-containing product, indicating the presence of unacetylated glucosamine in the trichomonad LPGs . Reductive radiomethylation has been applied to label free amino groups in the hexosamine or other free amine-containing residues of the trichomonad glycoconjugates . Treatment of the LPGs with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis liberates a ceramide substituent . Treatment of LPGs with nitrous acid releases a phospholipid moiety containing myo-inositol and ceramide, implying that the LPGs are anchored in the membrane via an inositol-phosphate-ceramide . Structural characterization of the ceramide by gas-liquid chromatography (GC) and GC-mass spectrometry indicated the presence of the major long-chain base sphinganine (d 18: 0 dihydrosphingosine) and a C 16:0 N-acyl group . Lipophosphoglycans from both parasites contain ceramide as their only lipid moiety . These results suggest that T . vaginalis and T . foetus anchor their LPG-like glycoconjugates on the cell surface via inositol-phosphoceramide and also the glycan inositol core of the macromolecule appears to be unique in nature.

Biochemistry, 1994 Mar 1, 33(8), 2297 - 305
Source of catalysis in the lactate dehydrogenase system . Ground-state interactions in the enzyme-substrate complex; Deng H et al.; The Raman spectra of both the NAD-pyruvate and the pyridine aldehyde adenine dinucleotide (PAAD)-pyruvate bound to pig heart, pig muscle, and Bacillus stearothermophilus lactate dehydrogenases were measured and are nearly the same, which is consistent with the conserved shell of residues surrounding the active-site cavity in these enzymes . The symmetrical stretching mode of the pyruvate carboxylate group, found at 1398 cm-1, is shifted only slightly when complexed to these enzymes, which shows that the group remains ionized in the ion pair complex with Arg-171 on the enzyme . The vibrational mode for the carbonyl stretch of the bound pyruvate moiety is shifted about 35 cm-1 to a lower frequency than observed for the carbonyl of unliganded pyruvate in the bacterial enzyme because of polarization of the carbonyl bond . Thus, the bacterial enzyme shows the same substrate activation because of the C(+)-O- charge separation that was seen previously with the mammalian enzymes . On the basis of an empirical Badger-Bauer relationship between frequency shift and interaction enthalpy, this shift in frequency is equivalent to an approximately -14 to -17 kcal/mol interaction between the enzyme and the adduct C = O coordinate, a substantial part of which is an electrostatic interaction (hydrogen bond) between the C V O and the protonated His-195 . Thus, while the C = O bond is polarized on the enzyme (which requires energy), the overall ground-state enthalpy of the carbonyl imidazolium part of the reaction coordinate is stability substantially relative to its value in solution, and this is the dominant enthalpic effect on the entire reaction coordinate since the other internal coordinates for the hydride transfer are not much affected during formation of the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Kyobu Geka, 1994 Mar, 47(3), 232 - 4
{A case of Bacillus cereus prosthetic valve endocarditis}; Yamamura M et al.; A rare case of prosthetic valve endocarditis by Bacillus cereus was reported . The patient was 43-year-old Japanese man, who had mitral valve replacement 5 months prior to this admission . Remitral valve replacement was immediately done successfully . His postoperative course was uneventful for 8 months.

J Bacteriol, 1994 Mar, 176(5), 1434 - 42
The Myxococcus xanthus dsg gene product performs functions of translation initiation factor IF3 in vivo; Kalman LV et al.; The amino acid sequence of the Dsg protein is 50% identical to that of translation initiation factor IF3 of Escherichia coli, the product of its infC gene . Anti-E . coli IF3 antibodies cross-react with the Dsg protein . Tn5 insertion mutations in dsg are lethal . When ample nutrients are available, however, certain dsg point mutant strains grow at the same rate as wild-type cells . Under the starvation conditions that induce fruiting body development, these dsg mutants begin to aggregate but fail to develop further . The level of Dsg antigen, as a fraction of total cell protein, does not change detectably during growth and development, as expected for a factor essential for protein synthesis . The amount of IF3 protein in E . coli is known to be autoregulated at the translational level . This autoregulation is lost in an E . coli infC362 missense mutant . The dsg+ gene from Myxococcus xanthus restores normal autoregulation to the infC362 mutant strain . Dsg is distinguished from IF3 of E . coli, other enteric bacteria, and Bacillus stearothermophilus by having a C-terminal tail of 66 amino acids . Partial and complete deletion of this tail showed that it is needed for certain vegetative and developmental functions but not for viability.

J Bacteriol, 1994 Mar, 176(5), 1427 - 33
The dsg gene of Myxococcus xanthus encodes a protein similar to translation initiation factor IF3; Cheng YL et al.; The dsg mutants of Myxococcus xanthus are defective in fruiting body development and sporulation, yet they grow normally . The deduced amino acid sequence of the dsg gene product is 50 and 51% identical to the amino acid sequence of translation initiation factor IF3 of both Escherichia coli and Bacillus stearothermophilus, respectively . However, the Dsg protein has a carboxy-terminal extension of 66 amino acids, which are absent from its E . coli and B . stearothermophilus homologs . The Shine-Dalgarno sequence GGAGG and 5 bases further upstream are identical in M . xanthus and several enteric bacteria, despite the wide phylogenetic gap between these species . The infC gene, which encodes IF3 in enteric bacteria, starts with the atypical translation initiation codon AUU, which is known to be important for regulating the cellular level of IF3 in E . coli . Translation of the Dsg protein overexpressed from the M . xanthus dsg gene in E . coli cells initiates at an AUC codon, an atypical initiation codon in the AUU class . The dsg mutants DK429 and DK439 carry the same missense mutation that changes Gly-134 to Glu in a region of amino acid identity.

Infect Immun, 1994 Mar, 62(3), 980 - 6
Improved purification and characterization of hemolysin BL, a hemolytic dermonecrotic vascular permeability factor from Bacillus cereus; Beecher DJ et al.; Bacillus cereus causes diarrheal and emetic food poisoning syndromes as well as a variety of mild to severe infections . A dermonecrotic vascular permeability (VP) factor has been implicated as a virulence factor in these illnesses . Hemolysin BL was previously identified as a unique tripartite hemolysin possessing VP activity . In this study, a high-yield purification scheme, which allowed quantitative characterization of hemolysin BL activity and determination of the molecular weight, pI, and N-terminal sequence of each component, was developed . Milligram quantities of the B, L1, and L2 components were highly purified by a combination of anion-exchange and hydroxylapatite chromatographies . The combined components had VP activity at low doses and were necrotic at higher doses . The toxin exhibited an unusual dose-response zone phenomenon in turbidometric hemolysis assays . Activity increased at doses up to 200 ng/ml, then decreased at doses up to 350 ng/ml, and was constant at doses up to at least 2,500 ng/ml . This behavior may provide an explanation for the unusual discontinuous pattern typical of hemolysin BL in gel diffusion assays . At high concentrations of one or two components, the presence of low amounts of the complementary component(s) resulted in full hemolytic activity . Erythrocytes were protected from lysis by Zn2+ at micromolar concentrations but not by Ca2+ and Mg2+ at concentrations up to 25 mM . These data provide guidelines for future work on this toxin and indicate that hemolysin BL is the dermonecrotic VP factor implicated as a B . cereus virulence factor.

Prikl Biokhim Mikrobiol, 1994 Mar-Apr, 30(3), 379 - 83
{Isolation of intracellular inhibitors of bacterial RNAses on a column with immobilized Bacillus intermedius RNAse}; Bannikova GE et al.; Intracellular inhibitors of RNases from Bacillus amyloliquefaciens and Bac . intermedius were isolated using affinity chromatography on covalently immobilized RNase from Bac . intermedius . The inhibitor of RNase from Bac . amyloliquefaciens was isolated from cells of E . coli HB 101 and purified to homogeneity . Proteins with molecular weights of 70, 36 and 20 kD possessing an inhibitory activity were found in the extract obtained from frozen cells of Bac . intermedius.

Protein Sci, 1994 Mar, 3(3), 467 - 75
Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase; Wakarchuk WW et al.; Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans . Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism . We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C) . In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme . On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.

J Biomol NMR, 1994 Mar, 4(2), 257 - 78
1H, 13C and 15N NMR backbone assignments and secondary structure of the 269-residue protease subtilisin 309 from Bacillus lentus; Remerowski ML et al.; 1H, 13C and 15N NMR assignments of the backbone atoms of subtilisin 309, secreted by Bacillus lentus, have been made using heteronuclear 3D NMR techniques . With 269 amino acids, this protein is one of the largest proteins to be sequentially assigned by NMR methods to date . Because of the size of the protein, some useful 3D correlation experiments were too insensitive to be used in the procedure . The HNCO, HN(CO)-CA, HNCA and HCACO experiments are robust enough to provide most of the expected correlations for a protein of this size . It was necessary to use several experiments to unambiguously determine a majority of the alpha-protons . Combined use of HCACO, HN(COCA)HA, HN(CA)HA, 15N TOCSY-HMQC and 15N NOESY-HMQC experiments provided the H alpha chemical shifts . Correlations for glycine protons were absent from most of the spectra . A combination of automated and interactive steps was used in the process, similar to that outlined by Ikura et al . {(1990) J . Am . Chem . Soc., 112, 9020-9022} in the seminal paper on heteronuclear backbone assignment . A major impediment to the linking process was the amount of overlap in the C alpha and H alpha frequencies . Ambiguities resulting from this redundancy were solved primarily by assignment of amino acid type, using C alpha chemical shifts and 'TOCSY ladders' . Ninety-four percent of the backbone resonances are reported for this subtilisin . The secondary structure was analyzed using 3D 15N NOESY-HMQC data and C alpha secondary chemical shifts . Comparison with the X-ray structure {Betzel et al . (1992) J . Mol . Biol., 223, 427-445} shows no major differences.

J Am Mosq Control Assoc, 1994 Mar, 10(1), 42 - 4
Fixed-wing, aerial application of liquid Bacillus thuringiensis H-14 (Acrobe) for control of spring Aedes mosquitoes in Michigan; Knepper RG et al.; Liquid Bacillus thuringiensis H-14 (Acrobe) was applied from fixed-wing aircraft at a rate of 4.68 liters of water-insecticide mixture (1.17 liter concentrate) per hectare to woodland pools in Michigan . A post-treatment larval survey indicated an 88.5% reduction in Aedes species larvae . A volume median diameter of 208 microns was determined.

Microbiology, 1994 Mar, 140 ( Pt 3), 637 - 42
Purification and properties of a new exo-(1-->3)-beta-D-glucanase from Bacillus circulans YK9 capable of hydrolysing resistant curdlan with formation of only laminari-biose; Kanzawa Y et al.; A (1-->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.6), capable of hydrolysing resistant curdlan, was purified chromatographically from the culture supernatant of Bacillus circulans complex YK9 on Toyopearl HW-55F and butyl-Toyopearl 650M columns . The purified enzyme had a specific activity of 190 units mg-1 on regenerated curdlan . The molecular mass was estimated to be about 70 kDa as judged by SDS-PAGE . The enzyme had a pH optimum of approximately pH 6.0 . It hydrolysed regenerated and resistant curdlans yielding predominantly laminari-biose, although the rate of hydrolysis of the former was much higher than the latter . This enzyme rapidly hydrolysed laminaran, curdlan and carboxymethyl-curdlan, but did not cleave schizophyllan and screloglucan, which have glucosyl side chains . The enzyme hydrolysed low molecular mass (1-->3)-beta-D-glucans-(mean degree of polymerization, DPn = 131, 49 and 14) and laminari-heptaose more efficiently than curdlan . It also hydrolysed laminari-hexaose and -pentaose effectively, but laminari-tetraose only slightly and it did not hydrolyse laminari-triose or -biose . The enzyme is an exo-hydrolase of curdlan and various oligomers composed of (1-->3)-beta-D-glucosidic linkages, liberating laminari-biose from their non-reducing terminals . The laminari-biose generated was in the alpha-form.

Bioorg Med Chem, 1994 Mar, 2(3), 147 - 51
Synthesis of optically-active hexadecyl thiophosphoryl-1-D-myo-inositol: a thiophosphate analog of phosphatidylinositol; Bushnev AS et al.; The synthesis of optically-active hexadecyl thiophosphoryl-1-D-myo-inositol 11 was accomplished from 2,3-O-(D-1',7',7'-trimethyl{2.2.1}bicyclohept-2'-ylidene)-4,5,6-O- tris(methoxymethyl)-D-myo-inositol 6 or 2,3,4,5,6-O-pentakis(methoxymethyl)-D-myo-inositol 14, using the Arbusov reaction of their dimethyl phosphite derivatives 7 and 15 with N-hexadecyl thiophthalimide 8 . This product was a substrate for phosphatidylinositol-specific phospholipase C from Bacillus cereus.

J Am Mosq Control Assoc, 1994 Mar, 10(1), 45 - 50
Incorporation of body components of diverse microorganisms by larval mosquitoes; Avissar YJ et al.; A pulse-purge schedule of exposure to labeled microorganisms was used to compare their digestibility by larval mosquitoes . Larvae were placed for an hour in suspensions of diverse axenically grown microorganisms that had been labeled with radioactive carbon (in the form of glucose or glycine) . The guts of these mosquitoes were then purged with nonlabeled Sephadex particles for 30 min, and retained radioactivity was measured . Larvae imbibed no dissolved material . Larval mosquitoes differ in their capacity to derive label from algae (sensu lato), and certain algae contribute more label to these mosquitoes than do others . The nature of any algal food, as well as the feeding habits and developmental stage of the larva, influence its capacity to derive label from algae . This pulse-purge method of analysis can assist in the selection of algal "vectors" suitable as vehicles for transgenic larvicide . Although larval mosquitoes fail to assimilate the contents of Palmellacoccus cells with which they are confined, as much as 1/3 of the body contents of a Euglena gracilis cells become incorporated into their bodies . Because larval mosquitoes internalize more material from Euglena than they do from various other algae, these microorganisms provide a promising candidate vehicle for transgenic Bacillus thuringiensis israelensis.

J Ind Microbiol, 1994 Mar, 13(2), 112 - 9
Fermentation and toxin studies of the molluscicidal strains of Bacillus brevis; Singer S et al.; Strain SS86-4 was one of 40 Bacillus brevis strains shown to be molluscicidal to the schistosomiasis snail vector Biomphalaria glabrata . When grown in mB4 medium in 2-L fermentors, SS86-4 was molluscicidal only if fructose or phenylalanine was present in the medium . This is reminiscent of secondary fermentation factor effects, in this case an antioxidant effect . In vivo proteases also were capable of reducing molluscicidal activity . The molluscicidal toxin has an LC50 of 1 microgram toxin protein ml-1 (approx . 1 p.p.m.) and may be described as a small proteinaceous, heat-stable, oxygen-sensitive entity associated with the particulate portion of the cell wall fraction of B . brevis that is formed prior to sporulation . Initial information indicates that its HPLC signature shows major peaks at 148.37 and 163.96 s and consists of two bands of approximately 5.3 kDa and 8.7 kDa on PAGE gel.

J Am Mosq Control Assoc, 1994 Mar, 10(1), 1 - 6
Control of Aedes albopictus larvae using time-release larvicide formulations in Louisiana; Nasci RS et al.; The ability of time-release formulations of larvicides and insect growth regulators (IGRs) to provide long-term control of Aedes albopictus was investigated in the field . Larvicides used in the study were Bactimos pellets (Bacillus thuringiensis var . israelensis, active ingredient) and Abate pellets (temephos, active ingredient) . The IGR Altosid (methoprene, active ingredient) was used in pellet and sand formulations . Application rates were higher than label recommendations . In a preliminary test, clay flower bowls were treated with 2 g of material . Bactimos pellets failed to provide control after 60 days . Abate pellets and the Altosid formulations provided essentially 100% control for 150 days . After 360 days in the field, the Abate pellets produced 100% larval mortality, and significant levels of control were provided by the Altosid formulations and the Bactimos pellets . In a small-scale operational trial of this technique, 1 g of Altosid pellets was applied to every container that could be located in 2 urban residential neighborhoods in Lake Charles, LA . Aedes albopictus biting populations were monitored weekly in the treated areas and in an untreated control area . Biting population densities declined significantly in treated areas compared with the control area . Results suggested that long-term control of Ae . albopictus populations can be achieved economically with one application of Altosid pellets or Abate pellets in containers.

FEBS Lett, 1994 Feb 28, 340(1-2), 89 - 92
Membrane interactions and surface hydrophobicity of Bacillus thuringiensis delta-endotoxin CryIC; Butko P et al.; The interaction between Bacillus thuringiensis insecticidal delta-endotoxin CryIC and phospholipid vesicles was studied by fluorescence spectroscopy . The toxin dissipates the diffusion potential across vesicular membranes, presumably by creating ion permeable channels or pores . This effect is pH-dependent and strongly increases under acidic conditions . The enhanced membrane-perturbing activity of CryIC at low pH correlates with the increased surface hydrophobicity of the toxin molecule . The membrane permeabilizing effect of the toxin is further increased by the presence of acidic phospholipids . These findings are discussed in relation to the mode of insecticidal action of the toxin.

FEBS Lett, 1994 Feb 28, 340(1-2), 39 - 44
Detection of 4'-phosphopantetheine at the thioester binding site for L-valine of gramicidinS synthetase 2; Stein T et al.; Biosynthesis of gramicidinS in Bacillus brevis is catalysed by a multienzyme system consisting of two multifunctional proteins, gramicidinS synthetase 1 and 2 codified by the grsA and grsB genes, respectively . GramicidinS synthetase 2 shows a modular architecture of four amino acid-activating domains each containing a thioester binding motif LGG H/D S L/I highly conserved in its C-terminal region, as demonstrated by sequence analysis of the grsB gene {W . Schlumbohm et al . (1991) J . Biol . Chem . 266, 23135-23141} . This multienzyme was specifically labeled at the thioester binding site of L-valine with {3H}N-ethylmaleimide using a substrate protection technique . After enzymatic digestion a labeled active site peptide was isolated in pure form by multistep methodology . This fragment was identified by gas-phase sequencing as the active site peptide of the thiotemplate site for L-Val by comparison with the grsB gene sequence . By mass spectrometry in combination with amino acid analysis it was demonstrated that a 4'-phosphopantetheine carrier was attached to the active serine in this motif . Our results give evidence that multiple peripheral 4'-phosphopantetheine carriers are involved in the formation of gramicidinS in contrast to a central carrier arm as assumed in the original version of the thiotemplate mechanism . A 'Multiple Carrier Model' of nonribosomal peptide biosynthesis is proposed.

J Biotechnol, 1994 Feb 28, 32(3), 283 - 8
Replacement of an amino acid residue of cyclodextrin glucanotransferase of Bacillus ohbensis doubles the production of gamma-cyclodextrin; Sin KA et al.; Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin (CD) from starch through an intramolecular transglucosylation reaction . To obtain a better understanding of the amylolytic and cyclization mechanisms of CGTase, and furthermore to improve the production of gamma-CD, mutant CGTases were constructed by site-directed mutagenesis of the CGTase gene of Bacillus ohbensis replacing Tyr at position 188 by 19 other amino acids . All mutant enzymes retained both starch-degrading and CD synthesizing activities to various extents . Among them, a mutant enzyme having Trp instead of Tyr-188 produced 15% of gamma-CD from soluble starch, which is about twice as much as the amount produced by the wild-type enzyme.

Med J Aust, 1994 Feb 21, 160(4), 197 - 201
The prevalence of tuberculosis infection among Year 8 schoolchildren in inner Sydney in 1992; Alperstein G et al.; OBJECTIVE: To determine the prevalence of tuberculosis (TB) infection in Year 8 schoolchildren (aged 12-14 years) in Sydney . DESIGN: Cross-sectional survey . SETTING: 22 inner city Sydney secondary schools . PARTICIPANTS: 2290 Year 8 school children enrolled in 1992 . OUTCOME MEASURES: Distribution of Mantoux test reaction size and proportion of children who were Mantoux positive (i.e., having Mantoux reaction > or = 15 mm with previous Bacille Calmette-Guerin vaccination; > or = 10 mm without) . RESULTS: Of the 2290 children, 1836 (81%) were screened and 1801 Mantoux reactions were read . Ten per cent of children were Mantoux positive--27% of foreign-born children and 2% of Australian-born children (relative risk 16.7, 95% confidence interval 10.6-26.4) . Two children were found to have active TB disease . CONCLUSION: There is a high prevalence of primary (non-contagious) TB infection in children aged 12-14 years in inner Sydney, mostly confined to children born overseas . Thus there is a large pool of infected children at risk of developing active (contagious) adult-type TB disease in the future . This public health problem should be addressed by identification and treatment of those infected.

J Mol Biol, 1994 Feb 18, 236(2), 663 - 5
Crystallization and quaternary structure analysis of the NAD(+)-dependent leucine dehydrogenase from Bacillus sphaericus; Turnbull AP et al.; The NAD(+)-dependent leucine dehydrogenase from Bacillus sphaericus has been crystallized by the hanging drop method of vapour diffusion, using ammonium sulphate as the precipitant . The crystals belong to the tetragonal system and are in space group I4, with unit cell dimensions of a = b = 138.4 A and c = 121.8 A . Considerations of the values of Vm, the space group symmetry and an analysis of a self-rotation function calculated on a preliminary data set collected to 3 A resolution show that the asymmetric unit contains a dimer with the twofold axis perpendicular to the crystallographic four fold, indicating that the quaternary structure of this enzyme is octameric . Leucine dehydrogenase belongs to a superfamily of amino acid dehydrogenases which display considerable differences in amino acid specificity and elucidation of its three-dimensional structure should enable the molecular basis of this differential specificity to be examined in detail.

J Mol Biol, 1994 Feb 18, 236(2), 590 - 600
Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form; Lawson CL et al.; The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus circulans strain 251 was cloned and sequenced . It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B . circulans strain 8 . The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution . The structure of the enzyme is nearly identical to the CGTase from B . circulans strain 8 . Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C . The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts . The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.

JAMA, 1994 Feb 16, 271(7), 531 - 5
Rochalimaea henselae infection . A new zoonosis with the domestic cat as reservoir; Koehler JE et al.; OBJECTIVE--To determine the reservoir and vector(s) for Rochalimaea henselae, a causative agent of bacillary angiomatosis (BA) and cat scratch disease, and to estimate the percentage of domestic cats with R henselae bacteremia in the Greater San Francisco Bay Region of Northern California . DESIGN--Hospital-based survey of patients diagnosed with BA who also had significant exposure to at least one pet cat, as well as a convenience sampling of pet or impounded cats for prevalence of Rochalimaea bacteremia . SETTING--Community and university hospitals and clinics; veterinary clinics treating privately owned or impounded cats . PATIENTS--Patients with or without human immunodeficiency virus infection, with biopsy-confirmed BA, who had prolonged exposure to pet cats prior to developing BA . MAIN OUTCOME MEASURES--Cultures and laboratory studies were performed on blood drawn from pet cats associated with patients with BA . The Rochalimaea species infecting pet cats and fleas and causing the BA lesions in human contacts of these cats was identified by culture, polymerase chain reaction-restriction fragment length polymorphism analysis, and DNA sequencing . The presence of R henselae bacteremia in pet cats was documented, and predictor variables for culture positivity were evaluated . RESULTS--Four patients diagnosed with BA who had prolonged contact with seven pet cats were identified . The Rochalimaea species causing BA lesions in these patients was determined to be R henselae . The seven pet cats were found to be bacteremic with R henselae; this bacterium was also detected in fleas taken from an infected cat by both direct culture and polymerase chain reaction . Blood samples were cultured from pet and impounded cats (N = 61) in the Greater San Francisco Bay Region, and R henselae was isolated from 41% (25/61) of these cats . CONCLUSION--We have documented that the domestic cat serves as a major persistent reservoir for R henselae, with prolonged, asymptomatic bacteremia from which humans, especially the immunocompromised, may acquire potentially serious infections . Antibiotic treatment of infected cats and control of flea infestation are potential strategies for decreasing human exposure to R henselae.

Biochim Biophys Acta, 1994 Feb 16, 1204(2), 164 - 8
Chemical modification of xylanase from alkalothermophilic Bacillus species: evidence for essential carboxyl group; Chauthaiwale J et al.; The role of carboxyl group in the catalytic action of xylanase (M(r) 35,000) from an alkalothermophilic Bacillus sp . was delineated through kinetic and chemical modification studies using Woodward's Reagent K . The kinetics of inactivation indicated that one carboxyl residue was essential for the xylanase activity with a second order rate constant of 3300 M-1 min-1 . The spectrophotometric analysis at 340 nm revealed that the inhibition was correlated with modification of 24 carboxyl residues . In the presence of protecting ligand, modification of one carboxyl group was prevented . The pH profile showed apparent pK values of 5.2 and 6.4 for the free enzyme and 4.9 and 6.9 for enzyme-substrate complex . The pH dependence of inactivation was consistent with the modification of carboxyl group . The kinetic analysis of the modified enzyme showed similar Km and lower kcat values than the native enzyme indicating that catalytic hydrolysis and not the substrate binding was affected by chemical modification . The chemical modification of xylanase from alkalothermophilic Bacillus revealed the presence of tryptophans in the active site (Deshpande, V, Hinge, J . and Rao, M . (1990) Biochim . Biophys . Acta 1041, 172-177) . This finding and present studies demonstrated the experimental evidence for the participation of carboxyl as well as tryptophan groups as essential residues of xylanase from alkalothermophilic Bacillus sp.

