Bacillus

Biochem Biophys Res Commun, 1993 Nov 15, 196(3), 1406 - 13
Cholesterol oxidase: a potent insecticidal protein active against boll weevil larvae; Purcell JP et al.; The discovery of proteins that control insects is critical for the continued growth of the agricultural biotechnology industry . A highly efficacious protein that killed boll weevil (Anthonomus grandis grandis Boheman) larvae was discovered in Streptomyces culture filtrates . The protein was identified as cholesterol oxidase (E.C . 1.1.3.6) . Purified cholesterol oxidase was active against boll weevil larvae at a concentration (LC50 = 20.9 micrograms/ml) comparable to the bioactivity of Bacillus thuringiensis proteins against other insect pests . Histological studies demonstrated that cholesterol oxidase lysed the boll weevil midgut epithelium, suggesting that this is the primary mechanism of lethality.

FEBS Lett, 1993 Nov 15, 334(2), 247 - 9
Primary structure and catalytic properties of extracellular ribonuclease of Bacillus circulans; Dementiev AA et al.; A complete amino acid sequence of extracellular Bacillus circulans RNase was established and compared with a structure of B . amyloliquefaciens RNase . Gln15, Gly65 and Gln104 in B . amyloliquefaciens RNase were found to be replaced by Leu, Ala and Lys, respectively, in B . circulans RNase . Catalytic properties of B . circulans RNase were studied.

Biochim Biophys Acta, 1993 Nov 10, 1203(1), 85 - 92
The role of acidic amino-acid residues in catalytic and adsorptive sites of Bacillus cereus sphingomyelinase; Tomita M et al.; By the modification of acidic amino-acid residues with Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate), the activity of sphingomyelinase of Bacillus cereus was decreased by 80-90% . Also, the reduction of Cys residues in the sphingomyelinase molecule by dithiothreitol caused a drastic decrease in enzymatic activity, whereas the sphingomyelinase activity was not affected by treatment with p-chloromercuribenzenesulfonic acid . Actually, no inactivation of sphingomyelinase activity was observed after selective modification of basic amino-acid residues such as Lys, His and Arg, and of the uncharged amino-acid residues Ser and Thr . The treatment of the sphingomyelinase molecule with Woodward's reagent K or dithiothreitol also brought about the inhibition of the specific adsorption of sphingomyelinase toward intact erythrocyte membranes . However, the extent of inhibition in the enzyme adsorption, 20-50%, was less than that observed in the sphingomyelinase activity . These results suggest that acidic amino-acid residues, such as Asp and Glu, in the sphingomyelinase molecule are involved in the catalytic sites and the adsorptive sites . Apparently, the disruption of disulfide linkage in the sphingomyelinase molecule by dithiothreitol destabilized its structure, resulting in a drastic decrease in sphingomyelin-hydrolyzing activity and specific adsorption of sphingomyelinase towards erythrocyte membranes.

Orv Hetil, 1993 Nov 7, 134(45), 2487 - 90
{Bacillary angiomatosis}; Torok L et al.; In a 78 years old patient with chronic lymphoid leukemia, diabetes mellitus a cat scratch induced disseminated angiomatous papules were observed . In the lesions great number of bacilluses were observed with light -and electron microscope . As a result of antibiotic treatment the lesions regressed without trace . This opportunist infection resulting general symptoms as well, may be regarded as a cutaneous manifestation of immunodeficiency . The adequate antibiotic treatment depends on the exact diagnosis.

J Mol Biol, 1993 Nov 5, 234(1), 209 - 21
Mapping the stability determinants of bacterial tyrosyl transfer RNA synthetases by an experimental evolutionary approach; Guez-Ivanier V et al.; The tyrosyl-tRNA synthetases from Bacillus stearothermophilus (Bst-TyrTS) and Escherichia coli (Eco-TyrTS) are 56% identical in amino acid sequence . To map and characterize the set of interactions that makes Bst-TyrTS more stable than Eco-TyrTS, a family of nine hybrid proteins was constructed between the two enzymes . The N-terminal part of each hybrid came from Eco-TyrTS and the C-terminal part from Bst-TyrTS . The stability and activity of these hybrids were estimated by experiments of thermal inactivation and tRNA charging . For all the hybrids, the temperature of half-inactivation in 30 minutes was above 44 degrees C and the rate of charging was at least 40% that of Bst-TyrTS . In general, the temperature of half-inactivation increased and the rate of charging decreased monotonically when the number of residues coming from the more stable and less active Bst-TyrTS increased . As a result, the rate of charging decreased when the temperature of half-inactivation increased . These results show that the sequences and structures of the two enzymes can replace each other locally and still give a stable and active TyrTS, and that the greater stability of Bst-TyrTS is due to cumulative changes of residues scattered along the sequence . They suggest that Bst-TyrTS is more rigid than Eco-TyrTS at low temperature . The existence of a few exceptional hybrids, having stabilities or activities lower than those of the neighbouring hybrids, shows that compensatory changes of residues have occurred between the two sequences during evolution . These exceptions could be explained by the systematic identification of the couples of residues that are in contact in the Bst-TyrTS structure and become heterologous in some hybrids.

J Mol Biol, 1993 Nov 5, 234(1), 179 - 87
Crystal structure of phospholipase C from Bacillus cereus complexed with a substrate analog; Hansen S et al.; We report the first crystal structure of a complex between PLC from Bacillus cereus (PLCBc) and a competitive inhibitor that is an analog of the natural phospholipid substrate . The structure has been determined at 1.9 A resolution and refined to a final R-factor of 15.7% . The inhibitor binds with its phosphonyl group to the three Zn ions in the active site of the enzyme and is also involved in a hydrogen bonded network including several water molecules and amino acid side-chains which appear to help orient the substrate for productive binding . The interactions within this complex provide some important information regarding the mechanism of PLC-catalyzed hydrolysis of membrane phospholipids . A water molecule, located approximately apical to the diacylglycerol leaving group, seems to be the most likely candidate for the attacking nucleophile which initiates the reaction.

J Urol, 1993 Nov, 150(5 Pt 1), 1498 - 1500
Corticosteroid-associated fatal mycobacterial sepsis occurring 3 years after instillation of intravesical bacillus Calmette-Guerin; Izes JK et al.; A case is reported of systemic Mycobacterium bovis infection that occurred 3 years after uneventful instillation of intravesical bacillus Calmette-Guerin (BCG) and after several months of oral prednisone therapy . The literature on delayed BCG infection and the systemic persistence of BCG after intravesical instillation is reviewed . We propose that rarely a reservoir of dormant M . bovis may become established after intravesical therapy . Reactivation infection may later develop in a manner that parallels the natural history of secondary tuberculosis.

J Virol, 1993 Nov, 67(11), 6596 - 604
Phosphatidylcholine hydrolysis activates NF-kappa B and increases human immunodeficiency virus replication in human monocytes and T lymphocytes; Arenzana-Seisdedos F et al.; We have tested whether breakdown of phosphatidylcholine (PC) initiated by exogenous addition of a PC-specific phospholipase C (PC-PLC) from Bacillus cereus or by endogenous overexpression of PC-PLC induces functional activation of NF-kappa B and increases human immunodeficiency virus (HIV) enhancer activity . PC-PLC-activated hydrolysis of PC was found to induce bona fide p50/p65 NF-kappa B binding activity in three different cell lines of human or murine origin . No significant changes in the turnover of other cellular phospholipids were detected in PC-PLC-treated cells . Induction of NF-kappa B by PC-PLC did not depend on de novo synthesis of proteins or autocrine secretion of either tumor necrosis factor or interleukin 1 . In human monocytic and lymphoblastoid T-cell lines, induction of NF-kappa B by PC-PLC resulted in clear induction of luciferase expression vectors placed under the control of synthetic kappa B enhancers or wild type, but not kappa B-mutated, HIV long terminal repeat constructs . HIV replication was increased by PC-PLC in chronically infected monocytes and T lymphocytes . NF-kappa B activation promoted by addition of exogenous PC-PLC correlated with an intense production of diacylglycerol . However, addition of a phosphatidylinositol-specific PLC from B . cereus also induced diacylglycerol but did not activate kappa B enhancer-directed vectors . PC-PLC-induced NF-kappa B activation could not be blocked by a specific inhibitor of phorbol ester-inducible protein kinases C . These results indicate that a cellular transduction pathway, dependent on specific PC breakdown, is functional in T lymphocytes and monocytes and may be used by various transmembrane receptors to activate HIV transcription through NF-kappa B-dependent induction of the HIV enhancer.

Biull Eksp Biol Med, 1993 Nov, 116(11), 504 - 5
{The ulcerostatic effect of the exopolysaccharide from Bacillus mucilaginosus and its possible mechanisms}; Rasulov MM et al.; The stimulatory action of Bacillus mucilaginosus exopolysaccharide on proliferative processes in experimental gastric ulcer healing . With the drug, the levels of collagen, noncollagen proteins, glycosaminoglycans, DNA, and RNA in the ulcer tissue.

Protein Eng, 1993 Nov, 6(8), 907 - 11
Zinc ions bound to chimeric His4/lactate dehydrogenase facilitate decarboxylation of oxaloacetate; Carlsson H et al.; A chemically synthesized DNA linker coding for a peptide fragment that contains four histidines was fused in-frame to the 5'-end of the Bacillus stearothermophilus lactate dehydrogenase gene . The gene product, His4/lactate dehydrogenase, could be purified to homogeneity using either immobilized metal (Zn2+)-affinity chromatography or affinity chromatography on oxamate agarose . The stability against heat and urea for the modified enzymes was decreased as compared to the native lactate dehydrogenase but could be increased if zinc ions were present during the denaturation . In the presence of zinc ions the His4/lactate dehydrogenase could catalyse the sequential reaction from oxaloacetate to L-lactate, hence operating as a semi-synthetic bifunctional enzyme . A small increase in the apparent second-order rate constant (kcat/Km) of the coupled reaction was observed as compared to a corresponding system with native lactate dehydrogenase.

Curr Genet, 1993 Nov, 24(5), 400 - 7
Regional sequence homologies in starch-degrading enzymes; Janse BJ et al.; The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched (amylopectin) glucose polymers, is catalyzed by alpha-, beta- and glucoamylases (gamma-amylases), cyclodextrinases, alpha-glucosidases, and debranching enzymes . Saccharomyces cerevisiae cannot utilize starch . Our laboratory has previously co-expressed the Bacillus amyloliquefaciens alpha-amylase (AMY) and the Saccharomyces diastaticus glucoamylase (STA2) genes in S . cerevisiae . A gene encoding a debranching enzyme (pullulanase) from Klebsiella pneumoniae ATCC15050 was cloned and its nucleotide sequence determined . This gene will be co-expressed with the alpha- and gamma-amylase to produce an amylolytic S . cerevisiae strain . Extensive data base comparisons of the K . pneumoniae pullulanase amino-acid sequence with the amino-acid sequences of other debranching enzymes and alpha-, beta- and gamma-amylases (from bacteria, yeasts, higher fungi and higher eukaryotes), indicated that these debranching enzymes have amino-acid regions similar to those found in alpha-amylases . The conserved regions in alpha-amylases comprise key residues that are implicated in substrate binding, catalysis, and calcium binding and are as follows . Region 1: DVVINH; region 2: GFRLDAAKH and region 4: FVDNHD . When comparing conserved regions, no similarity could be detected between debranching enzymes and beta- and gamma-amylases.

