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%