FEMS Microbiol Lett, 1994 Feb 15, 116(2), 195 - 9
Haemolytic activity associated with parasporal inclusion proteins of mosquito-specific Bacillus thuringiensis soil isolates: a comparative neutralization study; Ishii T et al.; Solubilized parasporal inclusions of the three mosquito-specific Bacillus thuringiensis isolates belonging to three different H serovars, co-isolated from a single soil microhabitat, showed haemolytic activity towards mammalian erythrocytes . Neutralization tests with antibodies against whole inclusion proteins resulted in crossed neutralization of haemolytic activity among the isolates and the type strain of B . thuringiensis serovar kyushuensis, indicating that the three soil isolates produce toxins related to the CytB toxin . No cross-neutralization occurred between the type strain of B . thuringiensis serovar israelensis and the three soil isolates.

Biochem J, 1994 Feb 15, 298 ( Pt 1), 45 - 50
Purification and properties of a phenol sulphotransferase from Euglena using L-tyrosine as substrate; Saidha T et al.; A purification procedure based on (NH4)2SO4 precipitation, and chromatography on Affi-Gel Blue, DEAE-cellulose, hydroxyapatite and Bio-Gel P-60 yields a stable 6400-fold-purified active monomeric phenol (tyrosine) sulphotransferase of 26 kDa from W10BSmL, an aplastidic mutant of Euglena gracilis var . bacillaris . The apparent Km for adenosine 3'-phosphate 5'-phosphosulphate (PAPS) is 15 microM (60 microM tyrosine as substrate); adenosine 5'-phosphosulphate is inactive . L-Tyrosine gave the lowest apparent Km (33 microM) (with PAPS at 30 microM), but tyrosine esters, tyrosinamide, L-p-hydroxyphenylglycine and a number of tyrosine dipeptides were also active, with higher Km values . Nitrophenols (m- and p-) and chlorophenols (o-, m- and p-) were active, with higher Km values than for tyrosine . D-Tyrosine was inactive as a substrate, as was D-p-hydroxyphenylglycine and a number of other tyrosine derivatives lacking the carboxy carbonyl or the amino group, or having extra ring substituents or the hydroxy group in the wrong position . Adenosine 3',5'-bisphosphate and tyrosine O4-sulphate, products of the enzyme reaction with PAPS and tyrosine as substrates, showed competitive (Ki = 20 microM) and uncompetitive (Ki = 500 microM) inhibition kinetics respectively . This appears to be the first phenol sulphotransferase to accept tyrosine as substrate . This membrane-bound enzyme may be involved in tyrosine transport as well as detoxification.

Eur J Biochem, 1994 Feb 15, 220(1), 97 - 104
Preparation of cyclic 2',3'-monophosphates of oligoadenylates (A2'p)nA > p and A3'p(A2'p)n-1A > p; Budowsky EI et al.; The action of the guanylyl-preferring RNase from Bacillus intermedius (binase) on a mixture of oligoadenylates with randomly distributed 2'-5' and 3'-5' internucleotide bonds {(A2'/3'p)n} under conditions sufficient for complete hydrolysis of poly(A) results in a mixture containing a single circular oligoadenylate and two series of linear oligoadenylates ending in cyclic 2',3'-phosphate . Individual compounds formed upon digestion of (A2'/3'p)n with binase have been isolated . Their structure was determined on the basis of their chemical and enzymatic conversions and confirmed by 1H-, 13C- and 31P-NMR spectra . According to these data, the circular triadenylate contains one 2'-5' and two 3'-5' internucleotide bonds, linear oligoadenylates of one series contain exclusively 2'-5' internucleotide bonds {(A2'p)nA > p}, while each compound of the other series contains a single 3'-5' internucleotide bond connecting the 5'-ultimate nucleotide residue with the penultimate one {A3'p(A2'p)n-1A > p} . The incubation of compounds of the former series A3' p(A2'p)n > p at pH 1.0 and the subsequent action of phosphatase results in successive formation of compounds of two other new series: A3'p(A2'p)nA2'(3')p and A3'p(A2'p)nA.

Biochem Biophys Res Commun, 1994 Feb 15, 198(3), 940 - 7
Altered protoxin activation by midgut enzymes from a Bacillus thuringiensis resistant strain of Plodia interpunctella; Oppert B et al.; Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B . thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B . thuringiensis subspecies kurstaki (HD-1, Dipel) . Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate alpha-N-benzoyl-DL-arginine rho-nitroanilide than extracts from susceptible or kurstaki-resistant insects . Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract . The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin . The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B . thuringiensis.

Biochem Biophys Res Commun, 1994 Feb 15, 198(3), 862 - 8
Protein-binding DNA regions inside and in the vicinity of the cytochrome P450bm-3 gene from Bacillus megaterium; Gaidamakova EK et al.; Protein-binding DNA-regions inside and in the vicinity of the phenobarbital-inducible P450bm-3 gene from Bacillus megaterium were characterized by gel-retardation technique and foot-printing analysis . Regions with induction-dependent protein binding capacity were shown to be situated in -279bp/-215bp, -215bp/+83bp and +236bp/+425bp fragments . Precise localization of DNA-protein contacts was established by foot-printing analysis for region -215bp/+83bp . The data obtained are discussed in line with previously reported information on the regulatory regions of various phenobarbital-regulated genes.

Arch Biochem Biophys, 1994 Feb 15, 309(1), 29 - 35
The gene for biotin synthase from Saccharomyces cerevisiae: cloning, sequencing, and complementation of Escherichia coli strains lacking biotin synthase; Zhang S et al.; Biotin synthase catalyzes the insertion of a sulfur atom between two carbon atoms of dethiobiotin to form biotin in the last step of the biotin biosynthesis pathway . In Escherichia coli, biotin synthase is coded for by bioB gene . We report here cloning, sequencing, and initial functional characterization of the yeast gene for biotin synthase in Saccharomyces cerevisiae . We have named this gene BIO2 . It consists of a 355-codon open reading frame near the ZUO1 gene . Analysis of the yeast protein encoded by the BIO2 gene reveals that it shares extensive homology with biotin synthases of E . coli and Bacillus sphaericus . The yeast and the two bacterial biotin synthase proteins have similar molecular weights, amino acid compositions, and hydropathies . The plasmid pUCBIO2 containing the yeast BIO2 gene completely complements E . coli bioB- and delta bio mutants and enables these mutants to grow on dethiobiotin . Although BIO2 is physically linked to ZUO1, which encodes the putative left-handed Z-DNA binding protein zuotin, it appears to be regulated independently from it . The yeast BIO2 and ZUO1 genes reside near ADE3 gene on chromosome VII . BIO2 is the first eukaryotic gene reported from the biotin biosynthetic pathway.

J Mol Biol, 1994 Feb 11, 236(1), 209 - 16
Structural dependence of post-translational modification and reductive acetylation of the lipoyl domain of the pyruvate dehydrogenase multienzyme complex; Wallis NG et al.; The lipoyl domain of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex is recognized specifically by the lipoylating enzyme(s) in the cell and by the pyruvate dehydrogenase (E1) component in the parent complex . Highly conserved aspartic acid and alanine residues flank the lipoyl-lysine residue, on the N and C-terminal sides, respectively, in the sharp beta-turn in which the lipoyl-lysine residue is prominently displayed . A sub-gene encoding the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase complex was subjected to mutagenesis in the vector M13mp18 . Aspartic acid 41 was changed to glutamic acid (D41E), alanine (D41A) and lysine (D41K), and alanine 43 was changed to methionine (A43M), lysine (A43K) and glutamic acid (A43E) . The double mutations D41KK42A and D41MA43M were also made . All mutant domains were capable of being lipoylated, apart from the D41KK42A domain where the lipoyl-lysine had been moved round the beta-turn by one position towards the N terminus . Neither the D41K nor the A43K mutants showed any doubly lipoylated domain and the single lipoyl group was found attached only to the correct lysine residue . Accurate positioning of the lipoyl-lysine in the beta-turn is thus an essential cue for lipoylation, but the conserved aspartic acid and alanine residues are not necessary for the domain to be recognized by the lipoylating enzyme(s) . No biotinylation of the D41MA43M mutant domain was observed, although the sequence motif MKM is highly conserved as the biotinylation site in the structurally homologous biotinyl domain of biotin-containing enzymes . The mutations at the aspartic acid 41 position all lowered the rate of reductive acetylation of the lipoyl domain by the E1 component of the pyruvate dehydrogenase complex, as did the mutations A43E and A43K . The A43M mutant was reductively acetylated at the same rate as the wild-type domain . Thus, both the alanine and aspartic acid residues are important for recognition of the domain by E1, but there is no absolute dependence on retention of the sequence surrounding the lipoyl-lysine residue.

J Biol Chem, 1994 Feb 4, 269(5), 3374 - 80
Successive inactivation of the force-generating units of sodium-driven bacterial flagellar motors by a photoreactive amiloride analog; Muramoto K et al.; Like amiloride, 6-iodoamiloride (6-IA) competitively and reversibly inhibits rotation of the Na(+)-driven flagellar motors of alkalophilic Bacillus cells . However, when 6-IA-treated cells are irradiated with UV light, motility is irreversibly inhibited . This treatment does not alter the membrane potential or affect Na(+)-coupled alpha-aminoisobutyrate transport . An increase in the Na+ concentration during UV irradiation substantially protects the motors from irreversible inhibition . Thus, photoactivated 6-IA seems to bind specifically and covalently at or around the Na(+)-interaction site of the force-generating units of the motors to inhibit motor rotation irreversibly . Rotation of each motor, which is monitored using tethered alkalophilic Bacillus cells, is also inhibited by photoactivated 6-IA . In this case, however, the rotation rate during UV irradiation decreases stepwise, suggesting the presence of several independently functioning force-generating units in a motor . From the data of 14 tethered cells, the number of units/motor is estimated to be 5-9.

Biochemistry, 1994 Feb 1, 33(4), 1017 - 22
Microheterogeneity in glycosylphosphatidylinositol anchor structures of bovine liver 5'-nucleotidase; Taguchi R et al.; In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS) . Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA {Suzuki et al . (1993) J . Biochem . (Tokyo) 113, 607-613} is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored . Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS . Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected . Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography . Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS . Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined . The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate . Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1994 Feb, 176(3), 848 - 60
Isolation of two physiologically induced variant strains of Bacillus stearothermophilus NRS 2004/3a and characterization of their S-layer lattices; Sara M et al.; During growth of Bacillus stearothermophilus NRS 2004/3a in continuous culture on complex medium, the chemical properties of the S-layer glycoprotein and the characteristic oblique lattice were maintained only if glucose was used as the sole carbon source . With increased aeration, amino acids were also metabolized, accompanied by liberation of ammonium and by changes in the S-layer protein . Depending on the stage of fermentation at which oxygen limitation was relieved, two different variants, one with a more delicate oblique S-layer lattice (variant 3a/V1) and one with a square S-layer lattice (variant 3a/V2), were isolated . During the switch from the wild-type strain to a variant or from variant 3a/V2 to variant 3a/V1, monolayers of two types of S-layer lattices could be demonstrated on the surfaces of single cells . S-layer proteins from variants had different molecular sizes and a significantly lower carbohydrate content than S-layer proteins from the wild-type strain did . Although the S-layer lattices from the wild-type and variant strains showed quite different protein mass distributions in two- and three-dimensional reconstructions, neither the amino acid composition nor the pore size, as determined by permeability studies, was significantly changed . Peptide mapping and N-terminal sequencing results strongly indicated that the three S-layer proteins are encoded by different genes and are not derived from a universal precursor form.

Fertil Steril, 1994 Feb, 61(2), 331 - 5
Bromocriptine reverses the inhibitory effect of macrophages on human sperm motility; Scommegna A et al.; OBJECTIVE: To determine the effects of PRL suppression on the activation of murine peritoneal macrophages and their subsequent effects on human sperm motility . DESIGN: Laboratory study . INTERVENTIONS: Mice were treated with subcutaneous implants of 2.5 mg bromocriptine pellets 7 days before bacillus of calmette and guerin (BCG), a strain of Mycobacterium bovis, injection for activation of macrophages . Bromocriptine treatment, which significantly suppressed circulating PRL levels, continued through the day of peritoneal macrophage collection . Macrophages were subsequently cultured for 4 days and culture supernatant was collected . MAIN OUTCOME MEASURES: Human sperm were incubated for 4 hours in the presence of culture medium or culture supernatant from control and treated mice . Motion analysis was performed at 0, 2, and 4 hours . RESULTS: A significant decrease in human sperm motility was observed in the presence of culture supernatant from activated murine peritoneal macrophages . After 4 hours of incubation, sperm motility decreased from 69% +/- 3% in the nonactivated macrophage control group to 37% +/- 6% in the BCG- activated macrophage group . The suppressive effect of soluble products of activated macrophages on human sperm motility was reversed when mice were rendered hypoprolactinemic with bromocriptine . Motility after 4 hours was 56% +/- 3% in the BCG-bromocriptine group . Simultaneous administration of bromocriptine and PRL (100 ng per mouse daily) restored the inhibitory effect of soluble products of activated macrophages on sperm motility (36% +/- 5% motile) . CONCLUSIONS: PRL may modulate the deleterious effects of activated macrophages on human sperm motility, thereby suggesting novel and useful methods for the modification of the immune response in early reproduction.

Virology, 1994 Feb, 198(2), 663 - 70
Splicing in a plant pararetrovirus; Futterer J et al.; Analysis of expression in rice cells of plasmids in which an ATG-less chloramphenicol acetyl transferase ORF was placed in frame with the coding sequence of the pararetrovirus rice tungro bacilliform virus (RTBV) ORF IV, and which contained much of the upstream full-length transcript sequence of RTBV, gave evidence to suggest that a splicing event occurred . Reverse transcription/PCR of RNA from transfected protoplasts and from RTBV-infected plants yielded a product which confirmed that the 5' end of ORF IV was spliced in frame to a short ORF (sORF) in the RTBV leader sequence removing an intron of about 6.3 kb . Most of the translation is initiated at the ATG codon in the sORF with only about 10% at the ORF IV ATG codon . The efficiency of splicing appears to be inversely related to the length of the intron . The finding of splicing in a pararetrovirus blurs the differences between them and retroviruses but is in accord with the hypothesis that retroelements acquire genes and sequences which adapt them to specific niches.

Ann Intern Med, 1994 Feb 1, 120(3), 190 - 8
The booster effect in two-step tuberculin testing among young adults in Montreal; Menzies R et al.; OBJECTIVES: No consensus exists regarding the definition and interpretation of a significant boosting reaction after sequential tuberculin testing . The booster phenomenon is thought to represent remote tuberculous infection where tuberculin reactivity has waned, but it has also been described among persons with previous exposure to other mycobacteria or Bacille Calmette-Guerin (BCG) vaccine . We studied the factors affecting the booster phenomenon among Canadian-born young adults to determine the definition that would maximize sensitivity and specificity of a positive booster reaction in these persons . DESIGN: Point-prevalence survey of initial tuberculin reactions and response to repeated tuberculin testing after 1 to 4 weeks . SETTING: Community-based study of all students entering health professional training programs at six post-secondary institutions . MEASUREMENTS: In 1989, 1990, and 1991, students completed self-administered questionnaires, underwent two-step tuberculin testing with purified protein derivative-tuberculin (PPD-T), and had their childhood BCG vaccination status verified . In 1991, students were also tested with purified protein derivative-Battey (PPD-B) (for Mycobacterium intracellulare) . RESULTS: Overall, 74 students (5.2%) had positive booster reactions, which were significantly associated with older age (P < 0.001), larger initial tuberculin reactions (P < 0.001), previous BCG vaccination (P < 0.001), older age when vaccinated (P < 0.02), longer interval from vaccination to testing (P < 0.01), and sensitivity to PPD-B (P < 0.001) . Boosting was not associated with the number of BCG vaccinations, sex, or risk factors for tuberculous infection . The pattern, mean, and mode of the frequency distributions of booster reactions among those with BCG vaccination and sensitivity to PPD-B were similar to those with assumed tuberculous infection . CONCLUSIONS: In young adults, booster reactions due to previous tuberculous infection are uncommon and cannot be distinguished from false-positive reactions due to past exposure to other mycobacteria.

An Med Interna, 1994 Feb, 11(2), 62 - 6
{The study of contacts of tuberculosis patients}; Fernandez Revuelta A et al.; The study and follow-up of contacts is one of the main goals of the battle against tuberculosis . We studied 640 contacts of 141 patients diagnosed of active pulmonary tuberculosis (PT) in our center between 1985 and June 1990 . The average per index case (IC) was 4.5 . Contacts were classified according to the IC bacteriology (positive bacilloscopy and culture: 448 cases; negative bacilloscopy and positive culture: 126 cases; and both tests negative: 66 cases) . PPD was positive in 342 cases (53.4%) and the number of infected contacts was significant when IC showed positive bacilloscopy and culture (251 cases), cough (328 cases) . Twelve new cases of tuberculosis (1.9%) were detected, with an average age of 29.6 years . Chemoprophylaxis was completed during one-year period by 121 contacts (43.5%) . The systematic study of contacts allow us to detect new patients and infected cases, helping to break the epidemiological chain of transmission of the disease.

Pediatr Infect Dis J, 1994 Feb, 13(2), 113 - 6
Osteitis after newborn vaccination with three different Bacillus Calmette-Guérin vaccines: twenty-nine years of experience; Kroger L et al.; Newborns in Finland have been vaccinated with Bacillus Calmette-Guerin (BCG) since the 1950s . Until the end of 1970 the vaccine was made from BCG strain Gothenburg by the Swedish BCG laboratory in Gothenburg and from 1971 on from the same strain in Copenhagen, Denmark . It was replaced by the Glaxo vaccine in 1978 . Complications caused by BCG vaccination have been under follow-up, and the data have been collected from nationwide registers . In this study we analyzed the incidence rates of BCG osteitis between the years 1960 and 1988 . From 1960 to 1970 the incidence rate was from 2.7 to 13.0/100,000 BCG-vaccinated infants (mean, 7.3; median, 6.9) . The incidence increased during the years 1971 to 1978 when it varied between 15.3 and 72.9/100,000 BCG-vaccinated infants (mean, 36.9; median, 30.4) . Since 1978 the incidence has varied between 1.7 and 10.1/100,000 BCG-vaccinated infants (mean, 6.4; median, 7.2) . In Britain no reports of BCG osteitis have been published despite the use of the same Glaxo vaccine . Our results indicate that the incidence of BCG osteitis in a given population depends on the BCG vaccine used . The follow-up of BCG complications is an essential part of BCG vaccination program.

Int J Biol Macromol, 1994 Feb, 16(1), 43 - 9
Enzymatic degradation of chitins and partially deacetylated chitins; Shigemasa Y et al.; The enzymatic (lysozyme, chitinase etc.) digestibility of chitins obtained from squid pen and shrimp shell, and of partially deacetylated chitins (DA-chitins) was investigated . The digestibility of various chitins by the chitinase from Bacillus sp . PI-7S was much higher than that by lysozyme, and beta-chitin was digested more smoothly than alpha-chitin . DA-chitin deacetylated under homogeneous conditions (DAC) was hydrolysed by lysozyme more rapidly than that deacetylated under heterogeneous conditions (DAC) . DACs from shrimp shell and squid pen showed the same degree of digestibility by lysozyme in spite of a difference in the crystal structure of the original chitins . The crystal structure of chitin and the degree of N-acetyl group aggregation among DA-chitin molecules affect the enzymatic digestibility of chitin and DA-chitin, respectively.

Int J Biochem, 1994 Feb, 26(2), 171 - 9
Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C--IV . The release from Cacia porcellus organs; Nakabayashi T et al.; 1 . Alkaline phosphodiesterase I release from organs of Cacia porcellus by the action of phosphatidylinositol-specific phospholipase C (PIPLC) of Bacillus thuringiensis was studied . 2 . A significant amount of alkaline phosphodiesterase I was released from both slices and homogenate of the kidney and small intestine but not from the liver or pancreas . 3 . The release of the enzyme from kidney brush border membranes was dependent on, or proportional to, the reaction time and the PIPLC concentration . The enzyme release by PIPLC was suppressed when the PIPLC was heat-inactivated before addition to the reaction mixture . This suggests that the enzyme release must be due to direct action of PIPLC on kidney brush border membranes . 4 . The released alkaline phosphodiesterase I had a molecular weight of 240,000 and was activated by Mg2+, but strongly inhibited by EDTA, thiol reagents and 5'-nucleotide-containing compounds.

Int J Dermatol, 1994 Feb, 33(2), 109 - 12
Papulonecrotic tuberculid secondary to disseminated Mycobacterium avium complex; Williams JT et al.; BACKGROUND . Papulonecrotic tuberculid is a rarely reported cutaneous reaction to the mycobacterium bacillus . It is most often encountered in association with tuberculosis . The clinical and histologic picture of the entity is a distinctive one, but the etiology of the disease process is uncertain . Therapy directed against the causative organism is dramatically successful . METHODS . A 35-year-old white man with AIDS was referred to the Dermatology clinic for evaluation of a widespread skin eruption . The skin lesions were biopsied for histopathology and culture . From the cutaneous cultures Mycobacterium avium complex (MAC) organisms were grown . RESULTS . We report the first case of papulonecrotic tuberculid manifestation in an AIDS patient with disseminated MAC . Unusual features seen in this case include the predominance of pruritic eschars rather than asymptomatic papules and the confirmation by special stains of mycobacterium organisms within the skin biopsy . Papulonecrotic tuberculid has not been previously associated with either MAC or AIDS . CONCLUSIONS . Papulonecrotic tuberculid should be a diagnostic consideration in immunocompromised patients with MAC whose clinical and histologic features are compatible with this rare entity.

Am J Trop Med Hyg, 1994 Feb, 50(2), 219 - 28
An ultrastructural study of vertical transmission of Rickettsia tsutsugamushi during oogenesis and spermatogenesis in Leptotrombidium pallidum; Urakami H et al.; Rickettsia tsutsugamushi in Leptotromibidium pallidum was observed by electron microscopy and rickettsiae were found in the various tissues and organs of both larvae and adults . Budding of rickettsiae, a manner of release from the host cells, was observed only in the rudiments of the reproductive organs in larvae . Oogonia and maturing oocytes in adult females and eggs after oviposition contained the microorganisms . In adult males, rickettsiae were also found in the spermatogonia, spermatocytes, and spermatids in the early stage of spermatogenesis, but were eliminated from these cells during maturation . Only the maturing spermatids, but not the eliminated rickettsiae, migrated to another rickettsia-free area of the testis, resulting in the separation of spermatids from rickettsiae and in the production of rickettsia-free spermatophores . Based on these observations, the mechanism of vertical transmission of the rickettsiae to the progeny occurs only in the female parents . Most rickettsiae in the somatic cells of larvae and adults were coccoid, but some rickettsiae in the ovary and the testis of adult mites showed bacillary forms and were enveloped by a membrane of unknown origin.

Urology, 1994 Feb, 43(2 Suppl), 2 - 5; discussion 6
Intravesical therapy in superficial bladder cancer; Witjes JA et al.; OBJECTIVE . To identify the appropriate characteristics of superficial bladder cancer that allow the physician to predict tumor behavior . METHODS . We analyzed 1,745 patients with primary superficial bladder cancer with regard to disease and patient characteristics . RESULTS . With the characteristics we found, the population can be described and prognostic factors can be found . Moreover, with the use of these factors, it appears to be possible to predict tumor behavior on a group level . Prediction on an individual level, however, remains far from ideal . CONCLUSIONS . Three treatment options can be chosen: follow-up after initial transurethral resection, additional instillations with intravesical chemotherapy, or instillations with bacillus Calmette-Guerin (BCG) . In case instillations are indicated, most (but not all) superficial bladder cancer trials show superiority of BCG over intravesical chemotherapy, although at the cost of more side effects . However, with intravesical BCG, it seems possible to influence the natural history of superficial bladder cancer, which means that we can delay progression and improve survival.