Anticancer Res, 1993 Nov-Dec, 13(6A), 2069 - 75
Effect of tumour growth on the macrophage response to the antitumour agent 5,6-dimethylxanthenone-4-acetic acid; Ching LM et al.; Peritoneal macrophages from C3H/HeN mice bearing subcutaneous M-16/C or Spon-2 mammary carcinomas had enhanced tumouricidal activity over control macrophages from non-tumour bearers in response to 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA), a novel antitumour agent which has been scheduled for clinical evaluation . The effect of a palpable M-16/C tumour growing in C3H/HeN mice was similar to that of Bacillus Calmette-Guerin (BCG) infection, with macrophages being fully activated and tumouricidal without any further stimulus being required in culture . Macrophages from Spon-2 tumour bearing mice behaved like "primed" thioglycollate-elicited macrophages and produced a tumouricidal response to 5,6-MeXXA which was significantly higher than that obtained from resident peritoneal macrophages from non-tumour bearing mice . Resident and thioglycollate-elicited macrophages from C3H/HeJ mice were hyporesponsive not only to lipopolysaccharide (LPS), but to 5,6-MeXAA as well . Hyporesponsiveness was abrogated by BCG infection or by the presence of the M-16/C tumour, but not by the presence of the Spon-2 tumour . In response to LPS at low concentrations, or to 5,6-MeXAA at all concentrations, tumouricidal activity from macrophages from Spon-2-bearing C3H/HeJ mice was severely depressed compared with activity from their C3H/HeN counterparts . However, 5,6-MeXAA induced similar levels of haemorrhagic necrosis of tumours implanted in either C3H/HeJ or C3H/HeN hosts . LPS-induced haemorrhagic necrosis was significantly lower in C3H/HeJ than in C3H/HeN hosts . The results show that the presence of subcutaneous tumours modulates the activity of peritoneal macrophages in mice.

Trans R Soc Trop Med Hyg, 1993 Nov-Dec, 87(6), 644 - 5
Epidemiology of Buruli ulcer in Amansie West district, Ghana; Amofah GK et al.; This paper describes 90 cases of Buruli ulcer in Amansie West district, Ghana . 49% were below 15 years of age while 20% were over 50 years . There was a significant difference in the age and sex composition, with more males among the younger age groups than females but the converse among adults . Seasonal variation is described, with peak incidence in September and October . Cases who had received bacillus Calmette-Guerin (BCG) vaccination had a shorter duration of the ulcer than those who were not vaccinated . No such association was found between BCG vaccination and the age of onset of the disease . There was no significant difference between cases and controls regarding their BCG vaccination status . There is an urgent need to regard Buruli ulcer in Ghana more seriously.

J Dairy Res, 1993 Nov, 60(4), 575 - 80
Influence of pH and sugars on the growth and production of diarrhoeagenic toxin by Bacillus cereus; Sutherland AD et al.; Broth cultures supplemented with high levels of sugars, particularly glucose at > 50 milligrams, did not support diarrhoeagenic toxin production by psychrotrophic Bacillus cereus despite growth to high counts (approximately 10(7)/ml) over a 4 d period of incubation at 21 degrees C . In contrast, starch levels of 10 and 50 milligrams actually enhanced toxin production . Toxin production was also affected by pH levels of broth cultures, and was concomitant with alterations in bacterial growth . These findings help to explain variations in toxin levels previously found in some dairy desserts, which were thought to be associated with pH and sugar content (Sutherland, 1993).

J Dairy Res, 1993 Nov, 60(4), 569 - 74
Toxin production by Bacillus cereus in dairy products; Sutherland AD; Spores of a known toxigenic and psychrotrophic dairy isolate of Bacillus cereus (HRM 44) were unable to grow and produce diarrhoeagenic toxin at 6 degrees C in creams and dairy-based products . These findings suggest that the production of B . cereus diarrhoeagenic toxin is unlikely to occur in creams and dairy-based products maintained within the cold chain . Growth and toxin production were readily demonstrated in creams and some desserts stored at 21 degrees C . Growth in creams was associated with obvious spoilage . However, in the flavoured desserts, spoilage was not always obvious before significant growth of B . cereus and toxin production had occurred . Dairy desserts with high sugar content and/or low pH did not support toxin production and these findings are discussed.

Int J Biochem, 1993 Nov, 25(11), 1625 - 9
Membrane-bound 5'-nucleotidase/nucleoside phosphotransferase from Bacillus cereus; Baiocchi C et al.; 1 . A search for nucleoside phosphotransferase activity in Bacillus cereus led to the following results: (i) The phosphotransferase activity was associated with a membrane bound 5'-nucleotidase . (ii) The enzyme phosphorylates both purine and pyrimidine nucleosides as well as 2',3'-dideoxyinosine . (iii) The enzyme was inhibited by adenylic nucleotide di- and triphosphates, and its nucleotidase activity was increased in the presence of inosine as phosphate acceptor . 2 . Bacterial and vertebrate 5'-nucleotidases with phosphotransferase activity differ for several characteristics, such as cellular location, substrate specificity, magnesium requirement and regulation.

Ann Pharmacother, 1993 Nov, 27(11), 1378 - 82
Bacillary angiomatosis in a patient with AIDS; Teague AC et al.; OBJECTIVE: To report the clinical presentation and response to antimicrobial therapy of presumed bacillary angiomatosis in an AIDS patient . DESIGN: Single case report . SETTING: A 1058-bed, university teaching hospital . PATIENT: 28-year-old HIV-positive man (T4 lymphocyte count < 3/mm3), who was diagnosed with AIDS in 1984 . RESULTS: The skin lesions responded promptly to treatment with doxycycline and erythromycin . CONCLUSIONS: Bacillary angiomatosis is an infection that occurs with endstage AIDS . Skin lesions have recognizable characteristics and respond promptly to appropriate antibiotic therapy.

Bioorg Khim, 1993 Nov, 19(11), 1065 - 72
{Primary structure and catalytic properties of extracellular ribonuclease from Bacillus circulans}; Dement'ev AA et al.; A comparative research of individual peptide structures obtained after hydrolysing of Bacillus circulans and B . amyloliquefaciens RNases by the Glu-specific staphylococcal protease was carried out by means of mass-spectrometry and Edman degradation methods . A complete amino acid sequence of B . circulans RNase was determined . Gln15, Gly65 and Gln104 residues in B . amyloliquefaciens RNase were found to be substituted by Leu, Ala and Lys residues in B . circulans RNase, respectively . Catalytic properties of the B . circulans RNase in transesterification reactions with poly- and oligonucleotides as substrates were investigated.

Appl Environ Microbiol, 1993 Nov, 59(11), 3928 - 30
Role of the CryIVD polypeptide in the overall toxicity of Bacillus thuringiensis subsp . israelensis; Poncet S et al.; The gene encoding the CryIVD protein of B . thuringiensis subsp . israelensis crystals was disrupted by in vivo recombination . The toxicity of the CryIVD protein-free inclusions was similar to that of the wild-type crystals on Anopheles stephensi larvae but was half the wild-type toxicity on Culex pipiens and Aedes aegypti larvae.

Appl Environ Microbiol, 1993 Nov, 59(11), 3889 - 93
A soluble Bacillus cereus cytochrome P-450cin system catalyzes 1,4-cineole hydroxylations; Liu W et al.; A cytochrome P-450-dependent monooxygenase system that catalyzes the stereospecific hydroxylation of the monoterpene substrate 1,4-cineole was demonstrated in cell-free preparations of Bacillus cereus UI-1477 . 1,4-Cineole hydroxylations were catalyzed by a 100,000 x g (1-h)-centrifuging soluble, hexane-inducible enzyme that activated and incorporated molecular oxygen into hydroxylated products; required NADH; was inhibited by SKF-525A, imidazole, metyrapone, and octylamine; and displayed a 452-nm peak in the carbon monoxide difference absorption spectrum . The constant 7:1 ratio of endo/exo alcohol products formed when 1,4-cineole was hydroxylated by normal cells, hexane-induced cells, and cell extracts suggested that a single enzyme designated cytochrome P-450cin was responsible for both reactions.

Br J Urol, 1993 Nov, 72(5 Pt 2), 744 - 8
Intravesical Bacillus Calmette-Guérin with the Danish strain for treatment of carcinoma in situ of the bladder; Ovesen H et al.; We report our experience of the treatment of carcinoma in situ (CIS) using intravesical therapy with the Danish Bacillus Calmette-Guerin (BCG) strain 331 (SSI) . Forty-two patients received treatment, 11 had primary and 31 secondary CIS . The median follow-up period was 26 months (range 3-68) . Patients received 6 weekly instillations (1 course) and non-responders an additional 6 instillations at 2-week intervals (2 courses) . The complete response rate was 59% for 1-course patients, 33% for the 2-course patients and 68% for the entire series . Patients were considered treatment failures if they suffered progression to invasive cancer, metastasis or died from transitional cell carcinoma . BCG treatment was more effective in primary than in secondary CIS, with a complete response rate of 80% versus 65% and with no failures versus 35% . Patients with persistent CIS after the first course of BCG had a greater risk of failure than responders: 50% versus 17% . Patients with persistent CIS after the second course had a 75% failure rate . This suggests that cystectomy should be considered for non-responders following a 6-week course and recommended to those not responding to 2 courses . Ten patients had CIS in the prostatic urethra . All responded to BCG treatment; 2 suffered from recurrent CIS 1 associated with invasive urethral tumour . The incidence and severity of side effects were similar to those reported with other strains of BCG . One patient with primary CIS failed to complete the treatment owing to "BCG-itis".

Proteins, 1993 Nov, 17(3), 325 - 8
Crystallization and preliminary X-ray investigation of barstar, the intracellular inhibitor of barnase; Guillet V et al.; Crystals of barstar, the intracellular inhibitor of the extracellular ribonuclease produced by Bacillus amyloliquefaciens (barnase), were obtained through vapor phase equilibration using the hanging drop technique . Three crystal forms have been characterized . Forms I and II, crystallized either in potassium phosphate or sodium citrate, are tetragonal; they exhibit a superstructure along the c-axis . Form III crystals, suitable for a high resolution structure determination, were grown from 55-65% ammonium sulfate . This crystal form is hexagonal and diffracts to at least 2 A resolution at a synchrotron radiation source . It belongs to the hexagonal space group P6, with unit cell dimensions a = b = 143.6 A, c = 35.6 A . There are four molecules of barstar in the asymmetric unit . X-ray data have been collected to 2.2 A Bragg spacing . The structure determination is underway in order to analyze conformational changes of barstar upon complexation with barnase.