Braz J Med Biol Res, 1994 Feb, 27(2), 395 - 9
Studies on the GPI-anchored enzyme, hepatic 5'-nucleotidase . Microheterogeneity of the anchor, processing and localization; Ikezawa H et al.; Hepatic 5'-nucleotidases of vertebrates were investigated for localization in the lysosomes and the plasma membrane, microheterogeneity of the glycosylphosphatidylinositol (GPI)-anchor moiety and minimal requirement of the C-terminal signal peptide for GPI attachment . Using PIPLC of Bacillus thuringiensis and subcellular fractionation by Percoll gradient centrifugation, we found that chicken liver 5'-nucleotidase can be transferred from plasma membrane to lysosomes in the GPI-anchored or soluble form . Bovine liver ecto 5'-nucleotidase was solubilized by PIPLC, purified to a homogeneous state, and analyzed for the structures of GPI-anchor isoforms by HPLC and ESI-MS in combination with glycosidase treatments, after peptide-bond cleavage by CNBr or trypsin . Several isomers of the GPI anchor were thus characterized; major components contained two phosphorylethanolamine residues, whereas the component containing three phosphorylethanolamine residues was present only as a small percentage of the total . The cleavage/attachment site of the GPI anchor in the C-terminal of 5'-nucleotidase was shown to be Ser523 . The peptide region cleaved off at the posttranslational processing has a length of 25 amino acid residues which contains a hydrophobic stretch of 17 amino acids . By site-directed mutagenesis, we determined the minimal length of the hydrophobic peptide to be 13 amino acids for expression of 5'-nucleotidase as a GPI-anchored form on the COS cell surface . When peptide length was shortened to less than 13 amino acids, the expressed enzyme was not sorted to the cell surface but present within, or secreted out of the cells.

Mol Microbiol, 1994 Feb, 11(4), 629 - 39
Isolation and characterization of the aspartokinase and aspartate semialdehyde dehydrogenase operon from mycobacteria; Cirillo JD et al.; Diaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall . The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic . The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction of in vivo selection systems . Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria . In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette-Guerin (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated . The M . smegmatis asd gene was isolated by complementation in Escherichia coli . This gene was then used to isolate the asd genes from other mycobacteria . The asd-complementing fragments from BCG and M . smegmatis were sequenced . An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask) . Primer extension analysis revealed that the only transcriptional start in this region is found 5' of the ask gene . This observation indicates that the mycobacterial ask and asd genes are in an operon.

Mol Microbiol, 1994 Feb, 11(3), 429 - 36
The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N; Knight PJ et al.; A 120 kDa glycoprotein in the larval midgut membrane of the lepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin, has been purified by a combination of protoxin affinity chromatography and anion exchange chromatography . In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin in the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin . N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2) . When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M . sexta brush border membrane vesicles . We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M . sexta is the metalloprotease aminopeptidase N.

Am J Trop Med Hyg, 1994 Feb, 50(2), 247 - 60
DNA typing of rickettsiae in naturally infected ticks using a polymerase chain reaction/restriction fragment length polymorphism system; Gage KL et al.; We used the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) rickettsial typing system of Regnery and others to rapidly identify rickettsiae in naturally infected ticks . Unlike previously described methods, our PCR assays type rickettsiae directly from tick tissues without first isolating the organisms . We collected 226 adult Dermacentor andersoni ticks in the Bitterroot Mountains of western Montana and analyzed them for possible rickettsial infection by hemolymph test using the Gimenez stain . Thirteen (5.8%) of these ticks were positive by hemolymph test and selected for further analysis using the above PCR/RFLP typing system . The PCR assays performed using the first primer set (RpCS) resulted in amplification of fragments of the predicted size from nine of the 13 hemolymph test-positive tick samples . Only four of these nine tick samples were also positive in similar PCR assays performed with a second primer set (Rr190) that is presumed to be spotted fever group specific . The RFLP analyses of material amplified from these four ticks indicated they were infected with Rickettsia rickettsii (one sample) and R . rhipicephali (three samples) . The PCR/RFLP analyses of the five PCR-positive tick samples that were positive only in assays performed with the RpCS primer set indicated that these ticks were infected with R . bellii . The remaining four of 13 hemolymph test-positive tick samples gave negative PCR results with both the RpCS and Rr190 primer sets . Infected hemocytes from these PCR-negative ticks contained organisms of distinctive bacillary morphology that appeared similar to those described previously as long forms, and it is possible that these organisms belong to a genus other than Rickettsia . We also examined established laboratory isolates of tick-borne rickettsiae from different regions of North America to determine whether this typing system produces consistent results . Multiple isolates of R . montana (nine isolates), R . bellii (five isolates), R . rickettsii (Hlp-like) (four isolates), and R . canada (two isolates) were tested and no significant variations in PCR/RFLP patterns were observed between members of the same serotypes . However, among the five isolates of R . rhipicephali tested, two slightly different RFLP patterns were noted . Our results suggest that this PCR/RFLP typing scheme has wide applicability for identifying rickettsiae directly from D . andersoni or D . variabilis tick tissues.

Mol Gen Genet, 1994 Feb, 242(3), 365 - 8
Use of an operon fusion to induce expression and crystallisation of a Bacillus thuringiensis delta-endotoxin encoded by a cryptic gene; Crickmore N et al.; A delta-endotoxin gene previously cloned from Bacillus thuringiensis subsp . galleriae has been shown by a combination of restriction mapping and DNA sequence analysis to be a cryIIB clone; in common with other cryIIB genes it was found to lack a functional promoter . Addition of a promoter resulted in expression of the gene in Bacillus thuringiensis but did not result in the formation of the crystalline inclusions normally associated with such toxins . Inclusion formation was only observed when the gene was incorporated into an operon containing a gene known to be involved in the crystallisation of another delta-endotoxin.

J Chem Technol Biotechnol, 1994 Feb, 59(2), 193 - 9
Convenience of immobilized Bacillus licheniformis alpha-amylase as time-temperature-integrator (TTI); De Cordt SF et al.; For the immobilization of Bacillus licheniformis alpha-amylase to porous glass beads, the performances of three possible linking agents, glutaric dialdehyde, benzoquinone and s-trichlorotriazine were assessed in respect of the protein yield, the enzymic activity and the thermostability of the immobilized enzyme . These three properties are to be evaluated in view of the possible use of the enzyme preparations as time-temperature-integrators (TTIs) for assessing the severity of heat pasteurization or sterilization processes of food or pharmaceuticals . All three linkers improved the enzyme's resistance to irreversible heat inactivation to a similar extent and in each case biphasic inactivation kinetics were observed, whereas the dissolved B . licheniformis alpha-amylase showed a simple first order decay . The immobilization yield, measured as protein per carrier weight, did not differ markedly for the three linkers, although the enzymic activity of the glutaric dialdehyde-linked enzyme was lower than that of the benzoquinone- and s-trichlorotriazine-linked preparations.

Enzyme Microb Technol, 1994 Feb, 16(2), 99 - 103
Functional changes of dextran-modified alkaline proteinase from alkalophilic Bacillus sp; Yamagata Y et al.; A serine alkaline proteinase (EC 3.4.21.62) from Bacillus sp . (ALPase I) was modified with the 2,4-dialdehyde derivative of clinical dextran (dialdehyde dextran) . The modified preparation was purified using an ion-exchange column and gel filtration . The modified enzyme contained 75% carbohydrate by weight . The isoelectric point (pI) of ALPase I was converted from 8.2 to approximately 5.0 by this modification . The specific activity of the dextran-modified ALPase I was 56% of that of the native enzyme when milk casein was used as a substrate . It also had some superior characteristics: the thermostability of the modified enzyme at pH 10.0 was about 10-15 degrees C higher than that of control . In organic solvents such as n-hexane, benzene, and toluene, the hydrolysis reaction of the modified ALPase I for the fluorogenic substrate, succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-4-methylcoumaryl-7-am ide (Suc-Ala-Ala-Pro-Phe-MCA), was several times higher than that of the native . This modification greatly improved the stability of ALPase I against nonionic and anionic surfactants . After exposure to lauryl benzene sulfonate and sodium lauryl sulfonate the modified enzyme retained over 95 and 90% of its activity, respectively, but the native enzyme lost its activity . We conclude that modification of serine proteinases with dialdehyde-dextran might be a useful method for improving enzyme character for enzyme technology.

Contraception, 1994 Feb, 49(2), 131 - 7
Comparative in vitro study of contraceptive agents with anti-HIV activity: gramicidin, nonoxynol-9, and gossypol; Bourinbaiar AS et al.; Gramicidin, a polypeptide antibiotic derived from Bacillus brevis, was compared in vitro with the established contraceptive virucidal agents nonoxynol-9 and gossypol for activity against human immunodeficiency virus (HIV) infection . The effective antiviral 10 ng/ml concentration of gramicidin required for complete HIV inactivation was a thousand-fold lower than the dose observed for nonoxynol-9 or gossypol . Gramicidin, routinely used as a contraceptive agent in the former Soviet Union, should be considered for in vivo trials as a spermicide with potent antiviral activityPIP: At the New York University Medical Center, researchers used an antiviral assay, p24 ELISA, and cytoxicity assays to compare the antiviral activity of the newly-discovered anti-HIV compound gramicidin with established spermicides that have demonstrated antiviral activity . A 10 ng/ml of gramicidin completely inhibited productive HIV infection in MT-4 lymphocytes, while a 1000-fold higher dose of nonoxynol-9 and gossypol (10 mcg/ml) was needed to achieve the same effect . A possible mechanism is impaired permeability for cations, resulting in inhibition of virus-induced fusion . The study did not try to determine the biochemical mode of gramicidin action, however . Gramicidin is used in the former USSR as an active component of spermicidal gels and foams and as a topical, non-irritating antibiotic often used to treat ocular infections . The results of this in vitro study call for more in vitro studies to determine the biochemical mechanism of gramicidin action and its clinical potential as a vaginal spermicide with antiviral activity .

Radiat Res, 1994 Feb, 137(2), 186 - 9
Comparative effects of gamma rays and electron beams on spores of Bacillus pumilus; Hayashi T et al.; The effects of gamma rays and electron beams on the germination, outgrowth and the synthesis of protein and RNA of Bacillus pumilus spores were investigated to clarify the difference in the effects of the two types of radiations on bacterial spores . Gamma irradiation facilitated the germination to a slightly larger degree than electron irradiation . The outgrowth, growth and the synthesis of protein and RNA were inhibited by gamma irradiation to a greater extent than electron irradiation, when the spores were irradiated at the same dose . However, the effects of the two types of radiations were the same when the spores were irradiated with electron beams at a dose 30% higher than gamma rays . The results indicate that the effects of electron beams on bacterial spores and those of gamma rays are qualitatively the same but quantitatively different.

Ugeskr Laeger, 1994 Jan 31, 156(5), 628 - 31, 634
{Intravesical Bacillus Calmette Guerin treatment of bladder tumors and carcinoma in situ}; Ovesen H; Intravesical Bacillus Calmette-Guerin (BCG) has been used in the treatment of transitional neoplasia of the bladder since 1976 . The indications for BCG-treatment are: 1 . Carcinoma in situ (CIS), 2 . Prophylaxis and 3 . Treatment of tumor(s) . This paper reviews the treatment schedules, results, side effects, mode of actions and the efforts at improvement of the treatment . In conclusion, the results in CIS are satisfactory, especially in primary CIS . In prophylaxis, efficacy has been demonstrated in small randomized trials against recurrence of tumor, rate of progression and survival . Unfortunately, the characteristics of the tumor(s) treated by BCG are heterogenous (stage, concomitant CIS) and the numbers of patients are small, making it difficult to determine whether there is an effect in all patients or only a subgroup . Tumor treatment with intravesical BCG is only indicated in patients not fit to undergo transurethral resection . Treatment of CIS with BCG is well established, but the exact indications for prophylaxis and tumor treatment are not known . The optimal treatment schedule has probably not been established, and further studies are necessary in an effort to improve the efficacy.

Biochem J, 1994 Jan 15, 297 ( Pt 2), 351 - 7
Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis; Lemassu A et al.; The cell envelope which surrounds pathogenic mycobacteria is postulated to be a defence barrier against phagocytic cells and its outermost constituents have a tendency to accumulate in the culture medium . The present work demonstrates that the exocellular material of Mycobacterium tuberculosis contains large amounts of polysaccharides with only traces, if any at all, of lipids . Three types of polysaccharides were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents . They consisted of D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 123, 13 and 4 kDa respectively . Their predominant structural features were determined by the characterization of the per-O-methyl derivatives of enzymic, acetolysis and Smith-degradation products and by 1H- and 13C-n.m.r . spectroscopy of the purified polysaccharides, using mono- and two-dimensional homonuclear chemical-shift correlated spectroscopy and two-dimensional heteronuclear (1H/13C) spectroscopy . The glucan which represented up to 90% of the polysaccharides was composed of repeating units of five or six-->4-alpha-D-Glcp-1--> residues and a -->4-alpha-D-Glcp substituted at position 6 with an alpha-D-Glcp, indicating a glycogen-like highly branched structure not related to the so-called polysaccharide-II previously identified in tuberculin . The arabinomannan consisted of a mannan segment composed of a -->6-alpha-D-Man-1--> core substituted at some positions 2 with an alpha-D-Manp . The arabinan termini of the arabinomannan were found to be extensively capped with mannosyl residues . The possibility that these polysaccharides contribute to the persistence of the tubercle bacillus in the macrophage by molecular mimicry is discussed.

Am J Ophthalmol, 1994 Jan 15, 117(1), 65 - 71
Microbial keratitis in children; Clinch TE et al.; In a five-year review, we identified 29 cases of microbial infection in 28 patients who were 16 years old or younger . Herpes simplex infections were excluded . Predisposing factors included trauma (ten cases, 34%), severe systemic illness (eight cases, 27%), contact lens use (seven cases, 24%), exposure keratopathy (seven cases, 24%), and previous ocular surgery (six cases, 21%) . A total of 24 microorganisms were identified in cultures of corneal scrapings from 22 of the 29 cases; two cases involved polymicrobial infections . Of the 24 identified pathogens, gram-positive cocci were the most common (12) . Other microorganisms included gram-negative bacteria (five) and fungi (four) . Isolated cases of Acanthamoeba species, Borrelia burgdorferi, and Bacillus species were also present . Therapy with intensive topical antibiotics was successful in this series . The rate of surgical intervention (6/29, 21%) was similar to that of previous reports.

J Mol Biol, 1994 Jan 14, 235(2), 760 - 2
Crystallization and preliminary crystallographic analysis of sfericase . A Bacillus sphaericus calcium-dependent serine proteinase; Almog O et al.; Sfericase is an important intracellular proteinase produced by Bacillus sphaericus in the stationary phase of growth . It is a Ca(2+)-dependent serine proteinase with optimal activity at pH 9.0 to 9.3 . The molecular mass of sfericase is 32 kDa, as determined by sedimentation equilibrium . It seems to be involved in the interplay of various elements of the mosquitocidal activity of B . sphaericus, and hence is important for biological mosquito control . Sfericase significantly reduces viscosity of human pathological bronchial secretions and has recently shown good clinical effects in treatment of bronchitis, pneumonia and sinusitis . This enzyme was isolated from B . sphaericus and single crystals were obtained by the hanging drop vapor diffusion method . The crystals belong to the monoclinic space group P2, with cell dimensions of a = 46.94 A, b = 64.55 A, c = 86.23 A and beta = 95.4 degrees . These crystals are mechanically strong, they are stable in the X-ray beam and they diffract to better than 1.8 A resolution . The cell dimensions are consistent with four molecules per unit cell and two molecules in the asymmetric unit . A complete native data set to 1.77 A resolution has been collected on a Rigaku R-AXIS-IIc Imaging Plate Detector system and a heavy-atom derivative search is presently in progress.

Biochemistry, 1994 Jan 11, 33(1), 126 - 33
Inhibition and inactivation of the F1 adenosinetriphosphatase from Bacillus PS3 by dequalinium and activation of the enzyme by lauryl dimethylamine oxide; Paik SR et al.; The F1-ATPase from Bacillus PS3 (TF1) hydrolyzes 50 microM ATP in three kinetic phases . An initial burst rapidly decelerates to a partially inhibited, intermediate phase, which, in turn, gradually accelerates to an uninhibited, final steady-state rate . Lauryl dimethylamine oxide (LDAO) stimulates the final rate over 4-fold . The stimulatory effect saturates at about 0.1% LDAO . Under these conditions, the intermediate phase is nearly absent . Dequalinium inhibits TF1 reversibly in the dark in the presence or absence of LDAO . The apparent affinity of TF1 for dequalinium increases in the presence of LDAO . Dixon plots of the initial rates of the intermediate phase and the final rates against dequalinium concentration at a series of fixed ATP concentrations in the presence and absence of 0.03% LDAO indicate noncompetitive inhibition in each case . Replots of the slopes of the Dixon plots for the initial rate of the intermediate phase and the final rate against 1/{ATP} reveal apparent Km values of 770 microM and 144 microM, respectively, when obtained in the absence of LDAO . The apparent Km values determined from the data obtained in the presence of LDAO for the same phases are 303 microM and 163 microM, respectively . These results suggest that LDAO stimulates ATPase activity either by increasing the affinity of noncatalytic sites for ATP, which promotes release of inhibitory MgADP from a catalytic site, or by directly promoting release of MgADP from the affected catalytic site . Dequalinium retards this process without affecting the affinity of noncatalytic sites for ATP . When irradiated in the presence of dequalinium, TF1 is rapidly inactivated with an apparent Kd of 12.5 microM in the presence or absence of LDAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Jan 11, 33(1), 106 - 15
Equilibrium unfolding studies of barstar: evidence for an alternative conformation which resembles a molten globule; Khurana R et al.; The folding of the small protein barstar, which is the intracellular inhibitor to barnase in Bacillus amyloliquefaciens, has been studied by equilibrium unfolding methods . Barstar is shown to exist in two conformations: the A form, which exists at pH values lower than 4, and the N state, which exists at pH values above 5 . The transition between the A form and the N state is completely reversible . UV absorbance spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy were used to study the two conformations . The mean residue ellipticity measured at 220 nm of the A form is 60% that of the N state, and the A form has some of the properties expected for a molten globule conformation . Fluorescence energy transfer experiments using 1-anilino-8-naphthalenesulfonate indicate that at least one of the three tryptophan residues in the A form is accessible to water . Surprisingly, high concentrations of denaturant are required to unfold the A form . For denaturation by guanidine hydrochloride, the midpoint of the cooperative unfolding transition measured by circular dichroism for the A form at pH 3 is 3.7 +/- 0.1 M, which is significantly higher than the value of 2.0 +/- 0.1 M observed for the N state at pH 7 . The unfolding of the A form by guanidine hydrochloride or urea is complex and cannot be satisfactorily fit to a two-state (A<==>U) model for unfolding . Fluorescence-monitored tertiary structure melts before circular dichroism-monitored secondary structure, and an equilibrium unfolding intermediate must be present on the unfolding pathway of A.

Nucleic Acids Res, 1994 Jan 11, 22(1), 88 - 93
The reversible DNA-alkylating activity of duocarmycin and its analogues; Asai A et al.; Intact drugs with spirocyclopropylhexadienone moieties can be regenerated from the covalent DNA adducts induced by antitumor antibiotics duocarmycin (DUM) A, SA and some DUMA analogues in neutral aqueous solution . We detected the reversible nature of DUMs by determination of the antimicrobial activity and cytotoxicity of DUM-DNA adducts . All of the adducts selectively inhibited the growth of a sensitive strain of Bacillus but not that of the wild type strain, a property of parent DUM and its analogues . Most of the DNA adducts were also cytotoxic to HeLa S3 . These results suggested that active drugs can be released from their covalent DNA adducts under these biological assay conditions . Regeneration of intact drugs was quantitatively analyzed by HPLC and the amount of free drug released from DNA adducts revealed that the rate and efficiency of this reversal were dependent on structural variables among the drugs . The differences in rates of reversibility were correlated with the biological activity of DUMs . The effect of pH, temperature and salt concentration on the regeneration of drugs from their DNA adducts suggest a catalytic role of double-helical DNA on the reversal pathway.

J Biol Chem, 1994 Jan 7, 269(1), 683 - 90
Characterization of BcgI, a new kind of restriction-modification system; Kong H et al.; The BcgI restriction enzyme from Bacillus coagulans is unusual in that it cleaves on both sides of its recognition site, CGAN6TGC, releasing a fragment that includes the site and several bases on each side . We report the organization and nucleotide sequences of the genes for the BcgI restriction-modification system and the properties of the proteins that they encode . The system comprises two adjacent, similarly oriented genes . The proximal gene, bcgIA, codes for a 637-amino acid protein (molecular mass = 71.6 kDa) that resembles certain m6A-specific DNA-methyltransferases, particularly those that constitute the modification subunits of type I restriction-modification systems . The distal gene, bcgIB, codes for a 341-amino acid protein (molecular mass = 39.2 kDa) that resembles none of the sequences in the sequence data bases . The two genes overlap by several nucleotides . Alone, neither protein restricts or modifies DNA, but, together, they form a complex in the proportion A2B that does both . DNA binding assays showed that the DNA-protein complex can be formed only in the presence of both subunits, suggesting that the association of inactive subunits generates the active BcgI enzyme that can bind DNA and then either cleaves or methylates at target site.

J Biol Chem, 1994 Jan 7, 269(1), 47 - 50
Temperature-induced inversion of allosteric phenomena; Braxton BL et al.; Two instances, involving the enzymes carbamoyl-phosphate synthetase from Escherichia coli and phosphofructokinase from Bacillus stearothermophilus, respectively, are described in which increasing temperature alone causes the actions of an allosteric ligand to change from inhibition to activation . In neither case are these effects due to a change in the activation energy of the enzyme catalyzed reaction induced by the allosteric ligand . Rather, they are due to temperature-dependent changes in the extent to which the binding of allosteric ligand modifies the affinity of the enzyme for substrate . The data can be readily explained by an analysis of the apparent delta H and delta S components of the coupling free energy, which quantitatively describe the actions of allosteric ligands that act in this manner . These observations underscore the shortcomings of expecting to explain the actions of an allosteric ligand solely by the structural perturbations that accompany the binding of an allosteric ligand such as those often revealed by x-ray crystallography.

J Biol Chem, 1994 Jan 7, 269(1), 238 - 42
PIM1 encodes a mitochondrial ATP-dependent protease that is required for mitochondrial function in the yeast Saccharomyces cerevisiae; Van Dyck L et al.; The PIM1 nuclear gene in the yeast Saccharomyces cerevisiae encodes a mitochondrial ATP-dependent protease that exhibits over 30% identity with ATP-dependent protease La from Escherichia coli, Lon from Bacillus brevis, and one from Myxococcus xanthus . In addition, Pim1 is 1133 amino acids long and has a putative mitochondrial import signal in the N-terminal region . Enzymatic comparisons of normal PIM1+ and deficient pim1-delta strains revealed that the ATP-dependent protease is located within the mitochondrial matrix . The pim1-delta strains are unable to utilize nonfermentable substrates as the sole carbon source and are unable to maintain functional mitochondrial DNA, indicating that the Pim1 protease is required for mitochondrial function . PIM1 mRNA is constitutively expressed but is increased after thermal stress, suggesting that Pim1 may play a role in the heat shock response.

Biochim Biophys Acta, 1994 Jan 3, 1210(2), 249 - 53
Hormone-sensitive lipase is closely related to several bacterial proteins, and distantly related to acetylcholinesterase and lipoprotein lipase: identification of a superfamily of esterases and lipases; Hemila H et al.; We have sequenced a gene from Bacillus acidocaldarius which encodes an open reading frame (ORF3) of 310 amino acids . The ORF3 was found to be related to the mammalian hormone-sensitive lipase (HSL) . Searching the protein data base revealed five other bacterial proteins related to the HSL . Upon further sequence comparisons this HSL-group was found to be related to the family of carboxylesterases, and to a family of lipases (lipoprotein, hepatic and pancreatic lipases) . The evolutionary relationship of these serine-dependent hydrolytic enzymes has not been studied previously, and it has not been known that these proteins belong to the same superfamily . Finally, the alignment of the HSL with the bacterial proteins allowed us to infer the location of the hormone-sensitive regulatory domain of the HSL-protein.

J Exp Biol, 1994 Nov, 196(1), 145 - 55
DETERMINANTS FOR THE ACTIVITY OF THE NEUTRAL AMINO ACID/K+ SYMPORT IN LEPIDOPTERAN LARVAL MIDGUT
Giordana B, Parenti P.
The columnar cells of lepidopteran larvae express, in their apical brush-border membrane, a class of symporters which in vivo couple the intracellularly directed amino acid and K+ fluxes . An analysis of the functional properties of the symporter for neutral amino acids along the anterior, middle and posterior regions of the larval midgut of Bombyx mori demonstrated the ability of a K+ gradient to drive leucine accumulation into brush-border membrane vesicles (BBMV) in all three preparations . However, marked differences are evident between the posterior (P) and the anteriormiddle (AM) regions . In P-BBMV, much higher intravesicular accumulations were observed, Vmax was six- to eightfold higher than in AM-BBMV, a lowering of external pH (pHe) from 8.7 to 7.2 caused a tenfold increase of Km, and the absence of a potential difference (delta psi) caused a threefold decrease of Vmax . In contrast, leucine uptake in AM-BBMV was poorly sensitive to both pH and delta psi . The kinetics of leucine uptake as a function of cis K+ concentration were hyperbolic in P-BBMV and sigmoidal in AM-BBMV . More than 50 amino acids and analogues were used in inhibition experiments to characterize the amino acid binding site . Branched-chain amino acids modified on the carboxyl moiety were recognized only by the P-BBMV symporter . In AM-BBMV, substrate affinity was increased by the presence of a heterocyclic sidechain, even in the presence of a modified carboxyl- or alpha-amino group . Together, these results suggest that isoforms of the neutral amino acid/K+ symporter are present . A natural inhibitor of amino acid symport has not yet been identified . However, several lines of evidence suggest that strong interactions exist between the amino acid/K+ symporter and the receptor for the lepidopteran-specific Bacillus thuringiensis delta-endotoxins . CryIA(a) toxin, highly toxic for B . mori larvae, produced a dose-dependent inhibition of leucine uptake into both BBMV populations . The toxin was able to block the symporter in its ternary and leucine-only forms.

Arch Biochem Biophys, 1994 Jan, 308(1), 226 - 30
Comparison of the inhibition by phospho(enol)pyruvate and phosphoglycolate of phosphofructokinase from B . stearothermophilus; Tlapak-Simmons VL et al.; A comparison between the inhibition by phospho(enol)pyruvate (PEP) versus the inhibition by phosphoglycolate (PG) of phosphofructokinase (PFK) from Bacillus stearothermophilus is presented . Both inhibitors act by decreasing the apparent affinity displayed by the enzyme for its substrate fructose 6-phosphate (Fru-6-P) while having little effect on Vmax . However, the two ligands differ in both their affinity for the enzyme and their effectiveness at antagonizing the subsequent binding of Fru-6-P . Although PG binds with approximately 10-fold lower affinity, it antagonizes the binding of Fru-6-P 3.5-fold more strongly than does PEP . Moreover, the enthalpy and entropy contributions to the coupling free energy between inhibitor and Fru-6-P, from which these antagonisms derive, reveal even greater differences between the ligands . These data indicate, therefore, that the changes in the structure of PFK from B . stearothermophilus that result from PG binding, which have been determined by X-ray crystallography (T . Schirmer and P . R . Evans, 1990 Nature 343, 140-145), may not be comparable to those that result from PEP binding and consequently do not represent the generic "T-state," as has been presumed.

Int Arch Allergy Immunol, 1994, 103(2), 166 - 74
Role of lymphatic drainage on the development of Calmette-Guérin bacillus-induced granulomas in the hamster; Sinhorini IL et al.; The influence of lymphatic drainage on Calmette-Guerin bacillus (BCG)-induced granulomas was investigated by comparing the time course of granuloma formation in two sites: the hamster footpad and the check pouch, an area deprived of lymphatic vessels . Typical epithelioid granulomas developed in both sites . Whereas in the footpad the size of granulomas increased and the volume of the lesion persisted, in the pouch the lesion decreased in volume . The inoculation of BCG into the footpad of animal with granulomas in the pouch, reactivated the pouch lesions . T lymphocytes were detected by an immunocytochemical technique at the edge of these lesions . Inoculation of the bacteria into the pouch induces suppressive mechanisms which hold down the volume of the lesions induced in the footpad . The cutaneous purified protein derivative (PPD) test, positive in animals inoculated in the footpad, was always negative in animals with granulomas in the pouch . The number of bacteria per microscopic field in pouch granulomas increased from 10 to 1,000 after PPD injection . Evidence that PPD has a direct effect on pouch granuloma cells is given.

J Bacteriol, 1994 Jan, 176(2), 520 - 3
Zinc, a structural component of adenylate kinases from gram-positive bacteria; Gilles AM et al.; The recent finding that Bacillus stearothermophilus adenylate kinase contains a zinc atom coordinated to four cysteines prompted us to investigate the metal-binding properties of the enzyme from various bacteria . We conclude that zinc was present only in adenylate kinase from gram-positive species and that this property is correlated with the presence of three or four Cys residues in the sequence Cys-X2-Cys-X16-Cys-X2-Cys/Asp, in which X stands for different amino acid residues.

Biochem J, 1994 Jan 1, 297 ( Pt 1), 137 - 43
The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus: preparation and characterization of its binding to the dihydrolipoyl dehydrogenase component; Hipps DS et al.; The peripheral subunit-binding domain of the dihydrolipoyl acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was released by limited proteolysis from a di-domain (lipoyl domain plus binding domain) encoded by a subgene over-expressed in Escherichia coli . The domain was characterized by N-terminal sequence analysis, electrospray m.s . and c.d . spectroscopy . It was found to be identical in all respects to a chemically synthesized peptide of the same sequence . The association of the di-domain and binding domain (both natural and synthetic) with dihydrolipoyl dehydrogenase was analysed in detail and a tight binding was demonstrated . As judged by several different techniques, it was found that only one peripheral subunit-binding domain is bound to one dimer of dihydrolipoyl dehydrogenase, implying that the association is highly anti-cooperative.

Mol Cell Biol, 1994 Jan, 14(1), 646 - 54
NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype; Johansen T et al.; In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus . We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition . The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine . Expression of B . cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody . The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum . Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state . Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.

J Urol, 1994 Jan, 151(1), 27 - 30
Intravesical mitomycin C and doxorubicin sequential therapy for carcinoma in situ of the bladder: a longer followup result; Sekine H et al.; A total of 43 patients with carcinoma in situ of the bladder (primary in 26 and secondary in 17) who underwent intravesical mitomycin C and doxorubicin sequential therapy for 2 multicenter studies were followed for a median period of 45 months (range 10 to 84) . Of the patients 32 (74%) achieved complete response after induction therapy and underwent maintenance therapy with either mitomycin C plus doxorubicin, mitomycin C alone or observation only . Of the complete responders 13 (41%) had a local recurrence, and subsequent repeat intravesical mitomycin C and doxorubicin sequential therapy as well as bacillus Calmette-Guerin was effective in a significant proportion (75% or greater) . The maintenance therapy did not have a favorable effect on the recurrence rate . In 8 patients (19%) (3 of 32 complete responders and 5 of 11 nonresponders) progression developed, including invasive cancers in 4, metastatic disease in 2 and both conditions in 2 . Initial complete responders had a significantly higher progression-free rate than initial nonresponders, although there was no difference in the sites of progression between them . At the last followup 35 patients (81%) remained free of disease with 31 (72%) having a normally functioning bladder . According to these results, intravesical mitomycin C and doxorubicin sequential therapy appears to be applicable as initial treatment for carcinoma in situ of the bladder.

J Urol, 1994 Jan, 151(1), 21 - 6
Megadose vitamins in bladder cancer: a double-blind clinical trial; Lamm DL et al.; Epidemiological and laboratory studies suggest that vitamin supplements may be helpful in the prevention of some cancers but clinical trials to date have failed to demonstrate protection with naturally occurring vitamins . Without substantiation of the highly touted benefits of vitamins, few physicians who care for cancer patients have recommended their use . A total of 65 patients with biopsy confirmed transitional cell carcinoma of the bladder enrolled in a randomized comparison of intravesical bacillus Calmette-Guerin (BCG) with or without percutaneous administration was also randomized by closed envelope to therapy with multiple vitamins in the recommended daily allowance (RDA) versus RDA multivitamins plus 40,000 units vitamin A, 100 mg . vitamin B6, 2,000 mg . vitamin C, 400 units vitamin E and 90 mg . zinc . The addition of percutaneous BCG did not significantly lessen tumor recurrence but recurrence after 10 months was markedly reduced in patients receiving megadose vitamins . The 5-year estimates of tumor recurrence are 91% in the RDA arm and 41% in the megadose arm (p = 0.0014, Mantel-Cox) . Overall recurrence was 24 of 30 patients (80%) in the RDA arm and 14 of 35 (40%) in the high dose arm (p = 0.0011, 2-tailed Fisher's exact test) . Megadose vitamins A, B6, C and E plus zinc decrease bladder tumor recurrence in patients receiving BCG immunotherapy . Further research will be required to identify which ingredient(s) provide this protection.

J Urol, 1994 Jan, 151(1), 13 - 5
Complications of intracavitary bacillus Calmette-Guerin after percutaneous resection of upper tract transitional cell carcinoma; Bellman GC et al.; Percutaneous resection and intracavitary instillation of bacillus Calmette-Guerin (BCG) is currently being used in treatment protocols in select patients in whom the standard nephroureterectomy for upper tract transitional cell carcinoma is undesirable . However, the complications of BCG in these patients are not well defined . Among 16 patients treated in this manner 4 (25%) had asymptomatic granulomatous involvement of the renal pelvis discovered during regular followup nephroscopy and biopsy: 2 had recurrent carcinoma at discovery, whereas the other 2 remain free of disease . The significance and therapeutic consequences of BCG granulomatosis are unknown . Continued followup is necessary to identify any complications of BCG therapy.

Ital J Biochem, 1994 Jan-Feb, 43(1), 29 - 35
Regulation of dihydrodipicolinate synthase and diaminopimelate decarboxylase activity in Bacillus stearothermophilus; Selli A et al.; The feedback inhibition of the enzymes dihydrodipicolinate (DHDPS) and diaminopimelate decarboxylase (DAPD) in the wild strain Zu 183 of Bacillus stearothermophilus and in its S-(2-aminoethyl)-cysteine resistant L-lysine overproducing strain AEC 12 was studied . The optimum temperature and pH of both enzymes were also evaluated . No inhibition of DHDPS by L-lysine, L-threonine, L-methionine and L-isoleucine was observed either in the wild strain or in the AEC 12 mutant . DAPD was completely inhibited by L-lysine and only partially by L-threonine and L-methionine in Zu 183 and AEC 12 strains, but the concentration required was found to be much higher in the AEC 12 strain . The regulation mechanism of L-lysine biosynthesis in Bacillus stearothermophilus Zu 183 was also discussed.

J Biochem (Tokyo), 1994 Jan, 115(1), 93 - 7
Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification; Kim DW et al.; The gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp . YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues . The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(beta-aminoethyl)cysteine (SAEC), S-(beta-aminopropyl)cysteine (SAPC), and S-(beta-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively . The modified mutant enzymes showed absorbance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0 . The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3 . This suggests that omega-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate . The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 10(6)-10(8) times more active than the K239C mutant enzyme . These results suggest that omega-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.

Urol Int, 1994, 52(2), 69 - 72
Trial with bacillus Calmette-Guérin and epirubicin combination in the prophylaxis of superficial bladder cancer; Erol A et al.; The efficacy and side effects of the prophylactic intravesical bacillus Calmette-Guerin and epirubicin combination therapy were evaluated in 14 patients who underwent transurethral resection for Ta-T1 superficial bladder cancer . Therapy was started 7-10 days after the operation and maintained weekly for 6 weeks and then monthly for 6 months unless withdrawal due to side effects . Instillation of 50 mg epirubicin (Farmorubicin, Farmitalia Carlo Erba) diluted in 50 ml saline was followed by 80 mg of the Connaught strain bacillus Calmette-Guerin (ImmuCyst, Pasteur Merieux) in 50 ml saline and the duration of instillation was 2 h for each drug . All the patients experienced moderate to severe cystitis and fever . Side effects necessitated discontinuation of the therapy in 5 patients (35.7%) . Therapy was delayed in 7 patients (50%) for reasons related or unrelated to the side effects . Of the 9 patients who completed the course, 8 (89%) had no tumor recurrence 10-16 months (mean 14 months) after therapy . Results obtained revealed that this treatment schedule cannot be tolerated by a considerable number of the patients . Mainly the side effects of the combination are emphasized in this report . The therapeutic efficacy of such a combination has yet to be determined and further clinical studies are warranted.

Plasmid, 1994 Jan, 31(1), 72 - 88
Complete nucleotide sequence of the Bacillus thuringiensis subsp . israelensis plasmid pTX14-3 and its correlation with biological properties; Andrup L et al.; The complete nucleotide sequence of the plasmid pTX14-3 from Bacillus thuringiensis subsp . israelensis has been determined . The circular DNA molecule was 7649 bp and had a G + C content of 35.1% . Twenty-two open reading frames larger than 50 codons were identified . Ten of these open reading frames are suggested to be protein coding regions . The existence of the polypeptides encoded by the mob14-3 and rep14-3 genes were verified by maxi-cells analysis in Escherichia coli . Even though the rep14-3 gene was expressed in E . coli the plasmid pTX14-3 was unable to replicate in this bacterium . The minimal region of the plasmid pTX14-3 required for replication in B . thuringiensis was identified . Potential secondary structures upstream of the rep14-3 gene indicated regulation by antisense RNA and transcription attenuation . Extensive sequence homology with the B . thuringiensis subsp . thuringiensis plasmid pGI2 was found in the last part of the mob14-3 gene, downstream of the rep14-3 gene, and in the region containing the single-strand origin of replication (i.e., the minus origin) of pTX14-3 . A sequence of 700 bp containing multiple direct repeats was found in an ORF encoding a glycine and proline rich protein of 35.9 kDa . 1.2 kbp upstream and 0.1 kbp downstream of this ORF was found a large direct repeat of 230 bp (87% identity) . The region between this direct repeat was often spontaneously deleted from plasmid derivatives containing the entire pTX14-3.

APMIS, 1994 Jan, 102(1), 67 - 71
A synergistic interaction between Bacillus Calmette-Guérin (BCG) and NADPH oxidase stimulants on the production of reactive oxygen metabolites by human mononuclear phagocytes; Nyberg PW et al.; Bacillus Calmette-Guerin (BCG) was added simultaneously with known NADPH oxidase stimulants to suspensions of human mononuclear leukocytes, and the subsequent production of reactive oxygen metabolites (ROMs) was studied by luminol-dependent chemiluminescence . BCG significantly amplified the ROM responses induced by zymosan, phorbol myristate acetate (PMA), and quartz, but not by concanavalin A and asbestos fibers . The stimulatory effect occurred rapidly when BCG was added to cells already phagocytosing zymosan, and vanished rapidly when extracellular BCG was removed from adherent monocyte cultures by washing prior to the addition of zymosan . The stimulatory effect of BCG could not be reproduced with recombinant interferon-gamma, tuberculin PPD, muramyl dipeptide, nor with the apathogenic Mycobacterium tuberculosis strain RV37 . BCG and zymosan or PMA that had been incubated together prior to addition to the mononuclear cell suspensions caused ROM production with faster kinetics than if the reagents were added separately without preincubation . In conclusion, the synergy between BCG and some of the NADPH oxidase stimulants seems to be due to an interaction between BCG and the NADPH oxidase stimulants rather than to an interaction between BCG and the ROM-producing cells . Such interactions between mycobacteria and NADPH oxidase stimulants may be of importance as a factor affecting the individual susceptibility to tissue damage in tuberculosis, for example in silicotuberculosis.

Microbiology, 1994 Jan, 140 ( Pt 1), 159 - 66
Different effects of N-glycosylation on the thermostability of highly homologous bacterial (1,3-1,4)-beta-glucanases secreted from yeast; Meldgaard M et al.; Genes encoding Bacillus amyloliquefaciens (1,3-1,4)-beta-glucanase (AMY), B . macerans (1,3-1,4)-beta-glucanase (MAC), and a series of hybrid enzymes containing N-terminal sequence segments of different length derived from AMY with the remaining C-terminal segment derived from MAC, were expressed in Saccharomyces cerevisiae . The cells secreted active enzyme into the medium . While the quantity of N-glycan linked to the different enzymes was similar, pronounced differences in thermotolerance were observed when the glycosylated enzymes were compared with the unglycosylated counterparts produced in Escherichia coli . Glycosylated AMY and hybrid enzyme H(A16-M), consisting of 16 N-terminal amino acids derived from AMY with the remaining C-terminal segment from MAC, exhibited a 7.5- and 1.6-fold increase in half-life at 70 degrees C, pH 6.0 . N-terminal sequencing established that only two out of three sites for potential N-glycosylation of H(A16-M) secreted from yeast were actually glycosylated . Removal of N-glycans by endoglycosidase H and peptide:N-glycosidase F from H(A16-M) resulted in a 16- and 133-fold decrease of thermostability, demonstrating that N-glycans are a major determinant for the resistance of this enzyme to thermal inactivation . Glycosylated MAC and hybrid enzymes H(A36-M), H(A107-M) and H(A152-M) had increased thermostability but hybrid enzyme H(A78-M) was less thermostable . N-Glycosylation thus changes thermostability of (1,3-1,4)-beta-glucanases with similar primary structure in a variable, so far unpredictable way.

Arch Microbiol, 1994, 161(3), 252 - 7
Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus; Budarina ZI et al.; To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B . cereus and fourteen strains of B . thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B . cereus hemolysin II gene . In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested . The presence (absence) of the hydridization response in the microorganisms's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production . Only four out of thirteen B . cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B . thuringiensis strains responded positively . DNAs from ten B . thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B . cereus hemolysin II gene probe . The 3.5 kb EcoRV DNA fragment from one of these strains (B . thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells . The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B . cereus hemolysin II . The obtained data clearly show limited distribution of hemolysin II among B . cereus strains and demonstrate that hemolysin II is more characteristic of B . thuringiensis than B . cereus.

J Antimicrob Chemother, 1994 Jan, 33(1), 83 - 9
Efficiency of oxetanocin-G, a novel nucleoside against the woodchuck hepatitis virus; Ikeda N et al.; Oxetanocin-G (OXT-G), a potent antiviral agent, is a novel nucleoside isolated from the culture filtrate of Bacillus megaterium . We investigated the antiviral effect of oral administration of OXT-G for five days on woodchuck hepatitis virus (WHV), in vivo, using 12 woodchucks . Woodchucks were randomized into each of four treatment groups according to the dose of OXT-G . Two out of six woodchucks treated with 1.0 or 2.0 mg/kg/day of OXT-G died . After treatment with OXT-G, serum levels of WHV-DNA significantly decreased in all woodchucks . However, the antiviral effect was only partial and levels of serum WHV-DNA returned after the cessation of treatment . The amount of viral replicative intermediates was decreased in livers of woodchucks treated with OXT-G . Although further study of the toxicity of this compound would be essential before studies in man can be carried out, OXT-G has potent antiviral activity against WHV and may deserve evaluation as an antiviral agent in the treatment of chronic hepatitis B infections in humans.

Support Care Cancer, 1994 Jan, 2(1), 66 - 70
Results of antibiotic treatment of Hickman-catheter-related infections in oncological patients; Simon C et al.; A group of 330 oncological patients were supported throughout a 7-year period with central venous catheters (Broviac/Hickman catheters) and underwent standard oncological chemotherapy, because of hematological malignancies or solid tumors (156 children), or a myeloablative conditioning regimen followed by bone marrow transplantation because of leukemia or lymphoma (174 patients: 110 adults, 64 children) . Of these, 17 patients (8 after bone marrow transplantation) developed a catheter-related bacteremia and were treated by at least two antibiotics according to the sensitivity of the bacteria . In 1 patient the catheter (infected by Bacillus cereus) was removed on day 25 of antibiotic treatment because of persistent high fever and further positive blood cultures . After bone marrow transplantation, 2 other patients, with a Pseudomonas or a Staphylococcus infection respectively, did not respond to the combined antibiotic treatment and died 1 week and 7 weeks later, respectively, from transplant-related severe graft-versus-host disease . In the other 14 patients antibiotic treatment was successful and removal of the central-vein catheter could be avoided.

Prikl Biokhim Mikrobiol, 1994 Jan-Feb, 30(1), 88 - 94
{Interaction of Bacillus intermedius 7P ribonuclease with ligand-free serum albumin}; Ponomareva RB et al.; The efficiency of the binding of RNase 7P molecules to albumin on cocondensation with the aim of producing the prolonged action forms of the enzyme can be increased by using ligand-free human serum albumin (LFHSA) . The CD method showed that LFHSA underwent changes of the cooperative character under the action of acid and urea . On potentiometric titration the number of titrated groups of LFHSA decreased with time . The GPC method demonstrated the RNase bound more efficiently to freshly dissolved LFHSA . In this case part of the enzymic activity was manifested only after proteolysis of the albumin carrier . Cocondensation with the aid of glutaraldehyde resulted in the formation of LFHSA-RNase conjugates composed of 1-2 moles of human serum albumin and 1-6 moles of RNase . More than 50% of transferase activity retained in the blood plasma for 2-3 days after intravenous injection of the conjugates with a molecular weight of 70-80 kD to rabbits.

Prikl Biokhim Mikrobiol, 1994 Jan-Feb, 30(1), 121 - 6
{Proteolysis of collagen by several species of micromycetes and spore-forming bacteria}; Iakovleva MB et al.; The capability of lysing collagen was studied in 4 species of spore-forming bacteria of the genus Bacillus and 9 species of micromycetes of the genera Aspergillus, Penicillium, Cladosporium, and Alternaria . A specific medium was developed for determining the degree of collagen proteolysis . The spore-forming bacteria possessed the higher index of collagen lysis as compared to micromycetes . The maximum value of the lysis index was 4 times higher in B . licheniformis than in C . herbarum . The latter showed the highest degree of collagen proteolysis among the micromycetes studied . The pH optima of collagen proteolysis were determined for 5 species of Aspergillus and Penicillium fungi . It is shown that a submerged culture of A . chrysogenum used fragments of the Achilles tendon as a sole source of carbon . The specific proteolytic activity of the collagen-containing medium was 0.8 units of proteinase activity.min.mg protein on the 7th day of cultivation.

Antimicrob Agents Chemother, 1994 Jan, 38(1), 61 - 5
Clinical trial of sparfloxacin for lepromatous leprosy; Chan GP et al.; Nine previously untreated patients with lepromatous leprosy were treated with 200 mg of sparfloxacin daily for 12 weeks to determine whether this drug is bactericidal for Mycobacterium leprae in humans . The efficacy of therapy was monitored both clinically and by measuring changes in morphological index, mouse footpad infectivity, and the radiorespirometric activity of M . leprae organisms obtained from serial biopsy specimens and also by determining titers of phenolic glycolipid-I in serum . Most patients showed clinical improvement within 2 weeks of treatment; this was accompanied by significant reductions in the morphological index, mouse footpad infectivity, and bacillary radiorespirometric activity . After 4 weeks of treatment, all patients had a morphological index of zero and specimens from most patients were noninfectious for mice, while the median decrease in radiorespirometric activity was > 99% . Overall results by the rapid radiorespirometric assay paralleled those of the mouse footpad and morphological index assays . Sparfloxacin given at 200 mg once daily appears to be rapidly bactericidal in humans, with activity similar to that observed in a previous clinical trial with 400 mg of ofloxacin.

FEMS Microbiol Lett, 1994 Jan 1, 115(1), 13 - 7
Phylogeny of spore-forming lactic acid bacteria based on 16S rRNA gene sequences; Suzuki T et al.; The phylogeny of spore-forming lactic acid bacteria was investigated on the basis of 16S rRNA gene sequences . Sixteen strains were separated into three lines of descent; one consisted of 14 strains assigned to Sporolactobacillus spp . and Bacillus spp., and the other two each consisted of "Sporolactobacillus dextrus" and Bacillus coagulans . Strains of all the first lineage but one composed a cluster of similarity values of 97.2% and higher, and were represented by the type strain of S . inulinus . The cluster was further separated into five subclusters, four catalase negative and one positive . The definition of the genus Sporolactobacillus should be amended to accommodate catalase positive strains . Spore-forming lactic acid bacteria originated at different phylogenetic positions, and would have evolved convergently in the area of Bacillus.

Crit Care Med, 1994 Jan, 22(1), 40 - 9
Selective decontamination of the digestive tract reduces gram-negative pulmonary colonization but not systemic endotoxemia in patients undergoing elective liver transplantation; Bion JF et al.; OBJECTIVE: To examine the effect of selective antibiotic decontamination of the digestive tract in patients undergoing elective orthotopic liver transplantation . DESIGN: Prospective, randomized, concurrent allocation to either selective decontamination or standard antibiotic prophylaxis . SETTING: Operating theater and intensive care unit at a tertiary referral, university teaching hospital . PATIENTS: Fifty-nine adult patients were recruited into the study and underwent liver transplantation . INTERVENTIONS: Thirty-two patients were randomized to standard treatment (control group) and 27 patients were randomized to receive selective decontamination . After early deaths and exclusions, 31 controls and 21 decontamination patients were available for analysis . MEASUREMENTS AND MAIN RESULTS: Portal and systemic endotoxemia, colonization and infection rates, severity of illness (organ system failures, Acute Physiology and Chronic Health Evaluation II score, Therapeutic Intervention Scoring System score), antibiotic costs, and hospital survival rates were measured . Selective decontamination significantly reduced pulmonary infections and enteric, aerobic, and Gram-negative bacillary colonization without facilitating the emergence of resistant organisms, but selective decontamination had no effect on endotoxemia or the development of organ system failures . The financial costs of the selective decontamination regimen outweighed the advantages gained from an associated reduction in antibiotic usage . CONCLUSION: The failure of selective decontamination to enhance survival rates in many studies of the regimen in critically ill patients may, in part, be related to the inability of selective decontamination to abolish endotoxemia.