J Dermatol Surg Oncol, 1993 Nov, 19(11), 985 - 90
Pooled analysis of the efficacy of bacille Calmette-Guerin (BCG) immunotherapy in malignant melanoma; Tan JK et al.; BACKGROUND . The trials of bacille Calmette-Guerin (BCG) as adjuvant therapy in malignant melanoma conducted over the preceding 2 decades have presented conflicting claims of efficacy . OBJECTIVE . Determination of the role of BCG immunotherapy in malignant melanoma . METHODS . Critical analysis of randomized clinical trials of stage I and II melanoma and all reported trials of intralesional and oral BCG in stage III melanoma was conducted . A literature search used the Medline data base (1966-1992);bibliographic reviews of relevant texts and pertinent articles . RESULTS . No significant benefit of BCG as postsurgical adjuvant therapy in stage I malignant melanoma was observed . Although two of seven trials in stage II melanoma demonstrated benefit with the addition of BCG, the trial with the greatest power in this series detected no difference in outcomes . In stage III malignant melanoma, there was no significant benefit with addition of BCG to various chemotherapeutic regimens . Oral BCG monotherapy produced complete responses in 6%, partial responses in 1%, and extended survival in 7% of patients . Objective responses were observed primarily in patients with intracutaneous non-visceral metastases . Pooled analysis of 15 non-controlled trials of intralesional BCG injections revealed complete responses in 19%, partial responses in 26%, and extended survival in 13% of patients with stage III melanoma . Objective responses to intralesional BCG were more likely in patients with solely cutaneous metastases, particularly intradermal lesions . CONCLUSION . Pooled analysis of non-placebo controlled trials of intralesional BCG for stage III malignant melanoma supports a trend to enhanced survival in patients with cutaneous non-visceral metastases.

Cell Immunol, 1993 Nov, 152(1), 72 - 81
Activation of murine lymphocytes by exogenous phosphatidylethanolamine- and phosphatidylcholine-specific phospholipase C; Isakov N; Enzymes of the protein kinase C (PKC) family have a decisive role in the activation process of T cells . Under physiological conditions, PKC undergoes activation by diacylglycerol (DAG) which is transiently produced following activation of an antigen receptor-coupled phosphatidylinositol 4,5-bisphosphate (PIP2)-specific phospholipase C (PLC) and hydrolysis of PIP2 . In vitro activation of distinct PKC isoenzymes is differentially regulated by various synthetic DAGs and membrane phospholipids . We tested whether exogenous PLC obtained from Bacillus cereus can affect PKC-dependent functions in mouse lymphocytes . The results indicated that B . cereus PLC, which preferentially hydrolyzes phosphatidylethanolamine (PE) and phosphatidylcholine (PC), induces the expression of functional cell surface IL2 receptors and that, in the presence of exogenous IL2, it leads to cell proliferation . The PE/PC-PLC reversed the inhibitory effect of PC on enzymatic activity of T cell-derived PKC in vitro . Furthermore, it augmented the activity of PKC over the background level suggesting that the PC-derived DAG may function as a positive regulator of T cell-specific PKC isoenzymes.

J Leukoc Biol, 1993 Nov, 54(5), 439 - 43
Particle-induced in vivo priming of alveolar macrophages for enhanced oxidative responses: a novel system of cellular immune augmentation; Myrvik QN et al.; A novel system for priming adult rabbit alveolar macrophages (AMs) in vivo for markedly enhanced oxidative responses is described . When adult rabbits were injected intravenously (i.v.) with 1- to 5-microns particles such as zymosan, latex particles, or heat-killed bacille Calmette-Guerin, AMs were primed in 1-3 days for greatly enhanced phorbol myristate acetate (PMA)- or opsonized zymosan (Op-zym)-elicited chemiluminescent (CL) responses . Intratracheal (i.t.) injection of zymosan particles also primed AMs for enhanced PMA- or Op-zym-elicited CL responses . AMs obtained from particle-injected rabbits showed up to 100-fold higher levels of PMA-elicited CL responses than AMs from normal rabbits . In contrast, Op-zym failed to prime normal AMs in vitro for enhanced CL responses . Whereas AMs could not be primed in vivo with an i.v . injection of particles of approximately 24 microns diameter . AMs could be primed if the particles were administered by the i.t . route . The priming appears to be independent of particle types . The priming effect was of short duration and declined after 5 to 7 days . The possibility that this system represents the primitive cellular immune response found in invertebrates is discussed . The potential use of this system as a means of immune augmentation prompts further investigation.

J Bacteriol, 1993 Nov, 175(22), 7383 - 90
Functional domains of the penicillinase repressor of Bacillus licheniformis; Wittman V et al.; The penicillinase repressor (PENI) negatively regulates expression of the penicillinase gene (penP) in Bacillus licheniformis by binding to its operators located within the promoter region of penP.penI codes for a protein with 128 amino acids . Filter-binding analyses suggest that the active form of the repressor is a dimer . Genetic analyses of PENI derivatives showed that the repressor carrying either a 6-amino-acid deletion near the N terminus or a 14-amino-acid deletion at the C terminus was functionally inactive in vivo . A repressor derivative carrying a 6-amino-acid deletion within its N-terminal region was extensively purified and used in DNA footprinting and subunit cross-linking analyses . The results of these studies showed that the repressor derivative had lost its ability to bind operator specifically even though it could dimerize effectively . In similar studies, we demonstrated that an N-terminal portion of PENI with a molecular mass of 10 kDa derived by digestion with papain was able to bind operator specifically but with reduced affinity and had completely lost its ability to dimerize . These data suggest that the repressor has two functional and separable domains . The amino-terminal domain of the repressor is responsible for operator recognition, and the carboxyl-terminal domain is involved in subunit dimerization.

Eur J Biochem, 1993 Nov 1, 217(3), 947 - 53
Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases; Schou C et al.; The catalytic activity of nine enzymes (endoglucanases I-III, V, VI and cellobiohydrolases I and II from Humicola insolens; endoglucanases A and C from Bacillus lautus), representative of cellulase families A-C, H, J and K, has been investigated using a series of reduced cellooligosaccharides (cellotriitol to cellohexaitol) as substrates . For each enzyme, the specificity of cleavage was determined by analytical HPLC while the kinetic constants were obtained from a kinetic assay involving a cellobiose dehydrogenase purified from H . insolens as a coupled enzyme using 2,6-dichloroindophenol as the electron acceptor . These data were used to estimate the number of subsites in the enzymes . The stereochemical course of hydrolysis by seven enzymes, representing the six different families, was assessed using 1H-NMR . The enzymes belonging to families which had already been investigated (A-C), showed results in agreement with previous studies . The three other families (H, J and K), for which no mechanistic data was previously available, gave results which indicated that enzymes in group H had retaining-type activity and enzymes in groups J and K had inverting-type activity . The retaining endoglucanases I and III displayed a high glycosyl-transferase activity under the conditions used during the NMR experiments resulting in precipitates of higher oligomers.

Clin Exp Immunol, 1993 Nov, 94(2), 322 - 9
Distinct H-2 complex control of mortality, and immune responses to tuberculosis infection in virgin and BCG-vaccinated mice; Apt AS et al.; We have studied the impact of distinct haplotypes and of different alleles at specific H-2 loci on: (i) the susceptibility to lethal form of experimental tuberculosis; (ii) the level of DTH to mycobacterial antigens; (iii) the efficacy of vaccination with bacille Calmette-Guerin (BCG); and (iv) the IgG production and T cell proliferative response to H37Rv antigens . On the basis of median survival time (MST) following primary inoculation with lethal dose of Mycobacterium tuberculosis, susceptibility to infection associated with I-Ab and Db alleles, host resistance associated with I-Ak and Dd alleles . Mice bearing a disease-resistant phenotype also developed a vigorous DTH response . Vaccination with BCG before H37Rv infection significantly prolonged the survival time of both resistant and susceptible animals, except in B10.M (H-2f) mice . The latter exhibited intermediate resistance to infection before but slight decrease in the MST following a high-dose BCG vaccination . Distinct H-2 regulation of susceptibility to lethal infection and of BCG vaccination efficacy was confirmed in another relatively resistant H-2f-bearing strain A.CA, in which mortality occurred more rapidly in vaccinated compared with primarily infected animals . The expression of the H-2f haplotype was associated with a low DTH response to tuberculin following vaccination and subsequent lethal infection . The lack of BCG protection against Myco, tuberculosis challenge in B10.M mice associated with the high titre of specific IgG . In addition, these mice exhibited a unique ability to respond to 65-kD antigen by both IgG synthesis and T cell proliferation.

Arch Biochem Biophys, 1993 Nov 1, 306(2), 342 - 9
Manganese(II) activation of 3-phosphoglycerate mutase of Bacillus megaterium: pH-sensitive interconversion of active and inactive forms; Kuhn NJ et al.; The effects of manganese(II) ions and of pH were studied on 3-p-glycerate mutase purified from Bacillus megaterium . Mn2+ ions converted the enzyme within a few minutes from a catalytically inactive form to one that was catalytically active even after Mn2+ had been removed . The enzyme reverted over 60-90 min to the inactive form, from which further activation-deactivation cycles could be elicited . The slow, temperature-dependent, activation, and deactivation is suggestive of change in protein conformation . No other metal ion was found that activated more than 4% as much as Mn2+ . Activation by Mn2+ was strongly pH-dependent in the physiological pH range, consistent with displacement of 2 H+ . Together with the pH dependence of the catalytic activity itself, the system displayed pronounced pH sensitivity in the pH range 6.5-8.0 . The findings suggest that pH changes, documented for forming and germinating spores of B . megaterium, can account for much of the mutase control associated with accumulation and later utilization of 3-phosphoglycerate depots.

Virology, 1993 Nov, 197(1), 445 - 8
Mapping the 5'-terminus of rice tungro bacilliform viral genomic RNA; Bao Y et al.; The initiation site of the major transcript of rice tungro bacilliform virus (RTBV) has been located by mapping the 5' end of the RNA to nucleotides 7404 and 7405 of the RTBV genome using an RNase protection method . This was confirmed by the 5' RACE PCR procedure which mapped the 5' end of the RNA to nucleotide 7405 . These results are consistent with data from our analysis of the strong-stop DNA of RTBV . A eukaryotic RNA polymerase II promoter sequence (TATATAA) was located at nucleotide 7373 of RTBV genome which is 31-32 nucleotides upstream from the proposed initiation site of the RTBV transcript.

C R Acad Sci III, 1993 Nov, 316(11), 1355 - 62
{Mycobacterium leprae: behavior in the adipocyte undergoing differentiation}; Godard CM; We have investigated the behaviour of M . leprae in murine preadipocyte cells (clone Ob17) undergoing the adipose cell conversion process in vitro . Actively differentiating Ob17 cells were infected with M . leprae . The morphological index (MI) of the acid-fast bacteria (AFB) present at day 12 and day 25 after infection was compared to the MI of the AFB inoculated . An increase of the MI was consistently observed . This increase is suppressed by rifampicin . Due to important cell loss, an increase of the number of the AFB per culture could not be obtained in monolayer tissue cultures . In order to prevent cell loss, we used a three-dimensional culture system . This cell culture system is an in vitro reconstitution of the human dermis, a main target organ for the leprosy bacillus . Adipocytes infected with M . leprae are incorporated in a condensed collagen lattice together with skin fibroblasts . Under such conditions, both an increase of the MI and an increase of the number of the AFB are obtained . This suggests that cellular functions related to the adipose cell differentiation process might complement the defective bacterial genome, leading to transient multiplication in vitro.

Mikrobiol Z, 1993 Nov-Dec, 55(6), 46 - 50
{The nature of the interrelations of different Bacillus thuringiensis serotypes between themselves and other species of the genus Bacillus}; Vasilevskaia IA et al.; Intraspecies interrelations between representatives of 7 serotypes of Bacillus thuringiensis, as well as antagonistic interrelations between B . thuringiensis and 130 strains of 16 species of Bacillus bacteria were studied . Antagonistic activity was determined for 18 strains, representatives of genus Bacillus, with respect to B . thuringiensis var . tuviensis . Antagonism was the most pronounced between the strains of the alesti serotype and representatives of other serotypes as well as between the strains V . thuringiensis var . tuviensis and most of other bacillus species . Strain B . thuringiensis var . tuviensis is slightly sensitive to the action of toxic metabolites from strains of this and other species of genus Bacillus.

Rev Inst Med Trop Sao Paulo, 1993 Nov-Dec, 35(6), 485 - 90
{Geographic distribution of Lutzomyia verrucarum (Townsend, 1913) (Diptera, Psychodidae, Phlebotominae), vector of human bartonellosis in Peru}; Caceres AG; Lutzomyia verrucarum (Townsend, 1913) (Diptera: Psychodidae); the natural vector of Bartonella bacilliformis, agent of human bartonellosis (peruvian verruga or Carrion's disease), is a native species of Peru; its geographic distribution occurrs between latitudes 5 degrees and 13 degrees 25' South: in the Occidental and Interandean valleys of the Andean . The altitudinal distribution of Lu . verrucarum in the different valleys is as follows: Occidental between 1100 and 2980 m sea level and Interandean from 1200 to 3200 m sea level . Some discrepancies between the distribution of Carrion's disease and Lu . verrucarum suggest the existence of secondary vectors in certain areas where Lu . verrucarum is not present.

J Appl Bacteriol, 1993 Nov, 75(5), 416 - 9
Extracellular serine protease synthesis by mosquito-pathogenic strains of Bacillus sphaericus; Dumusois C et al.; Nine strains of Bacillus sphaericus toxic to mosquito larvae produced haloes of hydrolysis when cultured on casein-nutrient agar . In batch culture, protease synthesis by B . sphaericus BSE 18 occurred during exponential growth and was repressed by high concentrations of peptone . Extracellular protease from this bacterium showed optimal activity at about pH 10.2, was inhibited by phenylmethylsulphonyl chloride and chymostatin but not soybean trypsin inhibitor or EDTA . Hydrolysis of N-CBZ-glycine-nitrophenyl ester was consistent with the major enzyme being a serine protease.

Int J Biochem, 1993 Nov, 25(11), 1615 - 23
Alkaline phosphodiesterase I release from eucaryotic plasma membranes by phosphatidylinositol-specific phospholipase C . III . The release from tumor cells; Nakabayashi T et al.; 1 . Alkaline phosphodiesterase I release from two tumor cell lines, KB III or AH-130 cells, by the action of phosphatidylinositol-specific phospholipase C (PIPLC) of Bacillus thuringiensis was studied . 2 . A significant amount of alkaline phosphodiesterase I was released from both the cell suspension and homogenate of KB III cells, but not from AH-130 cells . 3 . The release of the enzyme from KB III cells was dependent on, or proportional to, the reaction time and the PIPLC or cell concentrations . 4 . Alkaline phosphatase and 5'-nucleotidase were also released from KB III cells, while gamma-glutamyl transpeptidase and dipeptidyl peptidase IV were not solubilized . The enzyme release by the action of PIPLC was suppressed when purified anti-PIPLC antibody was added to the reaction mixture . This suggests that the enzyme release must be due to the direct action of PIPLC on KB III cells . 5 . The alkaline phosphodiesterase I released from KB III cells had a mol . wt of 240,000 and was activated by Mg2+, but strongly inhibited by EDTA and thiol reagents and by 5'-nucleotide-containing compounds . Although KB III cells were derived from Homo sapiens tumor, the released alkaline phosphodiesterase I appeared to be very similar to enzymes obtained from normal tissues of Rattus norvegicus.

J Gen Microbiol, 1993 Nov, 139 ( Pt 11), 2849 - 54
Diversity of Bacillus thuringiensis environmental isolates showing larvicidal activity specific for mosquitoes; Ishii T et al.; Seven mosquito-specific strains of Bacillus thuringiensis isolated in Japan were examined for their flagellar (H) antigenicities and the properties of parasporal inclusion proteins . They were assigned to six H serovars: serovar fukuokaensis (H 3ade); serovar canadensis (H 5ac); serovar darmstadiensis (H 10); serovar kyushuensis (H 11ac); serovar shandongiensis (H 22); and an undescribed serovar belonging to H serotype 20 . Purified parasporal inclusions exhibited moderate mosquito larvicidal activities with LC50 values ranging from 1 microgram ml-1 to 10 micrograms ml-1 . The inclusions of these strains consisted of highly heterogeneous multiple protein components, and five distinct patterns were evident in their SDS-PAGE profiles . Antibodies against kyushuensis inclusion proteins were reactive with all strains to varying degrees, while israelensis antibodies gave only relatively weak reactions with the seven strains . Similarity in antibody-binding profiles was associated with similarity in SDS-PAGE profiles . In a given strain, different antisera gave altered immunoblot profiles . Haemolytic activity was shown by solubilized parasporal inclusion proteins of five of the seven strains.

Mol Gen Genet, 1993 Nov, 241(3-4), 380 - 8
Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwlL, of Bacillus licheniformis; Oda Y et al.; We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli . Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B . licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38,994 . The enzyme purified from the E . coli clone is an N-acetylmuramoyl-L-alanine amidase, which has a M(r) value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B . licheniformis, B . subtilis and Micrococcus luteus cell walls . The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B . licheniformis MC14 . Moreover, the amino acid sequence homology of CwlL with the B . subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL . The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.

S Afr Med J, 1993 Nov, 83(11), 855 - 6
Bacillary angiomatosis . The first case reported in South Africa; Levy GR et al.; A 28-year-old white man, positive for HIV, who was admitted to hospital for pneumonia, had for 2 months had several fluctuating cutaneous purple nodules on his legs and abdomen . Cultures of two lesions were negative, but light and electron microscopy showed organisms characteristic of bacillary angiomatosis . The patient responded well to therapy with erythromycin . This is the first reported case of bacillary angiomatosis in South Africa.

Biosci Biotechnol Biochem, 1993 Nov, 57(11), 1935 - 7
Molecular cloning and sequencing of the gene for a thermostable N-carbamyl-L-amino acid amidohydrolase from Bacillus stearothermophilus strain NS1122A; Mukohara Y et al.; The gene for N-carbamyl-L-amino acid amidohydrolase was cloned from Bacillus stearothermophilus strain NS1122A into E . coli . This gene started with a TTG triplet and was predicted to encode a peptide of 409 amino acids, with a calculated molecular weight of 44,248 . The deduced amino acid sequence shared moderate homology with that of the corresponding enzyme of Pseudomonas sp . strain NS671.

J Bacteriol, 1993 Nov, 175(21), 6760 - 6
Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus; Heinrichs JH et al.; Previous evidence suggests that hemolysin BL, which consists of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal form of gastroenteritis caused by food-borne strains of Bacillus cereus . To prove that hemolysin BL and the enterotoxin are the same requires large amounts of these components free of other B . cereus proteins . For this purpose, we sought to clone the gene encoding the B component and to express it in Escherichia coli . A partial genomic library was constructed and a 29-base, 1,152-fold-degenerate oligonucleotide probe, designed from the N-terminal amino acid sequence of the B component, was used to identify recombinant clones containing the gene . Detection of gene products reactive with a monoclonal antibody specific for the B component and analysis of the nucleotide sequence confirmed that isolated clones, reactive with the oligonucleotide probe, did contain the gene encoding the B component . The protein, expressed in E . coli, apparently from the B . cereus promoter, produces a ring-shaped zone of hemolysis when combined with purified L components from B . cereus, a reaction typical of hemolysin BL . Northern (RNA) blot analysis of B . cereus RNA showed a large (5.1-kb) transcript which hybridized with a 500-bp probe internal to the B-component-coding sequence, suggesting that the hblA gene encoding the B component may be transcribed as part of a polycistronic message, possibly including the structural genes for the two lytic components . Higher levels of expression and disruption of the hblA gene are being pursued to resolve whether hemolysin BL is indeed the enterotoxin.

Hinyokika Kiyo, 1993 Nov, 39(11), 987 - 91
{Prophylactic combination therapy after TUR of superficial bladder cancer}; Ao T et al.; We evaluated 63 patients with superficial bladder cancer (pTa, pTl) who were treated with instillation of bleomycin +/- bacillus Calmette-Guerin (BCG) and administration of uraciltfutraful (UFT) for prophylaxis of tumor recurrence after transurethral resection (TUR) . The patients were randomly assigned to groups A and B after transurethral resection by the closed envelope method . Group A (34 cases) was designed as continuous diluted intravesical instillation of bleomycin (120 mg/2,000 ml saline solution/day repeated for 3 days), instillation BCG (40 mg/40 ml saline solution/weekly 6 times) and UFT (400 mg orally/day for 2 years maximum) . Group B (29 cases) was designed as the aforementioned minus BCG instillation . Cumulative non-recurrence rates in group A and group B were 80.1% and 72.1% at the time of three years, which revealed no significant difference between the two groups (p = 0.265, generalized Wilcoxon test) . The high recurrent incidence of superficial bladder cancer is primarily due to the multifocal nature of the cancer or implantation of tumor cells at the time of the subsequent transurethral resection . The procedure was performed safely with no severe side effects . Our method might be useful to reduce the recurrences of superficial bladder cancer after transurethral resection of bladder (TUR).

Gene, 1993 Oct 29, 133(1), 91 - 4
Cloning, purification and characterization of the BseCI DNA methyltransferase from Bacillus stearothermophilus; Rina M et al.; The gene (bseCIM) encoding the BseCI DNA methyltransferase (MTase; M.BseCI) from a Bacillus stearothermophilus species was cloned and expressed in Escherichia coli using plasmid vector pBR322 . Selection of transformants carrying bseCIM was based on the resistance of the modified plasmid to cleavage by BseCI . The MTase was purified to homogeneity and further characterized . Its size as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and size exclusion chromatography was 68 kDa, suggesting that the MTase exists as a monomer . When phage lambda DNA was used as a substrate, the optimum temperature for MTase activity was determined to be 50-55 degrees C and optimum pH approx . 7.4 . M.BseCI is inhibited by concentrations of NaCl and KCl greater than 50 mM, and it does not require Mg2+ for activity . Finally, M.BseCI methylates the 3' adenine residue in the sequence, 5'-ATCGAT-3', similarly to its isoschizomer M.ClaI.

Biochem Biophys Res Commun, 1993 Oct 29, 196(2), 921 - 6
Ion channel activity of N-terminal fragments from CryIA(c) delta-endotoxin; Walters FS et al.; Proteolytically-derived amino-terminal fragments from the Bacillus thuringiensis delta-endotoxin, CryIA(c), demonstrated ion channel activity in two in vitro assays in the absence of any membrane receptors . A 24.5 kDa fragment (from Spodoptera frugiperda digestive proteolysis) formed cation-selective channels (10mV for a 3:1 salt gradient) in planar lipid bilayers up to several hundred picoSiemen conductance . A 21.5 kDa fragment doublet (from alkaline hydrolysis) also showed large conductance in planar lipid bilayers and resulted in nearly 2-fold greater 86Rb(+)-K+ efflux from phosphatidylcholine vesicles than 55 kDa CryIA(c) . In preliminary screening bioassays, however, the 21.5 kDa fragments showed no insecticidal activity toward neonate Heliothis virescens . Ion channel activity of these putative alpha-helical region fragments support this region being responsible for ion channel properties of the parent delta-endotoxin.