Int J Syst Bacteriol, 1994 Jan, 44(1), 125 - 9
DNA relatedness among Bacillus thuringiensis serovars; Nakamura LK; The genetic relationships of Bacillus cereus and of the Bacillus thuringiensis serovars were assessed from measurements of DNA reassociation . A study of 8 to 10 strains each of 13 of the most commonly encountered serovars revealed that the levels of intragroup DNA relatedness for most serovars ranged from 90 to 100% . In contrast, B . thuringiensis serovars canadensis and kenyae consisted of two DNA relatedness groups, each of which exhibited levels of intragroup relatedness of 80% or higher and levels of intergroup relatedness of 60 to 70% . Analyses of DNA relatedness performed with all of the serovars revealed that the taxa were segregated into 11 phena differentiated from each other at about the 65% level; within each phenon the level of relatedness was 80% or higher . Three phena contained strains belonging to more than one serovar; B . thuringiensis serovars alesti and dendrolimus clustered in phenon 1, serovars aizawai, kurstaki, galleriae, and morrisoni clustered in phenon 7, and serovar darmstadiensis and some strains of serovar kenyae clustered in phenon 11 . The levels of DNA relatedness between B . cereus and B . thuringiensis strains ranged between 65 and 70% . My results suggest that many of the B . thuringiensis serovars are genetically distinct but closely related.

Vopr Med Khim, 1994 Jan-Feb, 40(1), 14 - 6
{Activation of protein biosynthesis in rat organs and tissues during stimulation of the mononuclear phagocyte system with prodigiosan}; Panin LE et al.; As shown earlier, the mononuclear phagocyte system (MPS) plays an important role in regulation of protein synthesis in different rat tissues . In vivo stimulation of MPS by lipopolysaccharide from Bacillus prodigiosum caused a significant increase of protein synthesis in the liver, lungs, adrenal glands, thymus, spleen and bone marrow . The results obtained suggest that MPS is involved in the regulation of tissue regenerative process at the level of physiological state.

FEMS Microbiol Rev, 1994 Jan, 13(1), 13 - 24
Taxonomic position of the rickettsiae: current knowledge; Drancourt M et al.; The term rickettsiae initially encompassed all intracellular bacteria . Early rickettsial taxonomy was based on a comparison of a few phenotypic characteristics and recently, molecular studies brought new bases for rickettsial taxonomy . All rickettsial species studied so far belong to the alpha and gamma groups of the Proteobacteria . Ehrlichiae complex groups Cowdria ruminantium, Anaplasma marginale and Wolbachia pipientis and the related parthenogenesis and cytoplasmic incompatibility bacteria, whereas Rochalimaea species group with Bartonella bacilliformis . Rickettsia tsutsugamushi may form an independent lineage, whereas molecular data allow to regroup serologically defined typhus and spotted fever group rickettsiae . The true scale of Rickettsia and Coxiella genera remain to be determined.

Appl Environ Microbiol, 1994 Jan, 60(1), 353 - 6
PCR analysis of the cryI insecticidal crystal family genes from Bacillus thuringiensis; Ceron J et al.; A method allowing rapid and accurate identification of different subgroups within the insecticidal crystal CryI protein-producing family of Bacillus thuringiensis strains was established by using PCR technology . Thirteen highly homologous primers specific to regions within genes encoding seven different subgroups of B . thuringiensis CryI proteins were described . Differentiation among these strains was determined on the basis of the electrophoretic patterns of PCR products . B . thuringiensis strains, isolated from soil samples, were analyzed by PCR technology . Small amounts of bacterial lysates were assayed in two reaction mixtures containing six to eight primers . This method can be applied to rapidly detect the subgroups of CryI proteins that correspond with toxicity to various lepidopteran insects.

Appl Environ Microbiol, 1994 Jan, 60(1), 243 - 7
Isolation and characterization of soybean waste-degrading microorganisms and analysis of fertilizer effects of the degraded products; Kubo M et al.; Two microorganisms which could degrade soybean lees efficiently were isolated and identified as Bacillus circulans and B . stearothermophilus . These two strains secreted thermostable proteases into the medium and could digest soybean lees rapidly and completely at 50 degrees C . Initially, the soybean lees were degraded to proteins in approximately 20 h by these two strains, after which time the concentrations of peptides in the medium gradually increased . The degraded products from soybean lees contained abundant nitrogen compounds, such as peptides, amino acids, and amides . Approximately 10 times more fresh plant weight was obtained (in the case of Brassica campestris) when these degraded products were applied than when water was applied for 42 days . These stimulatory effects of the soybean lees products were almost equal to those of a chemically synthesized fertilizer.

Nippon Koshu Eisei Zasshi, 1994 Jan, 41(1), 74 - 81
{A reevaluation of the criteria for initiating medical treatment of pulmonary tuberculosis patients}; Toyota M et al.; In Japan some registered tuberculosis (TB) patients have had medical treatment initiated for the purpose of preventing development of inactive TB into active stages, or due to differential diagnosis . A reevaluation of the necessity of commencing medical treatment was performed on 91 TB patients to elucidate the special characteristics of TB patients who do not necessarily need medical treatment . The results are as follows: 1) Among the 91 patients, 67 patients were judged to be 'confirmed cases', while 24 patients were judged to be 'suspect cases', with either inactive TB or possibly without TB . 2) The rates of 'suspect cases' were higher in the tubercle bacillus negative groups as well as the X-ray mild cases compared to the tubercle bacillus positive cases and X-ray severe cases . 3) The patient's self-diagnosed symptoms proved to be useful in evaluating the TB patient's severity . It is thought that this information should be used to judge the necessity of commencing medical treatment in TB patients.

Arch Microbiol, 1994, 162(1-2), 98 - 102
Purification of amoebolytic substances from Bacillus licheniformis M-4; Lebbadi M et al.; Three antibiotic peptides with amoebolytic activity have been purified from culture supernatants of Bacillus licheniformis M-4 (amoebicins m4-A, m4-B, and m4-C) . They were hydrophilic peptides consisting of six different amino acids (Asp, Glu, Ser, Thr, Pro, Tyr) . Their molecular weights ranged from 3,000 to 3,200 . Purified amoebicins were active against human pathogenic and non-pathogenic strains of Naegleria . They also showed a broad antifungal spectrum, but a narrow antibacterial activity.

Microbiol Immunol, 1994, 38(1), 25 - 30
Helicobacter pylori: a fickle germ; Cellini L et al.; The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied . These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum . The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded . At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro . The coccoid morphology was observed earlier when the culture of H . pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin . To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80 C for 10 min and sonicated . In the absence of these treatments the cultures did not show growth in vitro . The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.

Curr Opin Rheumatol, 1994 Jan, 6(1), 105 - 10
Rheumatic manifestations of malignancy; Conaghan PG et al.; The rheumatic associations of cancer therapy are highlighted in this review . Interleukin-2, interferon alfa, and Calmette-Guerin bacillus immunotherapies are related to an inflammatory arthritis, and septic arthritis can complicate breast-cancer therapy . In a large retrospective study, an increased incidence of cancer in systemic sclerosis was confirmed, especially lung and breast cancers . Lymphoproliferative associations of Sjogren's syndrome were explored in a study of non-Hodgkin's lymphoma patients in which clinical and histologic criteria were used to diagnose Sjogren's syndrome . B- and T-cell lymphomas continue to be reported with rheumatologic manifestations such as seronegative polyarthritis and sacroiliitis . Malignant angioendotheliomatosis, which mimics central nervous system vasculitis diseases, has been reported . Paraneoplastic associations of lung, ovarian, and renal-cell carcinomas are discussed.

Int Urol Nephrol, 1994, 26(1), 33 - 42
Total cystectomy after intravesical bacillus Calmette-Guérin (Tokyo strain) therapy for superficial bladder cancer: experience in Japan; Takashi M et al.; To clarify which patients might most require total cystectomy after intravesical bacillus Calmette-Guerin (BCG) therapy, we reviewed data for 111 individuals with superficial bladder cancer . Of the 111 patients, 73 received the BCG treatment for prophylaxis of intravesical recurrence after transurethral resection (group 1), 24 therapeutically for Ta or T1 tumours (group 2), and 14 for eradicating carcinomas in situ (CIS, group 3) . Although the BCG therapy significantly reduced frequencies of recurrence in group 1, 27 patients (37%) did develop tumours again . Tumours disappeared in 18 of 24 patients (75%) in group 2, and in 11 of 14 (79%) in group 3 . The rate of disease progression was 6% for all 111 patients: 3% (2/73) for group 1, 17% (4/24) for group 2, and 7% (1/14) for group 3 . A total of 16 of the 111 patients (14%) underwent total cystectomy, the respective figures being 7% in group 1, 29% in group 2, and 29% in group 3 . Indications for total cystectomy were progression in 7, recurrent multiple tumours in 5, persistent CIS in 2, a contracted bladder in 1, and occurrence of bilateral renal pelvic cancer in 1 . Thus, 4 of 6 patients (67%) who had tumours unresponsive to BCG therapy in group 2 demonstrated progression and necessitated total cystectomy . Because tumours persisting after BCG therapy are frequently of the muscle-invasive type, such cases should be regarded as candidates for immediate total cystectomy.

Wiad Parazytol, 1994, 40(1), 89 - 92
{Control of fly larvae in a pig farm by oral administration of thuridan to pigs}; Ramisz A et al.; The studies were carried out in an experimental pig farm on 402 animals with Thuridan (spore of delta-endotoxin complex of Bacillus thuringiensis kurstaki) . It was applied orally, as feed additive in a concentration of 0.1 and 0.5% for three current days . The efficacy of Thuridan was established with the use of fly-catchers which were hung in the same places--once before and three times after Thuridan had been used . After 3 application of Thuridan the number of flies decreased by 88%.

Eur J Ophthalmol, 1994 Jan-Mar, 4(1), 1 - 5
Post-traumatic endophthalmitis; Verbraeken H et al.; In a group of 615 cases of perforating trauma, 25 cases (4%) of proven endophthalmitis were seen . The percentage of Bacillus infections was unusually high compared to other types of endophthalmitis (3.8% for the whole group, 31% for the group with intraocular foreign bodies) . Bacillus cases have a very poor outcome and in fact the overall functional results in the post-traumatic endophthalmitis group were poorer than in other categories.

J Protein Chem, 1994 Jan, 13(1), 129 - 33
Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase; Nicholls DJ et al.; The Gln residue at amino acid position 102 of Bacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition . The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured by kcat/Km) than the wild-type enzyme . However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants . The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity . However, their activities are restored by the presence of an aromatic substrate . All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.

Cytobios, 1994, 77(308), 19 - 27
Effect of Bacillus thuringiensis beta exotoxin on ultrastructures of midgut cells of Culex sitiens; Weiser J et al.; In midgut cells of Culex sitiens intoxicated with beta exotoxin of Bacillus thuringiensis, the microvilli are greatly reduced . In the cytoplasm there is progressive disarrangement of the membranes of the rough endoplasmic reticulum, first to spherules, and later to a granular mass . Minute vacuoles appearing in the cytoplasm are parts of the distorted labyrinth and remains of the Golgi membranes . There is no disintegration of the outer cell membrane, and no hypertrophy of goblet cells . Nuclei remain in their posterior position and there are no cytoplasmic autophagic vacuoles in the centre . Cells in the same part of the midgut differ in the degree of disorganization.

Bull Soc Pathol Exot, 1994, 87(1), 28 - 32
{Leprosy in New Caledonia from 1983 to 1992 . Histopathological and epidemiological data}; Monchy D et al.; New Caledonia is a South Pacific Island inhabited by more than 170,000 people: most of them are melanesians or europeans . Multidrug therapy for Hansen disease has been employed since 1983; so we made an epidemiologic and histopathologic study of the new cases diagnosed for 10 years, from 1983 to 1992 . Local (clinical, histological, microbiological and immunological) means of diagnosis are described . In New Caledonia, the endemic level is lower than in the small neighbouring Pacific islands but there is still a native reservoir of Mycobacterium leprae with 40% multibacillary types . Nearly half of the new cases are less than 25 years old . They are often male melanesians . The diagnosis of indeterminate forms is debated when there is no acid-fast bacillus . Border-line forms are rare . Tuberculoid leprosy poses many differential diagnosis problems . Despite available multidrug therapy, one cannot yet consider that the incidence is decreasing significantly.

J Clin Dent, 1994, 5(2), 35 - 7
Effectiveness of sealed dental prophylaxis angles inoculated with Bacillus stearothermophilus in preventing leakage; Barnes CM et al.; It was the purpose of this in vitro investigation to evaluate the effectiveness of several brands of sealed, reusable prophylaxis angles to keep internal materials within the internal portions of the head of the prophylaxis angle, and not allowing contaminates to leak out . Three brands of sealed, reusable dental prophylaxis angles were autoclaved and then taken apart under a biocontainment flow hood . Testing conditions were designed to prevent a "worst case scenario" by inoculating dental prophylaxis angles with 10(6) of the heat resistant spores of Bacillus stearothermophilus and 20% bovine serum albumin to simulate the presence of human serum . The concentration of Bacillus stearothermophilus spores was verified before testing procedures were initiated . The internal portions of the sterile prophylaxis angles were inoculated with a 1:1 mixture of the Bacillus stearothermophilus spores and bovine serum albumin, at a concentration of 1.15 x 10(6) spores/inoculation . The prophylaxis angles were reassembled under sterile conditions, and a sterile rubber cup was inserted into each of the prophylaxis angles . The prophylaxis angles were attached to a sterile dental handpiece and then submerged in a 50 ml tube containing sterile trypticase soy broth and run at 3000 rpm for 30 seconds . The tube of medium was incubated for 7 days . No growth of Bacillus stearothermophilus spores could be cultured from one of the brands of prophylaxis angles at any time during the incubation period . The other two brands of prophylaxis angles did produce some leakage of the Bacillus stearothermophilus spores.

Chin J Biotechnol, 1994, 10(1), 1 - 9
Transfer of shuttle vectors containing Bacillus thuringiensis toxin gene into wild-type B . cereus, B . brevis and B . subtilis by electroporation; Sun L et al.; Gram positive and negative bacterial shuttle vectors carrying Bacillus thuringiensis (B.t.) toxin gene were introduced by electroporation into wild-type Bacillus cereus, B . brevis and B . subtilis . The transformation efficiencies for these bacteria were around 10(1)-10(4) transformants per micrograms DNA based on the numbers of neomycin- and ampicillin-resistant colonies produced . The structure of the transferred plasmids proved identical with the original ones both in size and restriction pattern . Toxicity assays showed that the transformants gave a mortality of 90-100% against caterpillar of Heliothis assulta, indicating that the gene function was not changed by electroporation.

Arch Virol, 1994, 135(3-4), 333 - 44
New Bacillus bacteriophage species; Ackermann HW et al.; Nine new species of tailed Bacillus phages, based on morphological and physicochemical properties, are defined . Phage P10 is one of the largest viruses known . The total number of tailed Bacillus phage species is presently 33.

Microbiol Immunol, 1994, 38(7), 519 - 25
The presence of low molecular weight germinative substances in Bacillus cereus T spore extract; Shibata H et al.; An extract from intact spores of Bacillus cereus T having a germination-inducing activity was studied . Two distinct germinative principles were found through dialysis of the extract . One was diffusible through the dialysis membrane and the other was non-diffusible . The activity of the former fraction was inhibited by the addition of 1 mM glycoletherdiamine-N,N,N',N'-tetraacetic acid (GEDTA), whereas the latter fraction was inactive unless GEDTA was added to the assay system . The diffusible principle maintained the major portion of the activity found in the crude spore extract . By means of high-performance liquid chromatography (HPLC) using a gel permeation chromatography column, 9 fractions were obtained from the deproteinized diffusible fraction . Of those fractions, two fractions (No . 1 and No . 8) were responsible for the germination-inducing activity, but no reconstituted activity was observed unless both fractions No . 1 and No . 8 were added to the assay system . Amino acid analysis of fraction No . 1 revealed that the fraction was rich in free amino acids, especially in alanine . On the other hand, by the use of reverse-phase HPLC and fast atom bombardment mass spectrometry, it was concluded that the effective substance in fraction No . 8 was inosine . Based on these findings, it was suggested that the active substances in fraction No . 1 might be a free amino acid such as L-alanine and/or Ca2+ and a Ca(2+)-binding substance.

Dev Biol Stand, 1994, 82, 171 - 8
Towards new mycobacterial vaccines; Gicquel B; BCG, the Bacillus of Calmette and Guerin, has been used as a vector for the presentation of foreign antigens . The nef gene of HIV was cloned and expressed in BCG . A specific cellular immune response against Nef was observed following inoculation of mice . As a model system beta-galactosidase has also been expressed in BCG . Antibody responses against beta-galactosidase in addition to cellular responses were observed . Mucosal immunity was demonstrated after oral immunization of mice . With the aim of isolating new vaccines against tuberculosis, transposon mutagenesis was investigated . By using Tn610, representative libraries of Mycobacterium smegmatis insertion mutants were constructed.

Nat Toxins, 1994, 2(4), 166 - 74
Genetic relatedness of toxic and nontoxic isolates of the marine pennate diatom Pseudonitzschia (Bacillariophyceae): phylogenetic analysis of 18S rRNA sequences; Douglas DJ et al.; The nuclear small subunit (SSU) rRNA genes from several marine diatoms, including 2 species that have been responsible for toxic blooms, were amplified from total DNA by the polymerase chain reaction (PCR), and the sequences analysed to determine their genetic relatedness . The isolates investigated include 2 morphologically similar forms of the pennate diatom Pseudonitzschia pungens: (1) P . pungens f . multiseries, a known producer of the toxin domoic acid, and (2) P . pungens f . pungens, which is not toxic . Strains of a second toxin-producing species, P . australis, and a nontoxic Thalassiosira species were also included in this study . Phylogenetic analyses of sequences by both distance and parsimony methods clearly distinguished the 2 forms of P . pungens and the 2 Pseudonitzschia species from other diatoms for which sequence data are available . Differences in the nucleotide sequences of the 2 forms of P . pungens permitted the design of PCR primers that allowed discrimination between them . This may prove a valuable tool in identifying toxic and nontoxic forms of closely related and morphologically similar diatom species.

Microbiol Immunol, 1994, 38(5), 337 - 43
The rice culture filtrate of Bacillus cereus isolated from emetic-type food poisoning causes mitochondrial swelling in a HEp-2 cell; Sakurai N et al.; Rice culture filtrates of Bacillus cereus SA-50, an emetic-type strain, produced a toxin which caused cytoplasmic vacuole formation in HEp-2 and HeLa cells . Electron microscopic observation revealed that the apparent vacuoles in HEp-2 seen under a light microscope were actually swollen mitochondria . The oxygen consumption of HEp-2 cells was accelerated by the addition of the rice culture filtrate as was measured with a polarographic oxymeter; a respiratory control ratio was 1.0 for control cells, while 1.4 for ones with the filtrates . The culture filtrates showed a similar effect on the isolated mouse liver mitochondria; respiratory control ratios for the mitochondria with and without the filtrates were 3.6 and 1.0, respectively . The affecting manner of the culture filtrates on the oxygen consumption of mitochondria was similar to that of 2,4-dinitrophenol, suggesting that the culture filtrate contains a toxin acting as an uncoupler of oxidative phosphorylation in mitochondria . It is likely that the culture filtrates containing the emetic toxin of B . cereus causes mitochondrial swelling with a close relationship to the uncoupling of the oxidative phosphorylation of mitochondria.

Clin Exp Obstet Gynecol, 1994, 21(3), 177 - 83
Colposcopy, colpocytology and the vaginal ecosystem . Oncological, bacteriological and hormonal evaluation in a series of 400 women; Torrisi A et al.; The Authors retrospectively considered colpocytological and colposcopic findings in a series of 400 women, aged 16 to 83 years, presenting for the first time at the Oncological Gynaecology Unit of the Institute of Gynaecology and Obstetrics of Padua University between 1991 and 1992 . In addition to oncological evaluation, the bacteriological profile and hormone status of cytological samples were formulated in all cases . The most common oncological finding was a cell morphology within normal limits (67%), followed by reactive and reparative changes (19%) and low-grade squamous intraepithelial lesions (SIL, 12%) . Histological findings correlated well with the cytological diagnosis, though low-grade SIL was over-estimated . As for the bacteriological profile, a mixed flora was most frequent (56.7%) followed, especially in fertile age, by Doderlein's bacillus (20%) and vaginosis (15.5%) . Colposcopy most frequently revealed ectopia and/or a normal transformation zone (50.7%) and dystrophic mucosa (21%) . An abnormal transformation zone was more common among women with a moderate-to-abundant flora . Fifteen male partners were also checked: cellular changes typical of human papilloma virus infection were found in 40% and colposcopic findings compatible with said virus were observed in 26.6% of cases . These results confirm that colpocytology provides a complete and simultaneous evaluation not only of cell morphology, but also of the bacterial population and hormones in the vaginal ecosystem . It is therefore the method of choice in screening for cervical and vaginal neoplasms and an effective means for simultaneously evaluating vaginal flora and hormone status.

Pneumonol Alergol Pol, 1994, 62(5-6), 280 - 6
{Evaluation of selected immunologic parameters in healthy persons infected with mycobacterium tuberculosis bacillus}; Zabuska-Jablonska K et al.; Selected immunological parameters of peripheral blood leukocytes in 30 tb contracts and control group consisting of 30 healthy blood donors in similar age were examined . All contacts showed the exudate type Mantoux reaction (over 20 mm in diameter) and all revealed normal chest X-ray . No differences between both groups in total T cells, CD4, CD8 counts, CD4/CD8 ratio and in proliferative response to mitogens (PHA and Con A) were found . Tb contacts had the similar serum IgM and elevated IgG and IgA concentrations as compared with the controls . Tb contacts showed depressed granulocyte metabolic activity as measured by CL response to chemostatic peptide N-FORMYL-MET-LEU-PHE (FMLP) in comparison with control subjects.

Arch Med Res, 1994 Summer, 25(2), 159 - 63
Histopathologic and humoral study of Balb/c mice inoculated with BCG by different routes; Acosta A et al.; With the aim of determining the distribution and humoral immunogenicity of the bacillus Calmette-Guerin (BCG) administered by the oral (O), intravenous (IV) and subcutaneous (SC) routes, we studied 54 male Balb/c mice weighing 17-22 g that had been inoculated with BCG (10(6) CFU) by the O (n = 18), IV (n = 18) and SC (n = 18) routes . At weekly intervals we determined the distribution of the microorganism using histopathological techniques including Ziehl-Neelsen staining . Serum samples of the same animals were analyzed by ELISA and Western blot to determine the antibody response to the microorganism . In all groups, distinctive histopathologic lesions harboring the microorganism were found . Using the SC route the lesions were located at the inoculation site, whereas there was systemic dissemination with the O and IV routes, being more prominent with the latter . Anti-BCG antibodies were detected by ELISA in all groups; this response was more intense in the IV group, followed by the SC and O groups . In the Western blot analysis, reactivity against multiple bands and the predominant recognition of a 65 kd band in all groups was observed.

Microbiol Immunol, 1994, 38(11), 843 - 50
Coccoid Helicobacter pylori not culturable in vitro reverts in mice; Cellini L et al.; An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori . Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the "dormant" forms . The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains . Five hundred microliters of culture (OD550 = 5 Mc Farland) of Helicobacter pylori with bacillary (2-5 x 10(9) CFU/ml) and coccoid (0 CFU/ml) morphology were inoculated intragastrically in BALB/c mice . The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures . Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation . After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori . These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.

World J Urol, 1994, 12(6), 337 - 44
Specific binding of bacillus Calmette-Guérin to urothelial tumor cells in vitro; Schneider B et al.; Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) against recurrences of superficial bladder cancer and carcinoma in situ is a highly effective regimen in urology . Despite intensive efforts to clarify the immunological mechanisms of the most successful immunotherapy known today, the cellular mechanism of its antitumor activity remains unknown . In our approach to elucidate the way of action of intravesical BCG, we applied an in vitro adhesion assay to investigate the interaction of radiolabeled BCG with urothelial bladder-tumor cells . We demonstrated a BCG dose-dependent binding to bladder-tumor cell lines derived from tumors of different gradings . The binding of BCG is apparently specific, since competition experiments showed an inhibition by nonradioactive BCG but not by Escherichia coli . We also found that there was no difference between the binding of living or heat-killed mycobacteria . Control experiments showed only a low affinity of BCG for fibroblasts, smooth-muscle cells, and endothelial cells in comparison with the tumor cells . Furthermore, we investigated the role of fibronectin as an adhesion molecule that is also present in the bladder wall . We demonstrated that BCG was capable of binding to fibronectin-coated surfaces in a dose-dependent manner . However, competitive binding assays failed to reveal an inhibition of the binding of BCG to bladder-tumor cells by anti-fibronectin . Furthermore, binding was not influenced by soluble fibronectin . These data suggest that the in vitro attachment of BCG to bladder-tumor cells appears not to be mediated by fibronectin . In electron microscope studies an adhesion of BCG to bladder-tumor cells was observed after an incubation period of ony 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Urol Res, 1994, 22(4), 239 - 45
Effects of sequential intravesical administration of mitomycin C and bacillus Calmette-Guérin on the immune response in the guinea pig bladder; Balemans LT et al.; It has been suggested that intravesical treatment with mitomycin C (MMC) before instillation of bacillus Calmette-Guerin (BCG) improves the antitumor activity of BCG in human bladder cancer . Therefore, we studied the immunological effects of sequential intravesical treatment with MMC and BCG in the guinea pig . Four weekly intravesical instillations with MMC preceded six weekly intravesical BCG instillations . The delayed-type hypersensitivity (DTH) skin reaction evoked by tuberculin purified protein derivative (PPD) in guinea pigs receiving BCG intravesically appeared slightly earlier in animals pretreated intravesically with MMC than in phosphate-buffered saline (PBS)-pretreated animals . However, after completing BCG instillations no differences in DTH reaction were observed between these treatment groups . The extent of the local inflammatory reaction in the bladder wall, as well as the parameters measured in the draining iliacal lymph nodes (i.e., the weight, the number of leukocytes, and the composition of leukocyte subpopulations), did not differ in animals treated with BCG alone or in combination with MMC . A slight increase in the MHC class II expression on the bladder urothelium was shown if MMC and BCG treatment was combined . The adherence of mycobacteria to the bladder wall, measured using 3H-labeled mycobacteria, dit not differ between MMC/BCG- and BCG-treated animals . We conclude that MMC does not enhance the immune response against mycobacteria . Therefore, we hypothesize that a possible increased antitumor activity by the combination of MMC and BCG might be due to separate, rather than synergistic, effects of the drugs, namely a cytostatic effect of MMC on tumor cells and a local immune response in the bladder evoked by BCG.

Acta Biochim Pol, 1994, 41(3), 281 - 92
Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: crystallization and site-directed mutagenesis; Koyama T et al.; The gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli . The synthase was purified to homogeneity and crystallized . The enzyme carried only two cysteine residues in contrast to its counterparts from other sources, which have four to six cysteine residues . Either or both of the cysteine residues can be replaced with serine without causing a loss of the catalytic activity . The conserved arginine residue that occupies the third position from the C-terminus was also replaced with valine without significant loss of activity, but the valine mutant showed a weakened affinity for isopentenyl diphosphate.

Ann Dermatol Venereol, 1994, 121(3), 240 - 1
{Allergic vasculitis in brucellosis}; Boudghene-Stambouli O et al.; Brucellosis is an anthropozoonosis caused by a Gram negative bacillus of the Brucella gender . Skin manifestations have been reported in 1.5 to 11 p . 100 of the cases . Allergic vasculitis is rare . Recently a 24-year-old man was hospitalized for signs of infection . He had been treated with tetracycline . The clinical picture was suggestive of brucellosis and the Wright test was positive at 1/1,280 . There were violet and purpuric papulae on the limbs, arthritis of the knee and ankle joints and renal involvement (haematuria, proteinuria) . Histology revealed fibrinoid and leukocytoclastic vasculitis of the small veinules of the subpapillary plexus . Outcome was favourable with rifampicin, doxycycline and adjuvant dapsone, together with bed rest . Several types of skin manifestations have been reported in brucellosis although cases of allergic vasculitis are rare.

J Fr Ophtalmol, 1994, 17(10), 548 - 54
{Bouchut tubercles and AIDS . Apropos of 3 cases}; Campinchi-Tardy F et al.; Tuberculosis choroidal granulomas ("tubercules de Bouchut") are rare, even in patients with AIDS and active tuberculosis . The authors report the clinical and angiographical findings of three cases . Choroidal involvement was bilateral, with multiple lesions, mostly located at the posterior pole . The size of the granuloma ranged from 1/8 to 1/2 of a disc diameter . Fluorescein angiography showed early prolonged hypofluorescence and late moderate hyperfluorescence . The lesions remained stable for months despite treatment of tuberculosis, and then gradually subsided . In two cases, the choroidal granulomas were discovered before the diagnosis of disseminated tuberculosis and guided the investigations, which allowed the identification of Mycobacterium Tuberculosis bacillus in the body . In the third case, pulmonary tuberculosis had already been diagnosed . The number of tuberculosis cases is increasing together with the number of AIDS cases . This should make the observation of tuberculosis choroidal granulomas more frequent . The discovery of the typical aspects of granulomas described above during fundus examination of AIDS patients can help in diagnosing tuberculosis.

Chin J Biotechnol, 1994, 10(2), 75 - 82
Coleopterancidal delta-endotoxin and constructing its genomic library; Li X et al.; The composition of crystal proteins and plasmid patterns in five new coleopterancidal strains of Bacillus thuringiensis were investigated . Larvae of Plagiodera versicolora were chosen as a model insect in bioassay . Strain YM-03 had the highest toxicity . Plasmid patterns were detected by rapid agarose gel electrophoresis . It was demonstrated that plasmid patterns of five Bt strains were quite different . The partial amino acid sequences of the N-terminal of crystal protein from YM-03 were analyzed by amino acid analyzer . A genomic library of delta-endotoxin of YM-03 was constructed . The EcoRI fragments (20-30 kb) from the total DNA were ligated with the vector DNA of pLAFRI . Thirteen clones which contained both of pLAFRI and foreign DNA fragments were obtained from 17 recombined plasmids . The frequency of recombinant clones was 76% . Three positive clones, LE392 (pBYM2), LE392 (pBYM3) and LE392 (pBYM4) were detected from 1200 resistant clones by using a synthesized 18bp probe from a coleopterancidal delta-endotoxin gene . It was shown that LE392 (pBYM3) and LE392 (pBYM4) had the same EcoRI digestion patterns and that they all contained coleoptera-specific delta-endotoxin gene but showed a different level of expression.

Chin J Biotechnol, 1994, 10(2), 135 - 44
Studies on isolation and purification of penicillin acylase by adsorption on bentonite; Sun W; When bentonite I as an absorbent according to 0.6% (w/v) was added to the supernatant of the fermentation broth for adsorption of penicillin acylase from Bacillus megatherium, 100% activity of penicillin acylase and about 10% protein in the supernatant were adsorbed . The adsorption of enzyme was not obviously changed with different pH and salt concentration of the supernatant . Various kinds of buffer with different pH were used to wash the enzyme-adsorbent complex . Only 1% enzyme activity adsorbed was washed out; however, it can wash out about 15% protein adsorbed . When phosphate buffer containing 10% PEG and NaCl as an eluent was used to elute the complex, 100% of enzyme activity adsorbed on the complex would be eluted, and purification and concentration times of enzyme could achieve about 25 and 6, respectively . The isolation and purification process can be carried out at room temperature . Its characters were very simple and showed a high recovery yield of enzyme activity, and it can be directly used for isolation and purification of penicillin acylase from the fermentation broth.

Acta Chir Iugosl, 1994, 41(2), 155 - 7
{Isolated tuberculosis of the liver--case report}; Colovic RB et al.; Tuberculosis of the liver is rare even in areas where tuberculosis is widespread . Isolated tuberculosis of the liver is extremely rare . It usually presents as a nodular - pseudo-tumorous form . Tuberculosis of the bile ducts is even less frequent . A 37 year old woman with nodular - pseudotumorous form of tuberculosis of the liver is presented . The diagnosis was established after surgical biopsy and histology . It may be caused by the bovine type of the bacillus . The patient had an excellent response to tuberculostatic drugs . Tuberculosis of the liver must be taken into account in the differential diagnosis of the liver masses.

Chin J Biotechnol, 1994, 10(4), 241 - 8
Expression and secretion of alpha-amylase and glucoamylase in Saccharomyces cerevisiae; Luo J et al.; alpha-Amylase genes of Bacillus licheniformis and glucoamylase cDNA of Aspergillus niger were ligated to a E . coli-yeast shuttle vector . The resultant plasmid was used to transform Saccharomyces cerevisiae to construct starch-degrading yeast strain . The results of enzyme activity assay and enzyme property analysis show that alpha-amylase and glucoamylase genes have been expressed simultaneously in yeast under the control of promoters and terminators of yeast MF-alpha 1 factor and PGK genes and over 99% of enzyme activities were secreted to the medium . The engineered yeast strain hydrolyses 97% of the starch (10%) in the medium after 6 days . The recombinant plasmid exists stably in yeast.

SAAS Bull Biochem Biotechnol, 1994, 7, 1 - 6
Current research to develop the entomopathogen Bacillus thuringiensis for horn fly control; Temeyer KB; Bacillus thuringiensis subspecies israelensis (Bti) has been shown to produce at least two sporulation-specific proteins which result in larvicidal activity when incorporated in bioassays against horn flies (Haematobia irritans L.) . Development of a new control technology for horn flies based on Bti appears to be feasible through the use of recombinant DNA technology . Substantial work remains however, to develop new research tools and techniques for this application.

Biosci Biotechnol Biochem, 1994 Jan, 58(1), 121 - 5
Transfer action of alpha-D-xylosidases from Aspergillus flavus MO-5 on p-nitrophenyl-alpha-D-xylopyranoside; Yoshikawa K et al.; The transfer action of alpha-D-xylosidase I and II from Aspergillus flavus MO-5 on p-nitrophenyl-alpha-D-xylopyranoside (alpha-p-NPX) was investigated, as compared with that of commercial alpha-D-xylosidase (from Bacillus sp.) . In reaction mixtures with various concentrations of alpha-p-NPX, both enzymes (I and II) liberated p-nitrophenol and xylose at low concentrations, and showed transfer products in addition to hydrolyzates at high concentrations . IP-1 and IP-3, IIP-1 and IIP-2, and CP-1 and CP-3 were main transfer products by alpha-D-xylosidase I, alpha-D-xylosidase II, and commercial alpha-D-xylosidase, respectively . They were isolated by active charcoal column chromatography and silica gel column chromatography . Their structures were analyzed by enzymatic digestion, methylation analysis, and 13C-NMR analysis . As the results, IP-1, IIP-2, and CP-1 were found to be the same structure, p-nitrophenyl-4-O-(alpha-D-xylopyranosyl)-alpha-D-xylopyranoside . Then, it was proved that the structure of IIP-1 was p-nitrophenyl-3-O-(alpha-D-xylopyranosyl)-alpha-xylopyranoside . On the other hand, IP-3 and CP-3 were found to be the same structure, D-xylopyranosyl-alpha-1,4-D-xylopyranose without a p-nitrophenyl group . We found that alpha-D-xylosidase I showed alpha-1,4 xylosyl transfer action as well as commercial alpha-D-xylosidase, and alpha-D-xylosidase II showed alpha-1,3 xylosyl transfer action in addition to alpha-1,4 xylosyl transfer action on alpha-p-NPX.

Curr Microbiol, 1994 Jan, 28(1), 15 - 9
Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain buibui toxic to scarabaeid insects; Sato R et al.; Recombinant Escherichia coli strains harboring pAG1, pAG2, pKBB100, and pKBB101 were cloned by using antiserum constructed against 130-kDa crystal protein antigen of Bacillus thuringiensis serovar japonensis strain Buibui . DNAs in the recombinant strains hybridized to the 26-base oligonucleotide probe corresponding to N-terminal amino acids of the 130-kDa crystal protein of strain Buibui . Cultures of the recombinant strains were toxic to larvae of the cupreous chafer, Anomala cuprea . Furthermore, the production of the 130-kDa polypeptide was demonstrated in the cells harboring pAG1 and pAG2 by immunoblot analysis with antiserum against the 130-kDa crystal protein . Southern hybridization analysis showed that the 130-kDa crystal protein gene is located on the chromosomal DNA of strain Buibui . On the other hand, DNA probes derived from cryIA(a) and cryIIIA genes did not hybridize to the DNA of strain Buibui.

Arch Anat Cytol Pathol, 1994, 42(6), 289 - 96
{Bacillary angiomatosis related to Rochalimaea quintana . Anatomoclinical and ultrastructural study of cutaneous localizations in AIDS}; Bost F et al.; We report a case of bacillary angiomatosis in a 53-year-old homosexual man with acquired immunodeficiency syndrome (AIDS) . Pathological and bacteriological studies of cutaneous nodules led to the identification of a rickettsia: Rochalimaea quintana . This observation prompted us to relate the clinical presentation of cutaneous and visceral forms of this disease . Histopathological patterns are also considered . They usually consist in a lobular proliferation of capillaries with plump and sometimes epithelioid endothelial cells . Polymorphonuclear cells, histiocytes and necrotic areas may be present . The most characteristic feature is the presence of interstitial, granular and amorphous clusters of bacteria . Diagnostic problems can be raised with Kaposi's angiosarcoma which can be associated with bacillary angiomatosis . Two types of Rochalimaea have so far been isolated in this disease i.e., R . henselae which is the most frequently involved, and R . quintana . The usefulness of making such a diagnosis resides in the sensitivity of bacillary angiomatosis to antibiotics, emphasing the need to carefully look for the presence of bacterial clusters when atypical angioproliferative lesion appears in patients with AIDS.

DNA Res, 1994, 1(1), 15 - 26
Characterization of cDNAs induced in meiotic prophase in lily microsporocytes; Kobayashi T et al.; To identify and analyze genes functioning during reproductive cell formation in higher plants, cDNAs harboring the messages induced in meiotic prophase were isolated and characterized . A cDNA library constructed from microsporocytes in meiotic prophase of Lilium longiflorum was screened with a subtraction probe specific to meiotic prophase . Clones selected were classified into 18 groups by cross hybridization and partial sequencing . Northern blot analysis revealed that the transcripts corresponding to the respective cDNA groups began accumulating at the early stages of meiosis and exhibited clone-specific profiles during meiosis and the spore formation process . The amino acid sequences of the predicted gene products showed similarity with known gene products, e.g . heat shock proteins, serine proteases in Bacillus, and RAD 51 gene product in yeast . Half of the putative gene products had hydrophobic N-terminal regions, suggesting that they may function as signal peptides.

Urol Res, 1994, 22(3), 185 - 90
On the mode of action of intravesical bacillus Calmette-Guérin: in vitro characterization of BCG-activated killer cells; Bohle A et al.; Previously we had shown that, upon activation with viable bacillus Calmette-Guerin (BCG), peripheral blood mononuclear cells (PBMNC) could be rendered cytotoxic against otherwise insensitive natural killer (NK)-resistant bladder cancer cell lines . This phenomenon had been termed the BCG-activated killer (BAK) cell phenomenon . By means of depletion and enrichment procedures of mononuclear cell subpopulations derived from BCG-activated PBMNC we further characterized the cytolytic BAK effector cells functionally in an in vitro cytotoxicity assay against the bladder carcinoma cell line BT-A and phenotypically in their pathway of activation . Neither macrophages nor CD4+ T-helper/inducer cells exerted cytotoxic BAK activity . This cytotoxicity was restricted to the CD8+CD56+ subpopulation of T-cytotoxic/NK cells . Furthermore, activation of BAK cells via interferon gamma (IFN-gamma) was evidenced by the complete inhibition of BAK cell generation with an IFN-gamma antibody.

Acta Clin Belg, 1994, 49(3-4), 158 - 67
{Rochalimaea henselae, Afipia felis and cat-scratch disease}; Thonnard J et al.; Several years ago, Rochalimaea henselae has emerged as an agent of bacillary angiomatosis, bacillary peliosis and recurrent septicaemia that generally occur in patients infected with human immunodeficiency virus . An aetiologic role in cat scratch disease is also suspected widely on the basis of a serologic survey . Its slow growth and its culture requirement explain that this pathogen, a gram-negative bacterium, could not be isolated until 1990 . Moreover, blood and tissue samples request lysis and crushing for recovering by culture . The clinical, histological, microbiological and pathogenic aspects of these infections are described and discussed.

Infect Immun, 1994 Jan, 62(1), 86 - 90
Sera of leprosy patients with type 2 reactions recognize selective sequences in Mycobacterium leprae recombinant LSR protein; Singh S et al.; Type 2 reactions (erythema nodosum leprosum {ENL}) are episodic, reactional states causing significant morbidity in lepromatous leprosy patients . With a view to defining the immunological differences between the stable and reactional forms of lepromatous leprosy, we determined antibody responses to LSR, a recombinant protein of Mycobacterium leprae previously described by us (S . Laal, Y.D . Sharma, H.K . Prasad, A . Murtaza, S . Singh, S . Tangri, R . S . Mishra, and I . Nath, Proc . Natl . Acad . Sci . USA 88:1054-1058, 1991), as well as to 10- to 15-mer overlapping peptides synthesized on the basis of the LSR amino acid sequence . We report here the selective recognition of B cell epitopes by sera from patients with ENL as compared with a control group with nonreactional lepromatous leprosy . Peptides 2 and 3, with the sequences GVTYEIDLTNKNAA and IDLTNKNAAKLRGD, respectively, were recognized by > 95% of sera from patients with active ENL . Peptide 3 in addition showed reactivity with sera taken from 91.6% of lepromatous leprosy patients who were apparently stable but who were recorded to have had ENL several weeks before or after the sample collection . The core sequence IDLTNKNAA common to both these peptides may be a major target of humoral responses in ENL . In addition, the RGD motif at the C terminus appeared to influence the antigenicity of peptide 3 in enzyme-linked immunosorbent assay . It would appear that humoral responses during ENL are directed to selective antigenic determinants of the leprosy bacillus . The use of such serological markers to identify lepromatous leprosy patients with a high risk for developing ENL would be of clinical and predictive value.

Clin Diagn Lab Immunol, 1994 Jan, 1(1), 115 - 6
Persistent generalized lymphadenopathy and non-Hodgkin's lymphoma in AIDS: association with Rochalimaea henselae infection; Peter JB et al.; Cat scratch disease, which is caused by infection with Rochalimaea henselae, is often manifested as lymphadenopathy . R . henselae has also been isolated from human immunodeficiency virus (HIV)-positive patients with bacillary angiomatosis . In order to determine the frequency of R . henselae-reactive antibodies in HIV-positive patients with persistent generalized lymphadenopathy (PGL) or non-Hodgkin's lymphoma (NHL), we tested a total of 124 HIV-positive patients for R . henselae-reactive immunoglobulin G (IgG), IgM, and IgA antibodies by an enzyme immunoassay procedure using whole R . henselae antigen . Of the patients, 7 had PGL, 17 had NHL, and 100 were HIV stage IV (Centers for Disease Control criteria) . A total of 86% of PGL patients (6 of 7) were positive for R . henselae antibodies (three were positive for IgG, IgA, and IgM, one was positive for IgG and IgA only, and two were positive for IgG only) . A total of 29% of NHL patients (5 of 17) were positive for R . henselae antibodies (two were positive for IgG, IgA, and IgM and three were positive for IgG only) . Only 5% of HIV Stage IV patients without adenopathy (5 of 100) were positive for R . henselae-reactive IgG, IgA, and IgM . The high prevalence of R . henselae-reactive antibodies in HIV-positive PGL and NHL patients suggests that R . henselae is a potential etiologic agent or cofactor in these patients.

Biochem Biophys Res Commun, 1993 Dec 30, 197(3), 1334 - 9
A 4-amino-2-hydroxybutyrate activating enzyme from butirosin-producing Bacillus circulans; Aubert-Pivert E et al.; An enzyme catalyzing a 4-amino-2-hydroxybutyrate dependent ATP-PP(i)-exchange reaction has been partially purified from butirosin-producing cells of B . circulans by ammonium sulfate fractionation, gel filtration or sucrose gradient centrifugation and ion exchange chromatography on QAE-sepharose . No reaction was found with 4-aminobutyrate and diaminobutyrate . The protein coeluted with GroEL, which has been identified by alignment of the internal sequence KDGVITVEESK . A molecular mass of about 1,5 MDa has been estimated for the complex by gradient centrifugation . In SDS-polyacrylamide gels a protein band with an estimated size of about 500 KDa correlated with the enzyme activity . The activity was not found in non-producing cells and a non-producing mutant of B . circulans with an insertional inactivation in the butB gene.

J Biol Chem, 1993 Dec 25, 268(36), 27039 - 45
Evidence for lysine 80 as general base catalyst of leucine dehydrogenase; Sekimoto T et al.; To elucidate the functional role of the lysyl residue highly conserved in NAD(P)(+)-dependent amino acid dehydrogenases, Lys-80 of leucine dehydrogenase from Bacillus stearothermophilus has been mutated into Ala, Arg, or Gln . All of the mutant enzymes had markedly reduced activities in the oxidative deamination, whereas the Michaelis constants for substrate and coenzyme did not change significantly upon the mutation, except for a 10-30-fold increase in Km values for alpha-keto-iso-caproate in the Ala and Gln mutants . The pH profiles of kinetic parameters of the mutants considerably differed from those of the wild type, in which two ionizable groups with pKa values of 8.9 and 10.7 must be unprotonated for catalysis and protonated for substrate binding, respectively . Combined with the analyses of solvent isotope effect and inhibition by substrate analogs, these results unequivocally show that the epsilon-amino group of Lys-80 participates in catalysis as a general base, assisting the nucleophilic attack of a water molecule to the substrate alpha-carbon atom . Furthermore, the Ala mutant was markedly stimulated by primary amines depending on the pKa and molecular volume, suggesting that in the Ala mutant the added amines can partially replace the general base function of Lys-80 in the wild type enzyme.

FEBS Lett, 1993 Dec 20, 336(1), 79 - 82
Primary structure of cryX**, the novel delta-endotoxin-related gene from Bacillus thuringiensis spp . galleriae; Shevelev AB et al.; A cry-related sequence, designated cryX (EMBL X75019), was localized upstream of cryIG, the delta-endotoxin gene cloned from spp . galleriae of Bacillus thuringiensis and sequenced earlier {(1991) FEBS Lett . 293, 25-28} . Analysis of the cryX complete nucleotide sequence enabled us to explain its virtual crypticity and to reveal the chimeric structure of the genes, cryX and cryIG . The amino acid sequence of 1,151 residues encoded by the continuous reading frame of cryX is similar to the other delta-endotoxins but differs essentially from them.

Carbohydr Res, 1993 Dec 16, 250(1), 79 - 86
Hydrolysis of glycosylpyridinium ions by anomeric-configuration-inverting glycosidases; Padmaperuma B et al.; The hydrolyses of five beta-D-xylopyranosylpyridinium ions by the beta-D-xylosidase of Bacillus pumilus proceed with kcat values 10(8)-10(9)-fold larger than the rates of spontaneous hydrolysis of the same compounds . Log(kcat) values correlate well with aglycon pK(a) {B1g(V) = -0.52, r = 0.99}, whereas the correlation of log(kcat/Km) is poor {r = 0.77; beta 1g(V/K) = approximately -0.6} . The (1-->3)-beta-D-glucanase of Sporotrichum dimorphosporum hydrolyses 4-bromo-2-(beta-D-glucopyranosyl)isoquinolinium ion with a rate enhancement of 10(8) . The amyloglucosidase II of Aspergillus niger hydrolyses three alpha-D-glucopyranosylpyridinium ions with rate enhancements of 10(5)-10(8) . The efficient hydrolysis of glycosylpyridinium ions by these three inverting glycosidases, the catalytic mechanism of which is unlikely to involve a nucleophile from the enzyme, makes it improbable that the hydrolysis of glycosylpyridinium ions by retaining glycosidases, discovered some years ago, is initiated by addition of a catalytic nucleophilic carboxylate group of the enzyme to the pyridinium ring.