J Mol Biol, 1993 Oct 20, 233(4), 787 - 8
Crystallization and preliminary X-ray diffraction studies of Bacillus stearothermophilus farnesyl diphosphate synthase expressed in Escherichia coli; Nakane H et al.; Thermostable farnesyl diphosphate synthase (EC 2.5.1.10) from Bacillus stearothermophilus, which was overexpressed in Escherichia coli, has been crystallized by the vapor-diffusion procedure . Tetragonal crystals were obtained using ammonium sulfate as a precipitant . The crystals diffracted X-rays to about 3 A resolution . The diffraction pattern indicated that the space group is I4(1)22 with unit-cell dimensions of a = b = 114 A and c = 247 A . It is thought that the asymmetric unit comprises two or three molecules of farnesyl diphosphate synthase.

Presse Med, 1993 Oct 9, 22(30), 1385 - 90
{Neuromeningeal listeriosis in adults . Clinical aspects and contribution of cotrimoxazole in monotherapy}; Pinede L et al.; A series of 28 patients suffering from neuromeningeal listeriosis is reported . This disease is consecutive to infection by Listeria monocytogenes--an ubiquitous and opportunistic Gram-positive bacillus--and has become a public health problem: its incidence is increasing and its prognosis is very severe despite the development of new bacteriological identification methods . Human beings are contaminated by food, which explains the frequent outbreaks of epidemics which are widely publicized, the infection being one of the consequences of the unprecedented development of the food industry and the cold food chain . The predominant clinical picture is one of non-specific meningoencephalitis . In about 50 percent of the cases the subjects infected are "immunodepressed" and/or more than 60 years' old . The diagnosis is difficult since the bacteriological identification is delayed (direct examination of the cerebrospinal fluid is rarely positive) and this fluid may be sterile (hence the value of blood cultures) . A probability treatment therefore must be initiated before the diagnosis is confirmed if an unfavourable outcome is to be avoided . In Listeria monocytogenes infection cotrimoxazole administered alone seems to be a better antibacterial therapy than the reference ampicillin-aminoside combination.

Biochim Biophys Acta, 1993 Oct 6, 1202(2), 200 - 6
Domain structure and multiplicity of raw-starch-digesting amylase from Bacillus circulans: extensive proteolysis with proteinase K, endopeptidase Glu-C and thermolysin; Kim CH et al.; Raw-starch-digesting amylase (RSDA) is a key extracellular enzyme of mesophilic Bacillus circulans F-2 which uses raw starch granules as a carbon source . Previous work has demonstrated that there are two domains of the enzyme during digestion with subtilisin, and that RSDA activity is selectively inactivated by limited proteolysis with subtilisin, which cleaves the enzyme between these hydrolytic and adsorption domains (Kim, C.-H., Kwon, S.-T., Taniguchi, H . and Lee, D.-S . (1992) Biochim . Biophys . Acta 1122, 243-250) . In this work we show that a similar phenomenon is observed during limited proteinase K, thermolysin and endopeptidase Glu-C proteolysis of the enzyme . Fragments resulting from proteolysis were characterized by immunoblotting with anti-RSDA . The proteolytic patterns resulting from proteinase K and subtilisin were the same, producing 63 and 30-kDa fragments . Similar patterns were obtained with endopeptidase Glu-C or thermolysin . All proteolytic digests contained a common, major 63-kDa fragment . Inactivation of RSDA activity results from splitting off the C-terminal domain . Hence, it seems probable that the proteinase-sensitive locus is in a hinge region susceptible to cleavage . Extracellular enzymes immunoreactive towards anti-RSDA were detected through whole bacterial cultivation . 93, 75, 63, 55, 38 and 31-kDa proteins were immunologically identical to RSDA . Of these, the 75-kDa and 63-kDa proteins correspond to the major products of proteolysis with Glu-C and thermolysin . These results suggest that enzyme heterogeneity of the raw-starch hydrolysis system might arise from the endogenous proteolytic activity of the bacterium.

Presse Med, 1993 Oct 2, 22(29), 1352 - 6
{Generalized BCG infection after intravesical instillations of Calmette-Guerin bacillus}; de Saint Martin L et al.; BCG has been disappointing as immunotherapy of numerous cancers, but it has been clinically successful in the intravesical treatment of bladder carcinomas sparing the muscle coat; it has indeed become the reference treatment for this type of cancer . However, complications are repeatedly reported, including generalized BCGitis . We report such a case with positive BCG culture . From the cases already published there emerges a homogeneous and often subacute clinical presentation suggestive of an ordinary pathogen . Bacteriology is not very helpful, even when recent techniques are used, and therefore the diagnosis rests on the context and, when samples are taken, on suggestive histological findings . To discuss the physiopathology of BCGitis--generalized immune reaction or multifocal BCG proliferation--is not useless since treatment depends on it . It is probable that these 2 mechanisms working together can be incriminated justifying the prescription of both antibiotics and corticosteroids . When this is done, the prognosis seems to be favourable in most patients . Yet a strict respect of contra-indications and a very careful subsequent radiotherapy should reduce the risks.

Proc Natl Acad Sci U S A, 1993 Oct 1, 90(19), 9041 - 5
Site-directed mutations in a highly conserved region of Bacillus thuringiensis delta-endotoxin affect inhibition of short circuit current across Bombyx mori midguts; Chen XJ et al.; Bacillus thuringiensis delta-endotoxins (Cry toxins) are insecticidal proteins of approximately 65 kDa in the proteolytically processed and active form . The structure of one of these toxins, CryIIIA, has been determined by Li et al . {Li, J., Carroll, J . & Ellar, D . J . (1991) Nature (London) 353, 815-821} and contains three domains . It is believed that other delta-endotoxins adopt similar three-dimensional structure . Li et al . proposed that the first domain is the membrane pore-forming domain . Previous work from our laboratory has shown that the second domain is the receptor binding domain, but the function of the third domain is unclear . Site-directed mutagenesis was used to convert the "arginine face" of one of five highly conserved regions, QRYRVRIRYAS of CryIAa (residues 525-535), to selected other residues . This sequence corresponds to the beta-sheet 17 of CryIIIA in the third domain . Mutations in the second and third arginine positions resulted in structural alterations in the protein and were poorly expressed in Escherichia coli . Toxins from genes mutated to replace lysine for the first and fourth arginines were unaltered in expression and structure, as measured by trypsin activation, CD spectra, and receptor binding, but were substantially reduced in their insecticidal properties and inhibition of short circuit current across Bombyx mori midguts . It is proposed that this region plays a role in toxin function as an ion channel.

J Pediatr, 1993 Oct, 123(4), 564 - 72
Severe combined immunodeficiency: a retrospective single-center study of clinical presentation and outcome in 117 patients; Stephan JL et al.; We carried out a retrospective analysis of 117 patients with severe combined immunodeficiency who were examined in a single center between Jan . 1, 1970, and Jan . 1, 1992, for the purpose of evaluating disease onset, progression, and outcome . The frequency of case referral increased from 8 from 1970 to 1975 to 56 from 1986 to 1991 . The most frequent phenotype was T-/B+ (absence of T lymphocytes and presence of B lymphocytes) (n = 51); there were 36 cases of alymphocytosis, 16 of adenosine deaminase deficiency, 13 of Omenn syndrome, and 1 of reticular dysgenesis . Protracted diarrhea and lung infections were the main infectious complications; infection with bacillus Calmette-Guerin occurred in 10 of 28 vaccinated patients, but none of the six recipients of oral polio vaccine subsequently had poliomyelitis . The presence of maternal T cells was suspected or proved in half the patients with alymphocytosis or T-B+ severe combined immunodeficiency but did not occur in the other forms of the disease . Of the 117 patients, 22 died before transplantation could be performed . Adenosine deaminase deficiency and Omenn syndrome were more frequently associated with death before hematopoietic stem cell transplantation was possible . Fetal liver transplantation was successful in 1 of 10 cases . The survival rate among the 30 recipients of bone marrow with identical human leukocyte antigens (HLA) was 80%, with a median follow-up of 129 months; 23 of 25 patients recovered full immune function . The survival rate among the 50 recipients of HLA-haploidentical T cell-depleted bone marrow was 56%, with a mean follow-up of 35 months . Of the latter patients, 10 (35%) still require immunoglobulin substitution . There has been a trend toward improvement in the survival rate of haploidentical bone marrow recipients, presumably because of more effective infection-control measures and better transplantation strategy.

J Leukoc Biol, 1993 Oct, 54(4), 296 - 9
Autocrine amplification of PAF-acether formation in immunologically activated murine macrophages; Ninio E et al.; When murine macrophages activated in vivo with bacille Calmette-Guerin were triggered with either acetyl-CoA or propionyl-CoA to form PAF-acether (PAF), similar amounts of platelet-aggregating product were recovered . Liquid chromatographic purification and reversed-phase analysis showed that the composition of PAF molecular species formed in the presence of acetyl-CoA was an equimolar mixture of PAF bearing C16:0 alkyl chain (57% +/- 7, mean +/- SD, n = 3) and PAF C18:1 . The PAF-like material obtained from the propionyl-CoA-supplemented macrophages was a mixture of the propionyl analogue of PAF (66% +/- 11, n = 3) and native PAF . The rate of lyso-PAF:acetyl-CoA acetyltransferase (EC 2.3.1.67) reaction in a macrophage lysate was similar for either substrate in the presence of an equimolar mixture of propionyl-CoA and acetyl-CoA . We conclude that the exogenously added propionyl-CoA is transferred to lyso-PAF acceptor to form propionyl-PAF by the PAF-forming acetyltransferase . Propionyl-PAF triggers the formation of native PAF probably from the endogenous acetyl-CoA pool . Two specific PAF antagonists, BN 52021 (60 microM) and WEB 2086 (3 microM), did not influence the rate of PAF synthesis in the presence of either acetyl-CoA or propionyl-CoA and did not prevent native PAF formation when propionyl-CoA was added alone, suggesting that the classical PAF receptors are not involved . This is the first description of a possible mechanism of autocrine amplification of PAF biosynthesis in macrophages.

J Bacteriol, 1993 Oct, 175(20), 6530 - 6
Mobilization of small plasmids in Bacillus thuringiensis subsp . israelensis is accompanied by specific aggregation; Andrup L et al.; Mobilizations of pBC16 and pAND006, containing the replicon of the Bacillus thuringiensis subsp . israelensis plasmid pTX14-3, between strains of B . thuringiensis subsp . israelensis were examined . Transconjugants appeared after a few minutes and reached a maximum frequency after approximately 2 h . Plasmid pBC16 was mobilized at a frequency approximately 200 times that of pAND006 . However, pAND006 was consistently transferred, suggesting that the replicon of pTX14-3 is sufficient to sustain mobilization in B . thuringiensis subsp . israelensis . A specific protease-sensitive coaggregation between strains of B . thuringiensis subsp . israelensis was found to be unambiguously correlated with plasmid transfer . Two aggregation phenotypes, Agr+ and Agr-, were identified in this subspecies . Aggregation disappeared when the optical density of the mating mixture at 600 nm exceeded approximately 1, and it did not reappear upon dilution . Aggregation was shown to involve interactions of cells with opposite aggregation phenotypes, and evidence of a proteinaceous molecule on the surface of the Agr- that is cells involved in aggregation formation is presented . Matings and selection for the presence of two antibiotic resistance plasmids followed by identification of the host cell revealed that mobilization was unidirectional, from the Agr+ cell to the Agr- cell . The aggregation phenotype was found to be transferred with high frequency (approximately 100%) in broth matings, and the appearance of Agr- isolates from Agr+ strains suggested that the loci involved in aggregation formation are located on a plasmid . No excreted aggregation-inducing signals were detected in the supernatant or culture filtrate of either the donor, the recipient, or the mating mixture.