Eur J Biochem, 1993 Dec 15, 218(3), 929 - 36
Hexadecylpalmitoylglycerol or ceramide is linked to similar glycophosphoinositol anchor-like structures in Trypanosoma cruzi; de Lederkremer RM et al.; The lipopeptidophosphoglycan from Trypanosoma cruzi is a glycosylated inositol-phosphoceramide isolated from epimastigotes at the stationary phase of growth (4-5 days) . We have now purified two similar glycoinositolphospholipids (glycoinositolphospholipid A and glycoinositolphospholipid B) from epimastigotes after the second day of culture growth . {3H}Palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in glycoinositolphospholipid A and into ceramide in glycoinositolphospholipid B . The lipids were released by incubation with glycosylphosphatidylinositol-specific phospholipase C from Bacillus thuringiensis or by chemical methods . After alkaline hydrolysis, the lipids were analysed by GLC/MS . In glycoinositolphospholipid A the resulting lipids corresponded to 1-O-hexadecylglycerol and palmitic acid . The ceramide components in glycoinositolphospholipid B are sphinganine, palmitic acid and lignoceric acid . The oligosaccharides could be degraded by nitrous acid and further enzymic treatment showed that the two glycoinositolphospholipids isolated from T . cruzi share the common core structure of the glycosylphosphatidylinositol membrane anchors . The microheterogeneity was determined, as well as the substitution by galactose, and was mainly in the furanose configuration as was previously described for lipopeptidophosphoglycan . However, methylation analysis indicated that 20% of the galactose is in the pyranose form . Both glycoinositolphospholipids mainly differ in the lipid moiety.

Biochem J, 1993 Dec 15, 296 ( Pt 3), 753 - 8
Stereochemical course and structure of the products of the enzymic action of endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis; Malet C et al.; The stereochemical course of the reaction catalysed by endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) has been determined by 1H n.m.r . The enzyme-catalysed hydrolysis of barley beta-glucan proceeds with overall retention of the anomeric configuration, indicating that the enzyme operates through a double-displacement mechanism . The structures of the final oligosaccharide products, 3-beta-O-cellobiosyl D-glucopyranoside and 3-beta-O-cellotriosyl D-glucopyranoside, have been completely assigned by 1H- and 13C-n.m.r . spectroscopy.

Proc Natl Acad Sci U S A, 1993 Dec 15, 90(24), 11473 - 7
Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guérin expressing the Leishmania surface proteinase gp63; Connell ND et al.; Leishmania parasites cause a spectrum of diseases that afflict the populations of 86 countries in the world . The parasites can survive within the lysosomal compartments of the host's macrophages, unless those macrophages are appropriately activated . Despite the fact that protective immunity can be induced by vaccination with crude parasite preparations, little progress has been made toward a defined vaccine for humans . In this study the gene encoding the Leishmania surface proteinase gp63 was cloned and expressed as a cytoplasmic protein in a bacille Calmette-Guerin (BCG) vaccine strain . BALB/c and CBA/J mice were inoculated with a single dose of recombinant BCG and challenged with infective Leishmania major or Leishmania mexicana promastigotes . Significant protection was observed in both mouse strains against L . mexicana and in CBA/J against L . major, whereas only a delay in L . major growth was seen in BALB/c mice . Recombinant BCG also engendered a strong protective response against challenge with amastigotes of L . mexicana, demonstrating that the induced immune response recognized the intracellular form of the parasite . The results support the view that recombinant BCG expressing gp63 may prove a useful vaccine for inducing protective cell-mediated immune responses to Leishmania species causing American cutaneous leishmaniasis.

Biochemistry, 1993 Dec 14, 32(49), 13651 - 7
Mutation of lysine 233 to alanine introduces positive cooperativity into tyrosyl-tRNA synthetase; First EA et al.; Tyrosyl-tRNA synthetase from Bacillus stearothermophilus is a dimeric enzyme which displays half-of-sites reactivity with respect to the binding of both tyrosine and ATP . The binding of both substrates follows Michaelis-Menten kinetics . Mutation of lysine 233 to alanine (K233A) decreases the affinity of the active subunit for ATP at both saturating and subsaturating tyrosine concentrations (from the Hill plot, kcat = 0.56 s-1, nH = 1.54, Kd = 372 mM at 50 microM tyrosine) . In addition, this mutant displays sigmoidal kinetics (characteristic of positive cooperativity) with respect to the binding of ATP . These two effects can be reversed by the addition of NaCl (0.5 m final concentration) or by a second alanine mutation at either position K230 or T234 . The effect of either NaCl or second site mutation is to increase the binding affinity of the K233A mutant for ATP (KATP values are 22 mM for the K233A mutant in the presence of 0.5 M NaCl, 0.16 mM for the K230A/K233A mutant, and 0.14 mM for the K233A/T234A mutant) . With the restoration of the tight binding of ATP, Michaelis-Menten kinetics are restored since the kinetic analysis of tyrosyl adenylate formation involves only binding of ATP to the active subunit . It is likely that the physical mechanism for the positive cooperativity present in the K233A mutant actually exists in the wild-type enzyme but is not observed kinetically due to the initial binding of ATP to the active subunit . These results indicate that, in some cases, a decrease in substrate affinity is sufficient to introduce cooperativity into a noncooperative enzyme.

Biochemistry, 1993 Dec 14, 32(49), 13644 - 50
Involvement of threonine 234 in catalysis of tyrosyl adenylate formation by tyrosyl-tRNA synthetase; First EA et al.; There is a mobile loop in the tyrosyl-tRNA synthetase that contains the KMSKS signature sequence of class I aminoacyl-tRNA synthetases . As it has not been possible to determine the role of the mobile loop in catalysis from X-ray crystallographic studies, we are investigating its importance by a series of site-directed mutagenic and kinetic studies . Here we examine the role of threonine 234 (T234) in the catalysis of tyrosyl adenylate formation by tyrosyl-tRNA synthetase from Bacillus stearothermophilus . This residue is the carboxy-terminal residue in the signature sequence and is either a serine or threonine in eight of the ten class I aminoacyl-tRNA synthetases isolated from Escherichia coli . Kinetic analyses of tyrosyl adenylate formation in the mutant enzymes indicate that k3, the forward rate constant for the formation of tyrosyl adenylate, is reduced 500-fold on mutation of T234 to alanine . In contrast, mutation of T234 to serine results in only a 4-fold decrease in k3, suggesting that the loss of the hydroxyl group in the T234A mutant is responsible for its decreased reaction rate . Deletion of the hydroxyl group destabilizes the transition state for the formation of tyrosyl adenylate by 2.7 kcal/mol . The transition state is also destabilized by 1.4 kcal/mol on the mutation of K230 to alanine . The effects of mutation of both T234 and K230 to alanine are less than additive; there is a coupling energy of -1.3 kcal/mol in the transition state . The effects of mutating K230 and T234 to alanine are also nonadditive in the E.Tyr-AMP complex (coupling energy = -1.9 kcal/mol).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Pharmacol, 1993 Dec 3, 46(11), 1887 - 92
Inhibitory effect of bisbenzylisoquinoline alkaloids on nitric oxide production in activated macrophages; Kondo Y et al.; Bisbenzylisoquinoline (BBI) alkaloids are anti-inflammatory constituents of plants of the families Menispermaceae and Ranunculaceae, which have been used as folk remedies in Japan and China . Five BBI alkaloids (cepharanthine, chondocurine, cycleanine, isotetrandrine and tetrandrine) were tested for suppressive effect on in vitro nitric oxide (NO) production by lipopolysaccharide-stimulated peritoneal macrophages, which were induced with thioglycollate or bacillus Calmette-Guerin in mice . All these BBI alkaloids significantly suppressed NO production at 5 micrograms/mL . Cepharanthine, isotetrandrine and cycleanine were slightly more inhibitory than tetrandrine and chondocurine . The suppression persisted for at least 48 hr . As NO is one of the critical mediators in inflammation, these results may explain some aspects of the anti-inflammatory mechanisms of BBI compounds.

Immunol Cell Biol, 1993 Dec, 71 ( Pt 6), 559 - 70
BCG vaccination in deer: distinctions between delayed type hypersensitivity and laboratory parameters of immunity; Griffin JF et al.; Groups of deer were vaccinated with live or killed Bacillus Calmette-Guerin (BCG), with and without oil adjuvant, to compare their immune responses with those found in naturally infected animals . Killed BCG in oil induced strong lymphocyte transformation (LT) and antibody (ELISA) responses specific for Mycobacterium bovis antigens . Serum inflammatory proteins (SIP) were also induced after these animals were skin tested . This pattern of reactivity mirrored that found in naturally infected deer with active tuberculosis . Animals vaccinated with live BCG without oil adjuvant also produced strong LT reactivity but this was directed at common mycobacterial antigens found on both M . bovis and M . avium, although no antibody or SIP were detected at any stage of the experiment . The pattern of immune responsiveness to live BCG was similar to that found in naturally infected, but non-diseased deer, and may represent the immunoprotective response to tuberculosis . Significant differences in specificity of lymphocyte transformation and intradermal skin test reactivity to mycobacterial antigens were also identified . Vaccination with BCG in various formulations provides an experimental probe to evaluate the immunological basis of immunity to tuberculosis.

Int J Pept Protein Res, 1993 Dec, 42(6), 560 - 4
Kinetic characterization of alkaline mesentericopeptidase . Comparison with serine proteinases from different origins; Dolaschka P et al.; Comparative studies of the hydrolysis of succinyl-Ala2-Phe-methylcoumarylamide with mesentericopeptidase, a mesophilic extracellular serine proteinase from Bacillus mesentericus, and proteinases produced by organisms representing different levels of evolutionary development, were performed . Drastic differences in the proteolytic coefficient kcat/Km were found . As regards their catalytic efficiency, the proteinases studied can be placed in the following order: mesentericopeptidase < subtilisin Novo << subtilisin DY < proteinase K < subtilisin Carlsberg < thermitase < alpha-chymotrypsin . The size of the substrate-binding site of mesentericopeptidase for synthetic peptides was studied by using chloromethyl ketones with the general formula benzyloxycarbonyl-Alan-Phe-CH2Cl (n = 1, 2, 3) . The presence of at least five binding subsites (S1 .. . S5) on the S-side of the hydrolysed bond was suggested . Studies of the primary specificity of mesentericopeptidase with a series of dipeptide chloromethyl ketones having the general formula benzyloxycarbonyl-Ala-Aa-CH2Cl (Aa = Ala, Val, Leu, Phe) revealed the following order of reactivity toward these inhibitors: Aa = Leu >> Ala > Phe > Val . Kinetically, mesentericopeptidase is similar to subtilisin BPN'/Novo.

Int J Pept Protein Res, 1993 Dec, 42(6), 527 - 32
Is an amphiphilic region responsible for the haemolytic activity of Bacillus thuringiensis toxin?
Szabo E, Murvai J, Fabian P, Fabian F, Hollosi M, Kajtar J, Buzas Z, Sajgo M, Pongor S, Asboth B.
The amino acid sequence of the 27 kDa protein responsible for the haemolytic activity of Bacillus thuringiensis subsp . israelensis toxin has been analysed by secondary structure prediction, helical wheel/net diagrams and molecular mechanics calculations . We found that segment 116-126 presumably forms a strongly amphiphilic alpha-helix . This is supported by the findings that the synthesized segment 116-126 (a) has a significant alpha-helical content in water, and (b) displays an in vitro haemolytic activity comparable to that of bee venom peptide melittin . As segment 116-126 is present in the haemolyzing, but not present in the non-haemolyzing proteins from B . thuringiensis toxins, we suggest that this segment is responsible for the lytic potential of the B . thuringiensis subsp . israelensis protein.

Br J Urol, 1993 Dec, 72(6), 897 - 906
Bacillus Calmette-Guérin sensitises fresh transitional carcinoma cells and T24 cell line to non-MHC-restricted cytotoxicity in vitro; Kaasinen ES et al.; The activity of lymphokine (interleukin-2) activated killer (LAK) cells from 9 patients with transitional cell carcinoma (TCC) was tested against autologous, freshly purified transitional carcinoma cells . Cytotoxicity was relatively low . Bacillus Calmette-Guerin (BCG) substantially augmented both LAK activity and the cytolytic activity of non-stimulated peripheral blood mononuclear cells (PBMC) against autologous TCC when tested with 6 additional TCC patients . A similar enhancing effect of BCG was noted with leucocytes obtained from normal donors when tested against an allogenic T24 cell line . Both natural killer (NK) cells and T cells appeared to be responsible for the increased cytolytic activity caused by BCG . No cytolytic activity was noted against normal transitional epithelial cells . Sensitisation of TCC cells to the immune system may explain the clinical effects of BCG.

Rinsho Ketsueki, 1993 Dec, 34(12), 1568 - 72
{Two cases of acute myelogenous leukemia with Bacillus cereus bacteremia resulting in fatal intracranial hemorrhage}; Yoshida H et al.; This manuscript reports Bacillus cereus sepsis in two cases with acute myelogenous leukemia (AML) who suffered complications of fatal intracranial hemorrhage during remission induction therapy . The first case was 43-year-old male with AML (M0) receiving first consolidation chemotherapy who developed sudden diarrhea, abdominal pain and spiking fever . Two days later, he died of intracranial hemorrhage . The second case was 15-year-old male with AML (M5b) who was receiving first induction chemotherapy . He developed headache and vomiting following spiking fever and diarrhea . He died of subarachnoid hemorrhage the next day . In both cases, Bacillus cereus was isolated from blood culture . Fatal intracranial hemorrhage due to severe bleeding tendency caused rapid to death in both cases . These bleeding tendencies might have been induced by B . cereus sepsis . In addition, we should not overlook B . cereus as contamination, but rather consider it as a potential pathogen, when isolated from blood culture.

Biokhimiia, 1993 Dec, 58(12), 1845 - 60
{Isolation of site-specific endonucleases and methylases from Bacillus cereus 83}; Matvienko NN et al.; The site-specific endonuclease R Bce83I and methylase M Bce83I were isolated from Bacillus cereus 83 by three consecutive chromatographies on blue agarose, hydroxyapatite and heparin-Sepharose . R Bce83I recognizes {formula: see text} sequences on the DNA and cleaves the DNA as indicated by the arrows . The endonuclease is stimulated by S-adenosyl-L-methionine and may consequently be referred to type IV restriction enzymes.

Appl Environ Microbiol, 1993 Dec, 59(12), 4313 - 6
Antimicrobial activity of a newly identified bacteriocin of Bacillus cereus; Naclerio G et al.; A bacteriocin-producing Bacillus cereus strain was isolated . The bacteriocin, here called cerein, was shown to be active specifically against other B . cereus strains and inactive against all other bacterial species tested . Cerein was detected in the culture supernatants of stationary-phase cells, and its appearance was inhibited by induction of sporulation . The bacterial activity of cerein was insensitive to organic solvents and nonproteolytic enzymes, partially stable to heat, and active over a wide range of pH values . Direct detection of antimicrobial activity on sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of about 9 kDa.

J Cell Biol, 1993 Dec, 123(6 Pt 2), 1789 - 96
Activation of actin polymerization by phosphatidic acid derived from phosphatidylcholine in IIC9 fibroblasts; Ha KS et al.; alpha-Thrombin induced a change in the cell morphology of IIC9 fibroblasts from a semiround to an elongated form, accompanied by an increase in stress fibers . Incubation of the cells with phospholipase D (PLD) from Streptomyces chromofuscus and exogenous phosphatidic acid (PA) caused similar morphological changes, whereas platelet-derived growth factor (PDGF) and phorbol 12-myristate 13-acetate (PMA) induced different changes, e.g., disruption of stress fibers and cell rounding . alpha-Thrombin, PDGF, and exogenous PLD increased PA by 20-40%, and PMA produced a smaller increase . alpha-Thrombin and exogenous PLD produced rapid increases in the amount of filamentous actin (F-actin) that were sustained for at least 60 min . However, PDGF produced a transient increase of F-actin at 1 min and PMA caused no significant change . Dioctanoylglycerol was ineffective except at 50 micrograms/ml . Phospholipase C from Bacillus cereus, which increased diacylglycerol (DAG) but not PA, did not change F-actin content . Down-regulation of protein kinase C (PKC) did not block actin polymerization induced by alpha-thrombin . H-7 was also ineffective . Exogenous PA activated actin polymerization with a significant effect at 0.01 microgram/ml and a maximal increase at 1 microgram/ml . No other phospholipids tested, including polyphosphoinositides, significantly activated actin polymerization . PDGF partially inhibited PA-induced actin polymerization after an initial increase at 1 min . PMA completely or largely blocked actin polymerization induced by PA or PLD . These results show that PC-derived PA, but not DAG or PKC, activates actin polymerization in IIC9 fibroblasts, and indicate that PDGF and PMA have inhibitory effects on PA-induced actin polymerization.

Eur J Biochem, 1993 Dec 1, 218(2), 623 - 8
Evidence for a glycolipid anchor of gp64, a putative cell-cell adhesion protein of Polysphondylium pallidum; Saito T et al.; The membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell-cell adhesion protein identified by adhesion-blocking antibody fragments . Since gp64 can be purified in a few days and in substantial yields, it is a good candidate for clarifying the structure of a cell-cell adhesion protein . This study reveals that gp64 possesses a glycolipid anchor which is sensitive to deamination but resistant to phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis . Although the anchor resistance to phosphatidylinositol-specific phospholipase C can be ascribed to the presence of an additional acyl chain on the inositol ring in the glycosyl phosphatidylinositol anchors, this was not the case . After a mild-base treatment that released an additional acyl chain on the inositol ring, only a trace amount of fatty acid was detected but, after strong acid hydrolysis, we detected both amide-linked fatty acids and a long-chain base . The long-chain base was further analysed by gas-chromatography/mass spectrometry and was found to be phytosphingosine . Both fatty acids and myo-inositol were also analysed by gas-chromatography/mass spectrometry . These data suggest that gp64 possesses a glycolipid anchor which contains ceramide and myo-inositol.

Mol Gen Genet, 1993 Dec, 241(5-6), 564 - 72
A nuclear mutation conferring thiostrepton resistance in Chlamydomonas reinhardtii affects a chloroplast ribosomal protein related to Escherichia coli ribosomal protein L11; McElwain KB et al.; We have isolated a nuclear mutant (tsp-1) of Chlamydomonas reinhardtii which is resistant to thiostrepton, an antibiotic that blocks bacterial protein synthesis . The tsp-1 mutant grows slowly in the presence or absence of thiostrepton, and its chloroplast ribosomes, although resistant to the drug, are less active than chloroplast ribosomes from the wild type . Chloroplast ribosomal protein L-23 was not detected on stained gels or immunoblots of total large subunit proteins from tsp-1 probed with antibody to the wild-type L-23 protein from C . reinhardtii . Immunoprecipitation of proteins from pulse-labeled cells showed that tsp-1 synthesizes small amounts of L-23 and that the mutant protein is stable during a 90 min chase . Therefore the tsp-1 phenotype is best explained by assuming that the mutant protein synthesized is unable to assemble into the large subunit of the chloroplast ribosome and hence is degraded over time . L-23 antibodies cross-react with Escherichia coli r-protein L11, which is known to be a component of the GTPase center of the 50S ribosomal subunit . Thiostrepton-resistant mutants of Bacillus megaterium and B . subtilis lack L11, show reduced ribosome activity, and have slow growth rates . Similarities between the thiostrepton-resistant mutants of bacteria and C . reinhardtii and the immunological relatedness of Chlamydomonas L-23 to E . coli L11 suggest that L-23 is functionally homologous to the bacterial r-protein L11.

Cell Immunol, 1993 Dec, 152(2), 614 - 22
Role of macrophage tissue factor in the development of the delayed hypersensitivity reaction in monkey skin; Imamura T et al.; Delayed-type hypersensitivity (DTH) reaction is a cell-mediated immune response, characterized by fibrin deposition . To determine whether macrophage tissue factor (TF), an initiator of blood coagulation, was associated with the DTH reaction, TF expression on macrophages and resulting fibrin deposition at the site of DTH, induced in Bacille-Calmette-Guerin (BCG)-sensitized Japanese monkeys with intradermal administration of pure protein derivative of tuberculin (PPD), were examined immunohistochemically using monoclonal antibodies (MoAbs) against TF and fibrin . Most mononuclear cells, but not polymorphonuclear cells, in DTH reaction sites were TF-positive and were identified as macrophages by double-immunostaining using anti-TF and anti-macrophage MoAbs . Fibrin deposition was only observed around TF-positive macrophages in the stroma of DTH reaction sites, and the deposition was not seen in the periphery of other TF-positive tissues (epidermis, trichoepithelium, and blood vessels) . The development of induration and the extent of fibrin deposition paralleled an increase of TF-positive macrophages in the DTH reaction sites . In PPD-injected skin sites of nonsensitized monkeys, neither TF-positive macrophages nor fibrin were seen but epidermis, trichoepithelium, and blood vessels were TF-positive . Moreover, the development of induration was inhibited by simultaneous administration of anti-TF MoAb and PPD into BCG-sensitized monkeys . These results suggest that TF expression on macrophages in the reaction site is a key factor for the DTH reaction, through the activation of blood coagulation, resulting in fibrin deposition.

J Bacteriol, 1993 Dec, 175(24), 8014 - 7
Listeria monocytogenes phosphatidylinositol (PI)-specific phospholipase C has low activity on glycosyl-PI-anchored proteins; Gandhi AJ et al.; The ability of the phosphatidylinositol-specific phospholipase C (PI-PLC) from Listeria monocytogenes to hydrolyze glycosyl phosphatidylinositol (GPI)-anchored membrane proteins was compared with the ability of the PI-PLC from Bacillus thuringiensis to hydrolyze such proteins . The L . monocytogenes enzyme produced no detectable release of acetylcholinesterase from bovine, sheep, and human erythrocytes . The cleavage of the GPI anchors of alkaline phosphatase from rat and rabbit kidney slices was less than 10% of the cleavage seen with the PI-PLC from B . thuringiensis . Activity for release of Fc gamma receptor IIIB (CD16) on human granulocytes was also low . Variations in pH and salt concentration had little effect on the release of GPI-anchored proteins . Our data show that L . monocytogenes PI-PLC has low activity on GPI-anchored proteins.

Br J Rheumatol, 1993 Dec, 32(12), 1096 - 8
Reiter's syndrome after intravesical Bacillus Calmette-Guérin treatment for superficial bladder carcinoma; Pancaldi P et al.; A 64-year-old woman developed acute Reiter's syndrome 4 weeks after the start of intravesical Bacillus Calmette-Guerin immunotherapy for a recurrent superficial bladder carcinoma . The absence of any other cause suggests that the bacillus played an etiopathogenic role in this HLA-B27-positive patient.

J Infect Dis, 1993 Dec, 168(6), 1553 - 8
Evidence of previous infection with Mycobacterium avium-Mycobacterium intracellulare complex among healthy subjects: an international study of dominant mycobacterial skin test reactions; von Reyn CF et al.; Skin tests with 0.1 mL of intermediate-strength Mycobacterium tuberculosis purified protein derivative (PPD) and 0.1 mL of Mycobacterium avium sensitin were conducted on 484 healthy subjects from diverse geographic sites . Reactions of > or = 5 mm to one antigen that exceeded the reaction to the other by > or = 3 mm were considered M . avium- or PPD-dominant . PPD-dominant reactions were more frequent at sites where routine Bacille Calmette-Guerin immunization is done or where there are high rates of tuberculosis: New Hampshire, 2%; Boston, 7%; Finland, 14%; Trinidad, 26%; and Kenya, 28% . However, rates of M . avium-dominant reactions ranged from 7% to 12% at all sites . Analysis of dominant reactions based on a more stringent 10-mm minimum reaction size showed similar trends . These data suggest that exposure to MAC is similar in developed and developing countries but that broad mycobacterial immunity is greater in developing countries and may contribute to the lower rates of disseminated MAC infections in AIDS in these areas.

J Urol, 1993 Dec, 150(6), 1932 - 7
Dissecting the immunobiological effects of Bacillus Calmette-Guérin (BCG) in vitro: evidence of a distinct BCG-activated killer (BAK) cell phenomenon; Bohle A et al.; Several immunobiological effects of intravesical bacillus Calmette-Guerin (BCG) during immunotherapy of superficial bladder cancer have been suggested as possible mediators of the mode of action . In an attempt to elucidate which of these effects is relevant to tumoricidal activity, an in vitro cytotoxicity assay was employed in which the direct effects of BCG and of cytokines against four transitional carcinoma cell lines were studied . Furthermore, peripheral blood mononuclear cells (PBMNC) were analyzed for their cytotoxic potential against these target cells . We found that none of the cytokines interleukin-1, interleukin-2, interferon-gamma, tumor necrosis factor alpha, lymphotoxin, or BCG alone were cytotoxic against the bladder carcinoma cell lines . However, a pronounced cytotoxicity against these targets resistant to natural killer cells could be induced in PBMNC by coincubation with viable BCG . We termed this the BCG-activated killer (BAK) cell phenomenon . In contrast to lymphokine-activated (LAK) cells, these BAK cells needed prolonged activation for 7 days and did not enhance the cytotoxicity against K562 target cells sensitive to natural killer cells . Nonviable, heat-inactivated BCG was significantly less effective, and sonificated fractions of BCG were not effective in stimulating PBMNC towards BAK cell activity . In vitro dissection of effects observed during BCG intravesical therapy may give more insight into the mode of action of BCG and may help to separate primary tumoricidal effector mechanisms from secondary concomitant phenomena . Further characterization of the BAK cell may result in an improvement of intravesical BCG immunotherapy.