J Bacteriol, 1993 Oct, 175(20), 6441 - 50
Cloning and sequencing of a cellobiose phosphotransferase system operon from Bacillus stearothermophilus XL-65-6 and functional expression in Escherichia coli; Lai X et al.; Cellulolytic strains of Bacillus stearothermophilus were isolated from nature and screened for the presence of activities associated with the degradation of plant cell walls . One isolate (strain XL-65-6) which exhibited strong activities with 4-methylumbelliferyl-beta-D-glucopyranoside (MUG) and 4-methylumbelliferyl-beta-D-cellobiopyranoside (MUC) was used to construct a gene library in Escherichia coli . Clones degrading these model substrates were found to encode the cellobiose-specific genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) . Both MUG and MUC activities were present together, and both activities were lost concurrently during subcloning experiments . A functional E . coli ptsI gene was required for MUC and MUG activities (presumably a ptsH gene also) . The DNA fragment from B . stearothermophilus contained four open reading frames which appear to form a cel operon . Intergenic stop codons for celA, celB, and celC overlapped the ribosomal binding sites of the respective downstream genes . Frameshift mutations or deletions in celA, celB, and celD were individually shown to result in a loss of MUC and MUG activities . On the basis of amino acid sequence homology and hydropathy plots of translated sequences, celA and celB were identified as encoding PTS enzyme II and celD was identified as encoding PTS enzyme III . These translated sequences were remarkably similar to their respective E . coli homologs for cellobiose transport . No reported sequences exhibited a high level of homology with the celC gene product . The predicted carboxy-terminal region for celC was similar to the corresponding region of E . coli celF, a phospho-beta-glucosidase . An incomplete regulatory gene (celR) and proposed promoter sequence were located 5' to the proposed cel operon . A stem-loop resembling a rho-independent terminator was present immediately downstream from celD . These results indicate that B . stearothermophilus XL-65-6 contains a cellobiose-specific PTS for cellobiose uptake . Similar systems may be present in other gram-positive bacteria.

J Bacteriol, 1993 Oct, 175(19), 6337 - 40
Cloning and nucleotide sequence of a plasmid-carried gene coding for a minor small, acid-soluble protein from Bacillus megaterium spores; Carrillo-Martinez Y et al.; The gene (termed sspG) coding for a small, acid-soluble protein (SASP) from spores of Bacillus megaterium QMB1551, termed SASP-G, has been cloned, and its nucleotide sequence has been determined . SASP-G is a 42-residue protein containing 2 tryptophan and 11 lysine residues, including a hexalysine sequence, and is not homologous to any previously described SASP . The sspG gene appears to be an additional member of the sigma G regulon . No gene homologous to sspG is present in B . cereus T or B . subtilis 168 . The reason for the absence of sspG from other Bacillus species appears to be that in B . megaterium, sspG is present only on a 111-kb plasmid; this plasmid is not present in B . cereus T or B . subtilis 168 . The sspG gene is the first forespore-expressed gene found to be on a plasmid.

J Bacteriol, 1993 Oct, 175(19), 6105 - 12
Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1; Meyers PR et al.; A cyanide-degrading enzyme from Bacillus pumilus C1 has been purified and characterized . This enzyme consisted of three polypeptides of 45.6, 44.6, and 41.2 kDa; the molecular mass by gel filtration was 417 kDa . Electron microscopy revealed a multimeric, rod-shaped protein approximately 9 by 50 nm . Cyanide was rapidly degraded to formate and ammonia . Enzyme activity was optimal at 37 degrees C and pH 7.8 to 8.0 . Activity was enhanced by Sc3+, Cr3+, Fe3+, and Tb3+; enhancement was independent of metal ion concentration at concentrations above 5 microM . Reversible enhancement of enzymatic activity by azide was maximal at 4.5 mM azide and increased with time . No activity was recorded with the cyanide substrate analogs CNO-, SCN-, CH3CN, and N3- and the possible degradation intermediate HCONH2 . Kinetic studies indicated a Km of 2.56 +/- 0.48 mM for cyanide and a Vmax of 88.03 +/- 4.67 mmol of cyanide per min/mg/liter . The Km increased approximately twofold in the presence of 10 microM Cr3+ to 5.28 +/- 0.38 mM for cyanide, and the Vmax increased to 197.11 +/- 8.51 mmol of cyanide per min/mg/liter . We propose naming this enzyme cyanide dihydratase.

Eur J Immunol, 1993 Oct, 23(10), 2440 - 7
Murine CD8+ T suppressors against mycobacterial 65-kDa antigen compete for IL-2 and show lack of major histocompatibility complex-imposed restriction specificity in antigen recognition; Khetan S et al.; The mechanism of antigen-specific suppression and reasons for aberrant major histocompatibility complex (MHC) class II restriction mediated by CD8+ T cells was investigated in a previously reported murine model of immunosuppression, generated by intraperitoneal priming with Mycobacterium vaccae . Both the CD4+ T helper cells (Th) and CD8+ T suppressor cell (Ts) of M . vaccae-primed mice recognized the 65-kDa antigen of the bacillus, presented by I-A and I-E, respectively . The CD8+ Ts could inhibit non-antigen-specific proliferation of primed CD4+ T cells induced by the exogenously added interleukin (IL)-2 (concanavalin A-stimulated culture supernatant) . For inhibition, the Ts had to be activated by the 65-kDa antigen . The degree of inhibition was dependent upon the amount of added IL-2 and the relative numbers of primed CD8+ and CD4+ T cells . On incubation with antigen-presenting cells, and the 65-kDa antigen, the primed CD8+ T cells absorbed IL-2 as efficiently as primed CD4+ T cells . Based on this, it was concluded that the primed CD8+ T cells induced suppression by competition for IL-2 . Employing the same model, the MHC restriction of recognition of the suppressor epitope of the 65-kDa antigen by the CD8+ Ts was investigated . The epitopes presented by diverse MHC class II molecules, such as self I-A, I-E and even allogeneic I-E were similar, because they were recognized by the same population of primed CD8+ Ts . Further, immunization of C57BL/6 mice with Ltk-cells expressing H-2 DkKk alloantigens, stimulated CD8+ T cells capable of recognizing M.vaccae 65-kDa antigen . Based on these data, it was proposed that recognition of the suppressor epitope of the 65-kDa antigen by the primed CD8+ Ts exhibits lack of restriction specificity imposed by MHC diversity.

J Exp Med, 1993 Oct 1, 178(4), 1435 - 40
Mice that lack the interferon-gamma receptor have profoundly altered responses to infection with Bacillus Calmette-Guérin and subsequent challenge with lipopolysaccharide; Kamijo R et al.; Mice with a targeted disruption of the interferon gamma receptor gene (IFN-gamma R0/0 mice) and control wild-type mice were inoculated with the Bacillus Calmette-Guerin (BCG) strain of Mycobacterium bovis . BCG infection was not lethal for wild-type mice whereas all IFN-gamma R0/0 mice died approximately 7-9 wk after inoculation . Histological examination at 2 and 6 wk after BCG inoculation showed that livers of IFN-gamma R0/0 mice had higher numbers of acid-fast bacteria than wild-type mice, especially at 6 wk . In parallel, the livers of IFN-gamma R0/0 mice showed a reduction in the formation of characteristic granulomas at 2 wk after inoculation . Injection of lipopolysaccharide (LPS) 2 wk after BCG inoculation was significantly less lethal for IFN-gamma R0/0 mice than for wild-type mice . Reduced lethality of LPS correlated with a drastically reduced production of tumor necrosis factor alpha (TNF-alpha) in the IFN-gamma R0/0 mice . Interleukin 1 alpha (IL-1 alpha) and IL-6 levels in the serum were also significantly reduced in the IFN-gamma R0/0 mice after BCG infection and LPS challenge . The greatly reduced capacity of BCG-infected IFN-gamma R0/0 mice to produce TNF-alpha may be an important factor in their inability to resist BCG infection . These results show that the presence of a functional IFN-gamma receptor is essential for the recovery of mice from BCG infection, and that IFN-gamma is a key element in the complex process whereby BCG infection leads to the sensitization to endotoxin.

J Infect Dis, 1993 Oct, 168(4), 1059 - 62
Minimal effect of advanced aging on susceptibility of mice to infection with Mycobacterium tuberculosis; North RJ; Young (3-month-old) and old (> or = 24-month-old) mice of a long-lived strain were compared in terms of their ability to resist infection with Mycobacterium tuberculosis . There was little overall difference in the ability of young and old mice to resist this infection . Mice in both age groups could control infection in their livers and spleens by a mechanism that was predominantly CD4+ T cell-mediated but not in their lungs . Again, old mice were almost as capable as young mice at being immunized by bacille Calmette-Guerin against a challenge infection with M . tuberculosis . The results argue against including M . tuberculosis among infectious agents to which mice become susceptible with advanced age.

Clin Investig, 1993 Oct, 71(10), 787 - 90
Pulmonary tuberculosis due to bacille Calmette-Guérin; Kirsten D et al.; Immunotherapy using bacille Calmette-Guerin (BCG) has gained increasing acceptance in the management of superficial bladder cancer . Systemic reactions after intravesical instillation of BCG are rare . However, when the therapy is complicated, the lung often becomes involved . Since the pathogenesis of lung infiltrates after immunotherapy is unknown, we report on a patient who developed a lung infiltrate after receiving BCG immunotherapy for bladder cancer . The infectious etiology was established by culture confirmation of a BCG strain in the bronchoalveolar lavage fluid.

J Biochem (Tokyo), 1993 Oct, 114(4), 522 - 7
Effect of single base substitutions at glycine-870 codon of gramicidin S synthetase 2 gene on proline activation; Tokita K et al.; The mutant gene coding for a proline-activating domain (grs2-pro) was cloned and sequenced from Bacillus brevis Nagano, BII-3 strain, which produces gramicidin S synthetase 2 defective in proline-activation . By comparison of the nucleotide sequence with the wild-type sequence, a single point mutation was found at the 2609th guanine, which was replaced with adenine, resulting in the change of the 870th glycine to glutamic acid . Homology search for the deduced amino acid sequence of grs2-pro gene revealed that the 870th glycine was conserved in adenylate-forming enzymes, and its flanking sequence was highly conserved among the aminoacyl adenylate-forming enzymes, such as antibiotic peptide synthetases: gramicidin S synthetase 1 and 2 (GS1, GS2), tyrocidine synthetase 1 (TS1), and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS); and other aminoacyl adenylation enzymes: alpha-aminoadipate reductase (LYS2), EntF, and AngR . On the other hand, this flanking sequence was not conserved in the other adenylate-forming enzymes lacking amino acid activation, such as acetyl-CoA synthetase, long-chain acyl-CoA synthetase, luciferase, and 4-coumarate CoA ligase . Single base substitutions at the 870th GGG codon were carried out by oligonucleotide site-directed mutagenesis . Four mutagenized clones were isolated, containing grs2-pro genes which exchange 870-Gly for alanine, valine, arginine, and tryptophan . The translated products from these clones could scarcely catalyze proline-dependent ATP-32PPi exchange reaction . The coil structure of 870-Gly region was lost in the mutants . These results suggest that the 870-Gly residue of grs2-pro protein is essential for aminoacyl-adenylation in the antibiotic peptide synthetase family.