Infect Immun, 1993 Dec, 61(12), 4962 - 71
Characterization of Bartonella bacilliformis flagella and effect of antiflagellin antibodies on invasion of human erythrocytes; Scherer DC et al.; Bartonella bacilliformis is the etiologic agent of Oroya fever in humans . Flagellum-mediated motility has been postulated as a major virulence factor for invasion of host cells . To address this hypothesis, we purified and characterized flagella from strain KC584 and then assessed their role in human erythrocyte association and invasion . Electron microscopy of the flagellar preparation showed a high concentration of filaments with a mean wavelength of 800 nm . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, and KBr density gradient centrifugation indicated that the flagellar filament is composed of a polypeptide of 42 kDa . The flagellin is partially (ca . 50%) resistant to treatment with trypsin . The first 17 amino acid residues of the N terminus of the mature flagellin protein are GAAILTNDNAMDALQDL and show approximately 46% sequence identity to the residues of the N termini of two Caulobacter crescentus flagellin proteins . A monospecific polyclonal antibodies to the flagellin protein was generated, and its specificity was verified by both immunoblot and immunogold analyses . Human erythrocyte invasion assays performed with bartonellae exposed to the antiflagellin antiserum showed a significant decrease in bacterial association with and invasion of human erythrocytes in comparison with that in bartonellae exposed to preimmune rabbit serum or phosphate-buffered saline (PBS) controls . These results suggest that flagella are an important component in the invasiveness of B . bacilliformis.

Microbiologia, 1993 Dec, 9(2), 142 - 8
Alpha-amylase production in lactose medium by Bacillus circulans ACB; Bandyopadhyay A et al.; Alpha-amylase production by Bacillus circulans ACB was studied in various cultural conditions . During nutrient optimisation, it was found that 2% lactose can be utilized by the strain as source of carbon providing better growth and enzyme yields than starch . Ammonium sulfate of the basal medium can be replaced by ammonium nitrate for better growth and alpha-amylase activity . The strain demonstrated significant enhancement in alpha-amylase production when grown at pH 6.6.

Int J Neurosci, 1993 Dec, 73(3-4), 287 - 98
Distribution of brain monoamines in left- and right-handed mice injected with bacillus Calmette-Guerin; Deleplanque B et al.; Concentrations of brain monoamines from various cerebral structures were determined in right and left sides of the brain from female mice selected for paw preference and injected or not with BCG 8 weeks before . BCG-induced changes in brain monoamine distribution in prefrontal cortex, medial hypothalamus and brain stem were only observed in right-handers . In the posterior hypothalamus, even though there was no BCG effect, norepinephrine asymmetry observed in right-handed controls was suppressed after BCG-injection . Moreover, BCG-induced brain monoamine changes in right-handers mainly involved the right hemisphere except the NE decrease in brain stem which was left-sided . This work demonstrates that the injection of BCG leads to long lasting asymmetrical changes in brain monoamine distribution that furthermore depend on behavioral lateralization of mice.

Trends Microbiol, 1993 Dec, 1(9), 333 - 8
Dynamics of Bacillus colony growth; Schindler J; A growing bacterial colony is not an amorphous mass but is a dynamic and differentiated system . It may appear chaotic in the details of its structure but it can be accurately described by simple models for fractal geometry . As Benoit Mandelbrot said: 'Surprise, simple rules can generate rich structures'.

Clin Investig, 1993 Dec, 72(1), 50 - 4
Bacillary angiomatosis in a German patient with AIDS; Schneider T et al.; A 52-year old male homosexual patient with acquired immunodeficiency syndrome (AIDS) presented in our clinic with multiple nodular papules (more than 100) spread over the whole body which had developed within 3 months . Bacillary angiomatosis was suspected, which is a bacterial infectious disease recognized recently mainly in patients with AIDS . Histological and immunohistochemical examinations of extirpated skin lesions were in agreement with the diagnosis, and the detection of rod-shaped bacteria in the lesions by Warthin-Starry silver stain confirmed it . The patient was treated with 2 x 100 mg doxycycline per day . The fever disappeared, and the cutaneous lesions showed a slight tendency to improve . However, after 5 days of therapy the patient showed increasing weakness, with muscle and bone pain . The patient died 10 days after the doxycycline therapy had been started . The cutaneous lesions in bacillary angiomatosis may resemble Kaposi's sarcoma and may therefore be misdiagnosed . The disease may be fatal, but timely antibiotic treatment is usually effective; therefore, the diagnosis of bacillary angiomatosis is important . Although many cases have been reported from the United States, only one case is known from Europe . Our finding of bacillary angiomatosis in a German AIDS patient supports the concept of a worldwide distribution of this bacterial agent.

Yonsei Med J, 1993 Dec, 34(4), 356 - 64
Production of tumor necrosis factor by intravesical administration of bacillus Calmette Guérin in patients with superficial bladder cancer; Kim CI et al.; Although an immune response to bacillus Calmette Guerin (BCG) has often been associated with antitumor activity, the action mechanism(s) of intravesical BCG therapy for prophylaxis and treatment of superficial bladder cancer is not clearly understood . In an attempt to evaluate the roles of tumor necrosis factor (TNF)-alpha and lymphotoxin (LT) in the antitumor activity, TNF-alpha productivities by peripheral blood monocytes, serum levels of TNF-alpha, and LT productivities by peripheral blood lymphocytes were studied in superficial bladder cancer patients after six intravesical administrations of BCG . TNF-alpha productivities by peritoneal macrophages of guinea pigs were also studied after six intravesical administrations of BCG . The maximum TNF-alpha productivities by peripheral blood monocytes of superficial bladder cancer patients were seen after the fourth week of administration of BCG, and the serum TNF-alpha levels were also slightly increased after intravesical BCG administration in the superficial bladder cancer patients . LT productivities by peripheral blood lymphocytes of superficial bladder cancer patients were significantly enhanced and the maximum LT productivity was also seen after the third or fifth BCG administration . TNF-alpha productivities by peritoneal macrophages of guinea pigs were significantly enhanced and the maximum TNF-alpha productivity was seen after the second or third BCG administration . Our data might suggest that six consecutive intravesical BCG administrations could induce the increased productions of TNF-alpha and LT, which might play an important role in the antitumor activity in superficial bladder cancer.

Ophthalmologe, 1993 Dec, 90(6), 754 - 64
{H2O2 low temperature plasma sterilization . New possibilities for use with eye surgery instruments}; Fortsch M et al.; The H2O2-low-temperature-plasma-sterilization (STERRAD 100) works with a temperature below 50 degrees C (140 degrees F) . This system is appliable for thermostabile materials as well as for thermolabile materials . The efficancy of this new system is shown by a biological test with Bacillus pumilus spores . 5 typical ophthalmic surgical instruments were contaminated . After sterilization the numerical reduction of the microorganisms had to be at least 6 log levels . Corrosion caused by hydrogene peroxide was excluded after exposing steal with a high quantity of this substrate . Electromicroscopy analysations of the surfaces of stainless steal after LTP, steam sterilization and hot-air sterilization are compared . Options and limitations of this new sterilization technique are discussed . A newly developed operating system with a complete instrumental box (OP-Set) will be introduced.

Ophthalmologe, 1993 Dec, 90(6), 726 - 36
{Endophthalmitis . Importance of microbiologic studies for therapy and prognosis}; Mino de Kaspar H et al.; The fast and precise identification of infectious agents and their characterization is of upmost importance for the diagnosis and therapy of infectious endophthalmitis . A preliminary estimate of the most probable predominant agents may improve the choice of an adequate antimicrobial therapy prior to definitive microbiological analysis in culture media . A prospective study was performed in 46 patients with endophthalmitis . During surgery mostly undiluted samples were inoculated in 10 different culture media . Microscopic investigation and laboratory processing were performed within the next 10-120 min . A presumptive classification based on immediate direct microscopy and a preliminary recommendation on treatment were achieved in 25 cases . Culture yielded positive results in 38 cases (83%) . In 8 cases the inoculates remained negative . Most of the positive cultures (92%) contained only gram-positive bacteria, including 10 cases of mixed gram-positive infections . In 2 samples bacteria and fungi were present, while 2 contained fungi only . Of 8 posttraumatic eyes, 4 were infected with Bacillus spp . The results show that specific therapeutic approaches are required . Direct inoculation of surgical culture media combined with immediate microscopic investigation permits a fast and precise microbiological diagnosis.

Hautarzt, 1993 Dec, 44(12), 803 - 7
{Bacillary epithelioid angiomatosis in advanced HIV infection}; Hettmannsperger U et al.; A patient with advanced HIV infection developed multiple angiomatous papules and nodules on the upper chest within a few days . At first sight the lesions resembled disseminated Kaposi's sarcoma; the differential diagnosis, however, included eruptive haemangiomas and pyogenic granulomas . Such distinct clinical characteristics as the collarette-like desquamation at the borders of the tumours led to the suspicion of bacillary epithelioid angiomatosis in HIV infection, which was then confirmed by histology and ultrastructural demonstration of bacillary colonies within the lesions . Under systemic antibiotic treatment, marked regression of the lesions was quickly observed within 1 week and complete regression occurred after 4 weeks . It is important to consider bacillary angiomatosis in HIV infection in the differential diagnosis of Kaposi's sarcoma, and it is a separate entity in the form of angioproliferation caused by bacteria.

Appl Biochem Biotechnol, 1993 Dec, 43(3), 163 - 76
Synthesis of cyclodextrin glucosyl transferase by Bacillus cereus for the production of cyclodextrins; Jamuna R et al.; A potent indigenous bacillus isolate identified as Bacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly . Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis . The organism had the highest specific growth rate (0.7 mu) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37 degrees C at 300 rpm, 1.5 vvm of agitation, and aeration . At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h . CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL . Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL) . This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing {1.75 kU/L/h}) . Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h-1 resulting in the productivity of 23.0 kU/L/h.

Pediatr Infect Dis J, 1993 Dec, 12(12), 993 - 7
Bacillus Calmette-Guérin infection after vaccination of human immunodeficiency virus-infected children; Besnard M et al.; The use of Mycobacterium bovis/Bacillus Calmette-Guerin (BCG) to vaccinate against tuberculosis remains controversial . The development of tuberculosis in human immunodeficiency virus (HIV)-infected children demands specific evaluation of the risk/benefit ratio of BCG vaccination in this situation . In our institution 9 of 68 HIV-infected children vaccinated with BCG before the diagnosis of HIV infection was suspected developed vaccine-related complications: 7 of these children had a large satellite adenopathy with or without skin fistulae, whereas the other 2 had disseminated BCG infection beyond the satellite ganglion (involvement of the spleen and mesenteric and mediastinal lymph nodes in one case and the liver and lungs in the other) . The children were vaccinated soon after birth; no particular problems were observed at that time, but complications appeared 3 to 35 months later . All but one of these children had a rapidly progressive form of HIV disease . The possibility of delayed local or disseminated BCG infection must be considered in analysis of the risk/benefit ratio of vaccination of HIV-infected children . The prognosis of HIV infection must be taken into account, even if the child is asymptomatic when vaccination is being considered.

Bioorg Med Chem, 1993 Dec, 1(6), 447 - 55
Synthesis and beta-lactamase inhibitory activity of 6-fluoropenicillanic acids; Danelon GO et al.; The benzyl 6-fluoro-penicillanate sulfides 4a, 6a, 7a; and sulfones 6c, 7d were synthesized . The conversion to their free acids 4b, 6b, 6d, 7b, 7e and potassium salts 7c, 7f are described . These acids and salt 7c were evaluated as beta-lactamase inhibitors using beta-lactamase I from Bacillus cereus . The data indicate that substitution of the 6 alpha-hydrogen by a 6 alpha-fluorine atom on 6 beta-bromopenicillanic acid (1), leads to loss of beta-lactamase inhibitory activity . In the case of the isomers 6 beta- and 6 alpha-fluoropenicillanic acids the 6 beta-enantiomer proved to be considerably more potent . Potassium salts of 6 beta-fluoropenicillanate sulfide and sulfone were unstable in solid state and in water solution . The fragmentation of the sulfone in two parts in water solution is consistent with the hydrolytic behavior to the penicillanic acid sulfone (2) with 0.5 N NaOH.

Rev Rhum Ed Fr, 1993 Dec, 60(12), 919 - 21
{Present status of Poncet's tuberculous rheumatism . A new case}; Beauvais C et al.; A case of noninfectious polyarthritis of over one year's duration with calcaneal enthesopathy in a patient with visceral tuberculosis is reported . This pattern, termed Poncet's disease, shares pathophysiologic mechanisms with Freund's complete adjuvant-induced arthritis . Future studies should include polymerase chain reaction studies to look for the tubercle bacillus in joint fluid or synovial biopsy specimens.

J Bacteriol, 1993 Dec, 175(24), 8049 - 52
Heat shock applied early in sporulation affects heat resistance of Bacillus megaterium spores; Sedlak M et al.; Cells of Bacillus megaterium 27 were challenged by a 30-min heat shock at 45 degrees C during various sporulation stages and then shifted back to a temperature permissive for sporulation (27 degrees C), at which they developed spores . Heat shock applied at 120 min after the end of the exponential phase induced synthesis of heat shock proteins (HSPs) in the sporangia and delayed the inactivation of spores at 85 degrees C . Several HSPs, mainly HSP 70, could be detected in the cytoplasm of these spores . An analogous HSP, the main HSP induced by increased temperature during growth, belongs to the GroEL group according to its N-terminal sequence . The identity of this protein was confirmed by Western blot (immunoblot) analysis with polyclonal antibodies against B . subtilis GroEL . Sporangia treated by heat shock immediately or 240 min after exponential phase also synthesized HSPs, but none of them could be detected in the spores in an appreciable amount . These spores showed only a slightly increased heat resistance.

Biosci Biotechnol Biochem, 1993 Dec, 57(12), 2039 - 42
Expression of an 87-kD-beta-1,3-glucanase of Bacillus circulans IAM1165 in Saccharomyces cerevisiae by low-temperature incubation; Nakajima H et al.; A DNA segment encoding a signal peptide from yeast invertase was fused in frame to bglH gene encoding 87-kD-beta-1,3-glucanase from Bacillus circulans IAM1165 and was expressed in the yeast Saccharomyces cerevisiae under the control of the GAL1 gene promoter . Yeast cells containing this fused gene produced active beta-1,3-glucanase in the medium after a long period of incubation at low temperature . The enzyme produced by yeast was heterogeneous in size, and larger than the enzyme produced by Escherichia coli.

Infect Immun, 1993 Dec, 61(12), 4947 - 54
Characterization of an avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis: reduced expression of flagellin and phospholipases; Zhang MY et al.; An avirulent pleiotropic mutant of the insect pathogen Bacillus thuringiensis subsp . gelechiae, isolated by Heierson et al . (A . Heierson, I . Siden, A . Kivaisi, and H . G . Boman, J . Bacteriol . 167:18-24, 1986) as a spontaneous phage-resistant mutant, was further characterized and found to lack the expression of phosphatidylcholine- and phosphatidylinositol-degrading phospholipase C, beta-lactamase, and flagellin because of the absence of corresponding mRNAs . The avirulent mutant was also found to be less efficient in killing insect cells in vitro than the wild type and to have altered behavior in vivo; wild-type B . thuringiensis does not circulate in the insect hemolymph after injection, whereas the avirulent mutant and nonpathogenic control bacteria remain in circulation . Flagella and motility may be important for virulence in the early stages of an infection; mutants with decreased motility appear less virulent when fed to Trichoplusia ni but not when injected . The 50% lethal doses of wild-type strain Bt13 and avirulent mutant strain Bt1302 were estimated to be 0.52 +/- 0.25 and 2,600 +/- 1,300 CFU per injected larva, respectively.

J Bacteriol, 1993 Dec, 175(24), 7951 - 7
Transcriptional regulation of the Bacillus thuringiensis subsp . thompsoni crystal protein gene operon; Brown KL; The two predominant polypeptides of the Bacillus thuringiensis subsp . thompsoni crystal are encoded by the cry40 and cry34 genes . These crystal protein genes are located in an operon . Western analysis (immunoblotting) demonstrated that the operon promoter activity was located in the region upstream of the cry40 gene . The Cry34 protein was expressed only when the upstream promoter region was present . The crystal protein genes are the only cistrons in the operon, and they are expressed during sporulation, with the highest transcript levels detected early in sporulation (1.5 to 3 h after the onset of sporulation) . Transcription initiates from two adjacent sites located 84 and 85 bases upstream of the cry40 translational start codon . The B . thuringiensis subsp . thompsoni crystal protein gene operon promoter aligned with other crystal protein gene promoters, which are activated from early to midsporulation and transcribed in vitro by the B . thuringiensis RNA polymerase E sigma 35.

Clin Exp Immunol, 1993 Dec, 94(3), 500 - 6
Identification of human T cell epitopes in the Mycobacterium leprae heat shock protein 70-kD antigen; Adams E et al.; In a number of pathogens, heat shock proteins (hsp) stimulate humoral and cellular immune responses despite significant sequence identity with host hsp . The 70-kD hsp of Mycobacterium leprae, which shares 47% identity with human hsp70 at the protein level, elicited a T cell response in most Myco . bovis (bacille Calmette-Guerin (BCG)) vaccinees as well as leprosy and tuberculosis patients and their contacts . In order to locate T cell epitopes, DNA fragments encoding portions of the 70-kD hsp were expressed in the vector pGEX-2T and tested for T cell reactivity in an in vitro proliferative assay . Cultures of peripheral blood mononuclear cells (PBMC) from BCG vaccinees indicated that the C-terminal half of the molecule contained multiple T cell epitopes, as the T cells from a majority of Myco . leprae hsp70-reactive individuals responded to C-344 . Lower proportions of patients with paucibacillary leprosy (36%) and tuberculosis patients (16%) responded to C-344 . The smaller C-142 fragment which includes the terminal 70 residues unique to Myco . leprae and is the target for the human antibody response elicited a cellular response in few patients and no vaccinees . In order to map T cell epitopes, two series of synthetic peptides encompassing the region 278-502 were prepared . Using overlapping 12mer and 20mer peptides, this region of the molecule was found to contain several potential T cell epitopes . The longer peptides gave a clearer indication of reactive sequences including regions of the molecule which were not identified with the 12mer peptides . Fine mapping of reactive peptide pools using the 12mer peptides identified two T cell epitopes . Although both were located in regions of the molecule shared with Myco . tuberculosis, one appeared to be cross-reactive with the equivalent human sequence, and thus has the potential to initiate autoimmune responses.

Biochemistry, 1993 Nov 30, 32(47), 12812 - 20
Ribosomal protein S17: characterization of the three-dimensional structure by 1H and 15N NMR; Golden BL et al.; The structure of ribosomal protein S17 from Bacillus stearothermophilus was investigated by two-dimensional homonuclear and heteronuclear magnetic resonance spectroscopy . The 1H and 15N chemical shift assignments are largely complete, and a preliminary structural characterization is presented . The protein consists of five beta-strands that form a single antiparallel beta-sheet with Greek-key topology . The beta-strands are connected by several extended loops, and two of these contain residue types that are frequently seen in the RNA-binding sites of proteins . Additionally, two point mutations that affect antibiotic resistance, translational fidelity, and ribosome assembly are located in these two regions of the protein . Since these potential RNA-binding sites are distributed over a large surface of the protein, it appears that the molecule may interact with several regions of 16S rRNA.

Biochemistry, 1993 Nov 30, 32(47), 12730 - 5
Threonine 246 at the active site of the L-lactate dehydrogenase of Bacillus stearothermophilus is important for catalysis but not for substrate binding; Sakowicz R et al.; Threonine 246 is an active site residue that is conserved in all known L-lactate dehydrogenase (LDH; EC 1.1.1.27) sequences . In order to investigate the role of Thr246 in Bacillus stearothermophilus LDH, this residue was altered by site-directed mutagenesis to valine, alanine, leucine, and serine, respectively . The effects of these mutations, as observed in both steady-state and single-turnover kinetic measurements with different substrates, demonstrated the importance for catalysis of a hydroxyl group in the 246 amino acid residue . In contrast, no significant contribution of the OH group of Thr246 to productive pyruvate binding was observed . Instead, it is proposed that the role of Thr246 may be to facilitate hydride transfer from the nicotinamide ring of the NADH cofactor to the pyruvate carbonyl group.

Biochemistry, 1993 Nov 23, 32(46), 12363 - 71
Structural characterization, membrane interaction, and specific assembly within phospholipid membranes of hydrophobic segments from Bacillus thuringiensis var . israelensis cytolytic toxin; Gazit E et al.; The Bacillus thuringiensis var . israelensis (Bti) cytolytic toxin is hypothesized to exert its toxic activity via pore formation in the cell membrane as a result of the aggregation of several monomers . To gain insight into the toxin's mode of action, 2 putative hydrophobic 22 amino acid peptides were synthesized and characterized spectroscopically and functionally . One peptide corresponded to the putative amphiphilic alpha-helical region (amino acids 110-131, termed helix-2), and the other to amino acids 50-71 (termed helix-1) {Ward, E . S., Ellar, D . J., & Chilcott, C . N . (1988) J . Mol . Biol . 202, 527-535} of the toxin . Circular dichroism spectroscopy revealed that both segments adopt high alpha-helical content in a hydrophobic environment, in agreement with previous models . To monitor peptide-lipid and peptide-peptide interactions, the peptides were labeled selectively with either 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD) (to serve as donor) or tetramethylrhodamine (to serve as an acceptor), at their N-terminal amino acids . Both segments bind strongly to small unilamellar vesicles, composed of zwitterionic phospholipids, with surface partition coefficients on the order of 10(4) M-1 . The shape of the binding isotherms indicates that helix-2 forms large aggregates within phospholipid membranes . Resonance energy transfer experiments demonstrated that the segments self-associate and interact with each other, but do not associate with unrelated membrane-bound peptides . Functional characterization demonstrated that helix-2 permeates phospholipid SUV with a potency similar to that of naturally occurring pore-forming peptides . Thus, the results support a role for helices-1 and -2 in the assembly and in the pore formation by Bti toxin.

Biochem Pharmacol, 1993 Nov 17, 46(10), 1861 - 3
Suppression of lipopolysaccharide-induced fulminant hepatitis and tumor necrosis factor production by bisbenzylisoquinoline alkaloids in bacillus Calmette-Guerin-treated mice; Kondo Y et al.; The bisbenzylisoquinoline (BBI) alkaloids chondocurine, cycleanine, tetrandrine and berbamine were tested for their capacity to suppress hepatic injury and production of tumor necrosis factor (TNF) induced by lipopolysaccharide (LPS) in mice primed with bacillus Calmette-Guerin (BCG) . When administered for three consecutive days before LPS injection, chondocurine, cycleanine and tetrandrine (10 mg/kg/day) strongly suppressed serum alanine aminotransferase (EC 2.6.1.1.) and aspartate aminotransferase (EC 2.6.1.2.); however, berbamine gave only slight protection . Chondocurine, cycleanine and tetrandrine but not berbamine significantly reduced the level of TNF which peaked 2 hr after LPS injection . This study shows that BBI alkaloids prevent BCG/LPS-induced hepatitis at least in part by suppressing TNF production.

FEMS Microbiol Lett, 1993 Nov 15, 114(1), 23 - 9
Identification of a cryptic gene associated with an insertion sequence not previously identified in Bacillus thuringiensis; Hodgman TC et al.; After screening several Bt strains with a cryII toxin probe, clones from two strains were found to contain a cryptic cryIIB gene associated with an insertion sequence element belonging to the IS2/IS3 family . The lack of expression of this gene appears to result from mutation of the upstream orf2 gene which has been shown to be necessary for cryII expression.

FEMS Microbiol Lett, 1993 Nov 15, 114(1), 17 - 22
Characterization of a Bacillus thuringiensis strain which is toxic to the housefly Musca domestica; Hodgman TC et al.; A Bacillus thuringiensis isolate has been discovered which is toxic to the common housefly (Musca domestica) as well as other Diptera and Lepidoptera . Crystal delta-endotoxins purified from this isolate killed 50% of Musca larvae at a concentration of 10.2 micrograms/ml, and beta-exotoxin was not detected . Sodium dodecyl polyacrylamide gel electrophoresis of the purified crystals revealed three protein species which were related to CryIA(b), CryIB and CryIIA toxins on the basis of immunoreactivity and amino-terminal sequence determination . Southern blot and DNA restriction analyses suggested that the strain has sequences related to one cryIA(b), one cryIIA, and two cryIIB genes.

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