Laryngorhinootologie, 1993 Oct, 72(10), 478 - 84
{HIV-associated Kaposi sarcoma in the head and neck area: a clinical, morphologic and therapeutic review}; Riederer A et al.; Since 1987 233 HIV-infected patients have been treated at the Department of Otorhinolaryngology, Head and Neck Surgery of the Ludwig-Maximilians-University of Munich . 70% of these patients had advanced immunodeficiency disease (ARC and AIDS) . 46 presented a Kaposi's sarcoma (KS) in the head and neck region . 91% were homosexual men . KS was most often located in the mouth (67%), oropharynx (65%) and skin (39.1%), while the larynx (10.9%), hypopharynx (8.7%), lymph nodes (6.5%) and nasopharynx (4.3%) were rarely involved . In 15 patients, a KS of the head and neck region was the initial symptom for the HIV-infection . Although the clinical features of this disease are typical, histological examination is required because differential diagnosis can show other rare diseases, such as bacillary angiomatosis, which are easily cured . The morphology of early plain or elevated KS exhibits more irregular vascular components while the nodular KS is dominated by sarcomatous cell lines . Immunohistochemical studies with antibodies to viral components revealed no reactivity to HIV-, HPV-, HSV-, EBV- and CMV-antigens . The best local treatment proved to be CO2- or ND:YAG-laser therapy . Cutaneous lesions were treated with camouflage or by fractionated radiotherapy . Advanced disease showed best response to systemic chemotherapy . Despite the advanced stage of immunodeficiency syndrome, an adequate local or systemic therapy can obviously improve the quality of life in HIV-infected patients.

Tuber Lung Dis, 1993 Oct, 74(5), 310 - 6
Turning intermittent regimens into daily regimens using blister-packs . An exploration in murine tuberculosis; Guy A et al.; SETTING: Blisterpacks might be used to present low cost intermittent regimens while maintaining an easily remembered daily frequency of opening blisters, by alternating blisters containing 2 of the drugs of a 4-drug regimen with blisters containing the remaining 2 drugs . OBJECTIVE: The efficacy of the alternating regimens was examined in murine tuberculosis . DESIGN: 2 weeks after infection with Mycobacterium tuberculosis H37Rv, groups of mice were treated with rifampicin (R) 15 mg/kg, isoniazid (H) 25 mg/kg, pyrazinamide (Z) 300 mg/kg, ethambutol (E) 100 mg/kg three times weekly (RHZE3); RH/ZE, an alternating regimen of RH on days 1, 3 and 5 of the week and ZE on days 2, 4 and 6, RZ/HE or RE/HZ . RESULTS: Spleen and lung bacillary counts at 7 and 12 weeks indicated large differences in the efficacy of the regimens: RZ/HE > RE/HZ > RHZE3 > RH/ZE . Serum assays showed that Rifampicin (RMP) levels were much lower after HRZE and slightly lower after RZ, RE and RH than after R alone, whereas levels were similar when R was given before the remaining drugs; also, the absorption of Z was slightly increased by R . A second experiment used the same 4 regimens but gave R before other drugs . The organ colony forming units counts at 6 and 12 weeks were then similar . A third experiment examined continuation phase regimens of R3, R2, R2H2 and R2H6 given after daily RHZ treatment for 4 weeks . It found R2H2 only slightly superior to R2H6 and R3 much better than R2 . CONCLUSION: 1 . Alternating initial phase regimens were as effective as conventional intermittent regimens . 2 . R3H6 might be an optimal continuation phase regimen for blisterpacks.

J Gen Microbiol, 1993 Oct, 139 ( Pt 10), 2365 - 9
Formation of melanin pigment by a mutant of Bacillus thuringiensis H-14; Hoti SL et al.; A mutant of Bacillus thuringiensis H-14 produced a dark brown pigment during sporulation . Production of the pigment depended on the nutritional properties of the growth medium . The pigment was identified as melanin, based on chemical tests and its infra-red spectrum . Incorporation of L-tyrosine in the culture medium enhanced the level of melanin production, and L-3,4-dihydroxyphenylalanine (L-DOPA) was detected in the culture broth during the late-exponential phase of growth . This indicates that the pathway of melanin synthesis is from L-tyrosine, via L-DOPA, to melanin.

Int J Biol Macromol, 1993 Oct, 15(5), 317 - 8
Does the increased hydrophobicity of the interior and hydrophilicity of the exterior of an enzyme structure reflect its increased thermostability?
Janecek S.
The values of hydrophobicity of internal and external elements of the secondary structure of three Bacillus alpha-amylase (beta alpha)8 barrel domains have been calculated in order to investigate whether there is some correlation between the values and the enzyme stability . All the values have been referred to the number of amino acids in the given beta-sheet or alpha-helix to eliminate the differences caused by non-equal length of the sheet or helix . Hydrophobicity units obtained have been averaged according to the number of internal (all beta-strands and helix alpha 7) and external (helices alpha 1-alpha 6 and alpha 8) elements of secondary structure of the alpha-amylase (beta alpha)8 barrel . The averaged hydrophobicity units have been found to correlate with the thermal stability of the three Bacillus alpha-amylases in terms of the increased hydrophobicity of the interior as well as the increased hydrophilicity of the exterior of the (beta alpha)8 barrel domain for the alpha-amylase with increased thermostability.

Int J Food Microbiol, 1993 Oct, 20(1), 23 - 36
Properties of Bacillus cereus spores in reference materials prepared from artificially contaminated spray dried milk; in 't Veld PH et al.; A reference material for Bacillus cereus was developed based on spray drying of milk artificially contaminated with B . cereus spores . Various properties of the B . cereus spores in the milk powder were determined . The stability of the materials was good with no detectable decrease in the contamination level during 1 1/2 years storage at -20 degrees C or 4 weeks at 22, 30 or 37 degrees C . The homogeneity of the material was found acceptable for use as a reference material . Heat treatments (10 min at 70 or 80 degrees C) and addition of lysozyme to the enumeration medium did not influence the number of spores counted . The germination of the spores depended on the type of medium in which the milk powder was reconstituted, and on the storage period of the material . The suitability of the material was confirmed in a collaborative study . From the results obtained it was concluded that the material developed meets the general requirements set for reference materials and can therefore be used for, among others, testing laboratory performance.

Int J Syst Bacteriol, 1993 Oct, 43(4), 777 - 86
Proposals to unify the genera Bartonella and Rochalimaea, with descriptions of Bartonella quintana comb . nov., Bartonella vinsonii comb . nov., Bartonella henselae comb . nov., and Bartonella elizabethae comb . nov., and to remove the family Bartonellaceae from the order Rickettsiales; Brenner DJ et al.; DNA hybridization data (hydroxyapatite method, 50 to 70 degrees C) indicate that Rickettsia prowazekii, the type species of the type genus of the family Rickettsiaceae, is substantially less closely related to Rochalimaea species than was previously thought . The levels of relatedness of Rickettsia prowazekii to Rochalimaea species and to Bartonella bacilliformis under optimal conditions for DNA reassociation were 0 to 14%, with 25.5% or greater divergence in related sequences . When stringent reassociation criteria were used, the levels of relatedness were 0 to 2% . The genera Bartonella and Rochalimaea are currently classified in different families (the Bartonellaceae and the Rickettsiaceae) in the order Rickettsiales . On the basis of DNA relatedness data, previous 16S rRNA sequence data, guanine-plus-cytosine contents, and phenotypic characteristics, neither Bartonella bacilliformis nor Rochalimaea species are closely related to other organisms currently classified in the order Rickettsiales . In fact, the closest relative of these organisms is Brucella abortus . It is therefore proposed that the family Bartonellaceae should be removed from the order Rickettsiales . Previous 16S rRNA sequence data and DNA hybridization data revealed high levels of relatedness between Bartonella bacilliformis and the four Rochalimaea species, indicating that these species are members of a single genus . It is proposed that the genus Rochalimaea should be united with the genus Bartonella in the family Bartonellaceae . The name Bartonella is retained as the genus name since it has nomenclatural priority over the name Rochalimaea . This means that new combinations for the Rochalimaea species must be created . Proposals are therefore made for the creation of Bartonella quintana comb . nov., Bartonella vinsonii comb . nov., Bartonella henselae comb . nov., and Bartonella elizabethae comb . nov.

Mol Gen Genet, 1993 Oct, 241(1-2), 170 - 6
Cloning sequencing and expression of the gene for cytochrome P450meg, the steroid-15 beta-monooxygenase from Bacillus megaterium ATCC 13368; Rauschenbach R et al.; A 4.3 kb EcoRI fragment carrying the gene for cytochrome P450meg, the steroid-15 beta-monooxygenase from Bacillus megaterium ATCC 13368, was cloned and completely sequenced . The gene codes for a protein of 410 amino acids and was expressed in Escherichia coli and B . subtilis . Protein extracts from the recombinant E . coli strains were able to hydroxylate corticosteroids in the 15 beta position when supplemented with an extract from a P450- mutant of B . megaterium ATCC 13368 as a source of megaredoxin and megaredoxin reductase . In contrast, 15 beta-hydroxylation was obtained in vitro and in vivo without the addition of external electron transfer proteins, when cytochrome P450meg was produced in B . subtilis 168 . Protein extracts from nonrecombinant B . subtilis 168 could also support the in vitro hydroxylation by cytochrome P450meg produced in E . coli.

J Dairy Sci, 1993 Oct, 76(10), 3041 - 53
Evaluation of milk antibiotic residue screening tests in cattle with naturally occurring clinical mastitis; Van Eenennaam AL et al.; Milk from 172 commercial cows with mild to moderate clinical mastitis was tested with five antibiotic residue detection assay systems . One hundred cows were treated with one of two intramammary beta-lactam antibiotics, and the remaining 72 cows were treated with intramuscular oxytocin . Milk samples were collected pretreatment, twice after therapy, and again 21 d following the initiation of treatment . Presumptive false-positive assay results were tabulated from all pretreatment and 21-d milk samples and from samples collected following oxytocin therapy . The percentage of false-positive results was 43.6, 37.7, 81.7, 2.6, and 18.8% for the CITE probe (beta-lactam), Delvotest-P, Charm Farm, LacTek (beta-lactam), and Bacillus stearothermophilus var . calidolactis disk assay, respectively . In four of the assay systems, average SCC were significantly higher in samples yielding false-positive results than in those with negative results . Specificity and sensitivity were estimated for each assay system, and, based on these estimates, positive and negative predictive value curves were graphed as the prevalence of milk samples containing detectable concentrations of exogenous antibiotic residues in the sample population was varied from 0 to 100%.

Eur J Biochem, 1993 Oct 1, 217(1), 361 - 9
Immunoelectron microscopic localization of ribosomal proteins BS8, BS9, BS20, BL3 and BL21 on the surface of 30S and 50S subunits from Bacillus stearothermophilus; Schwedler G et al.; The locations of ribosomal proteins BS8, BS9 and BS20 on the 30S subunit of Bacillus stearothermophilus ribosomes, and of BL3 and BL21 on the 50S subunit, were determined by immunoelectron microscopy . BL3 was found to lie half-way down the body of the 50S subunit on the interface side, below the L7/L12 stalk, in agreement with the placement of the corresponding protein in Escherichia coli by neutron-scattering; BL21 was located at a similar position on the solvent side of the subunit, as predicted by cross-linking experiments with E . coli ribosomes . Similarly, BS8 was found in the upper region of the body of the 30S subunit on the solvent side, and BS9 on the top of the head of the subunit, also on the solvent side, both positions being in good agreement with neutron-scattering data and other immunoelectron microscopy results . In contrast, BS20 was found to lie at the extreme base of the body of the 30S subunit; this placement is not compatible with the location of E . coli S20 by neutron-scattering but fits very plausibly with other biochemical data, such as sites of RNA-protein footprinting on 16S RNA, relating to the location of S20 in E . coli.

Arch Biochem Biophys, 1993 Oct, 306(1), 125 - 32
Release of carcinoembryonic antigen from human tumor cells by phosphatidylinositol-specific phospholipase C: highly effective extraction and upregulation from LS-174T colonic adenocarcinoma cells; Gouin E et al.; Carcinoembryonic antigen (CEA), produced by gastrointestinal tumor cells, is anchored to cell membrane by a glycosyl-phosphatidylinositol moiety which can be cleaved with phosphatidylinositol-specific phospholipase C (PI-PLC) . We studied the extraction of CEA from living human colon carcinoma (LS-174T, HT-29, COLO-205, and HRT-18) and pancreatic carcinoma (CAPAN) cells by PI-PLC from Bacillus cereus . The total CEA content of LS-174T cells, quantitated by Triton X-114 extraction followed by radioimmunoassay or by immunohistochemistry, was 3.5-fold higher than that of other cells (P < 0.001) . The spontaneous release of CEA from LS-174T cells into culture medium was also higher than from other cells (P < 0.001), reaching 620 ng/10(7) cells (approximately 28% of cellular content) after 24 h . Overall, living LS-174T cells were highly susceptible to CEA extraction by PI-PLC, which was dependent on PI-PLC dose and on treatment time, leading in optimal conditions to the solubilization of 4100 ng/10(7) cells after 24 h (approximately 75% of total CEA) . After 24 h treatment at the highest PI-PLC dose, cell lines remained viable and growing, and membrane CEA expression was not exhausted but only reduced as compared to untreated cells . At the same time, the amount of CEA solubilized by PI-PLC exceeded the CEA reduction in membranes, suggesting that enzyme treatment increased CEA turnover . This was particularly true for LS-174T cells which maintained 54% of the expression of untreated cells, whereas the amount of CEA extracted by PI-PLC reached 190% of this expression . Growing LS-174T cells thus constitute an effective material for producing high quantities of CEA by PI-PLC cleavage, especially since these cells probably "regenerate" because of enhanced turnover during PI-PLC action, thus allowing continuous CEA production . These experimental conditions also provide an interesting model for studying the modulation of CEA expression and release.

Am J Med Sci, 1993 Oct, 306(4), 236 - 40
Case report: bacillary angiomatosis with massive visceral lymphadenopathy; Haught WH et al.; Bacillary angiomatosis is a newly characterized infectious disease occurring mainly in patients with AIDS . Most patients have cutaneous angiomatosis lesions resembling Kaposi's sarcoma or pyogenic granuloma . Although the disease may be life-threatening if not treated, it is curable with appropriate antibiotic therapy . A patient had a fever, nightsweats, abdominal pain, pleural effusions, and asymmetric peripheral lymphadenopathy . Computed tomography of the chest and abdomen revealed a unique pattern of enhancement of lymph nodes that, to this research team's knowledge, has not been reported previously with this condition . Appropriate antibiotic therapy resulted in a complete resolution of the disease . Included is a discussion of the clinical presentation, etiology, histology, and treatment of bacillary angiomatosis.

Wei Sheng Wu Xue Bao, 1993 Oct, 33(5), 383 - 6
{Nucleotide sequence of the toxic domain of an insecticidal protein gene from B . thuringiensis subsp . kurstaki HD-1}; Qiao L et al.; Two crystal protein genes, the 5.3kb and 6.6kb class respectively, from Bacillus thuringiensis subsp . kurstaki HD-1 (B . t HD-1) had been cloned previously . Based on the classification system of Hofte and Whiteley, these two genes should belong to Cry I A (b) and Cry I A (c) gene type respectively . The nucleotide sequence of the toxic domain of this Cry I A (c) gene from B . t kurstaki HD-1 is firstly reported here and compared with that of Cry I A (c) gene from B . t HD-73 and Cry I A (b) gene from B . t HD-1.

Pathology, 1993 Oct, 25(4), 398 - 401
Bacillary angiomatosis of the spleen; Mulvany NJ et al.; Bacillary angiomatosis is a recently described vasoproliferative lesion associated with infection by a newly characterized rickettsial organism, Rochalimaea henselae . Most previous reports have described skin lesions in immunocompromised patients infected with human immunodeficiency virus . This is the first case report detailing the features of bacillary angiomatosis of the spleen occurring in a patient undergoing cytotoxic chemotherapy for disseminated ovarian carcinoma.

Can J Physiol Pharmacol, 1993 Oct-Nov, 71(10-11), 840 - 7
Phosphatidylcholine metabolism in hypoxic and phospholipase C exposed rat ventricular myocytes; Myrmel T et al.; A phospholipase C specific for choline and ethanolamine acyl and plasmalogen glycerophospholipids (PC-PLC) has been described in myocardial tissue . In the present study we investigated whether an endogenous PC-PLC is activated in hypoxic, substrate-free incubations of rat ventricular myocytes . The phosphatidylcholine pool of the myocytes was prelabelled with {14C}choline during a 4-h preincubation (pulse) period . The myocytes were subsequently washed and incubated for another 2 h (chase period) in normoxic, hypoxic, or hypoxic buffer supplemented with PC-PLC from Bacillus cereus . We hypothesized that an increase in the total (intracellular plus extracellular) content of {14C}phosphocholine (one of the products resulting from PC-PLC action on phosphatidylcholine) throughout the chase period would indicate PC-PLC activity . Instead, an apparent decrease was observed for this parameter in all myocyte groups (17-29%), even in the one exposed to exogenous PC-PLC . However, 60 min after the start of the chase period, the level of total {14C}phosphocholine was higher in hypoxic (p = 0.022) and hypoxic + PC-PLC exposed (p = 0.013) myocytes compared with normoxic controls . The total content of {14C}choline increased significantly (p < 0.017) in all myocyte groups during the incubation period (98-153%) as a result of an increment of this metabolite in the buffer . Furthermore, the values measured in hypoxic and hypoxic + PC-PLC exposed myocytes during the first hour of the chase period were significantly (p < 0.017) higher than the corresponding values in normoxic myocytes . The present results do not allow firm conclusions regarding endogenous PC-PLC activation in energy-depleted rat cardiac myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Genet, 1993 Oct, 31(9-10), 343 - 62
Genetic structure and phyletic relationships of eastern Mediterranean Bacillus atticus brunner (Insecta Phasmatodea): a biochemical study; Mantovani B et al.; The allozymic characterization of several new Croatian, Greek, and Turkish samples thought to belong to different subspecies of Bacillus atticus or to atticus-like taxa is given . Several allelic combinations (zymotypes) were observed among both diploid and triploid samples; the occurrence of highly different levels of heterozygosity for the same locus among populations is also common . The biochemical-genetic features of the numerous zymotypes are interpreted on the basis of the recently assessed cytology of their parthenogenetic reproduction . Biochemical and meiotic features also allow one to suggest that both diploid and triploid cytotypes of B . atticus are more likely interracial hybrids in origin . The new triploid Greek samples show only small genetic distances from the Turkish triploid and diploid ones; also, they do not show clear-cut morphological differences, so that all triploids and Turkish diploid samples are together referred to as B . a . carius . On the other hand, all Croatian, Greek, and Italian diploids appear to belong to the same electrophoretic cluster, biochemically differentiated at a subspecific level from B . a . carious . This newly defined comprehensive group of diploid samples, which also morphologically show gradual patterns of variation, is referred to as B . a . atticus.

Rev Rhum Ed Fr, 1993 Oct, 60(9), 617 - 20
{Tuberculous arthritis and chondrocalcinosis . Apropos of 2 cases}; Pointud P et al.; Two cases of tuberculous arthritis in a joint affected with chondrocalcinosis are reported . No similar cases have been published . Diagnosis was established by demonstration of the tubercle bacillus and calcium pyrophosphate crystals in the joint fluid . Both patients were elderly French females . One patient with involvement of a knee required amputation . The other patient had involvement of a shoulder and developed inferior dislocation of the humeral head and drooping shoulder despite antituberculous therapy . Concomitant occurrence of the two conditions was apparently coincidental but may have adversely affected prognosis . Despite its rarity, tuberculous arthritis should be looked for in patients with arthritis and chondrocalcinosis to allow early specific therapy.

Zhonghua Jie He He Hu Xi Za Zhi, 1993 Oct, 16(5), 270 - 1, 318-9
{Clinical application of the single radial immunodiffusion (SRID) technic to detect antitubercle bacillus antibody}; Hu J et al.; Using the Tubercle bacillus Danish D1331 atoxic species as antigen and by single radial immunodiffusion (SRID) technic, the serum anti-TB-Ab of 454 patients with tuberculosis or other diseases were assayed and the results were reported . It was demonstrated that the sensitivity, specificity and accuracy of this method to diagnose tuberculosis were 81.78%, 97.92% and 90.31%; the positive and negative predictivities of this method were 97.22% and 85.77%, and the diagnostic efficiency was 80.08% . It was suggested this method had significant clinical value for diagnosing of tuberculosis.

Wei Sheng Wu Xue Bao, 1993 Oct, 33(5), 354 - 60
{Extraction and homogeny of larvicidal toxin in Bacillus sphaericus strain C3-41}; Zhou Z et al.; Spore-crystal toxin and spore-wall protein of Bacillus sphaericus C3-41 were extracted respectively from spore-crystal complex . When subjected to SDS-PAGE, spore-crystal toxin might give two toxic protein bands (43 and 40 kilodaltons), but spore-wall protein had only a protein band (MW 104 kD), which was degraded into toxic 43 and 40 kD proteins by using NaOH . The LC50 values of spore-wall protein and spore-crystal toxin purified by Sephadex G-75 were 267 and 10 ng/ml respectively against the third instar larvae of Culex quinquefasciatus at 48 hours . The results of immunodiffusion showed that sodium hydroxide-solubilized spore-crystal toxins from spore-crystal complex of strain 2362, 1593, Bs-10 (H5a5b) and 2297 (H25) revealed cross reactions with 43 and 40 kD antiserum of strain C3-41, but those of strain K (H1), SS II-1, 1404 (H2) and 2315 . (H26) without cross reaction with the same antiserum.

Appl Environ Microbiol, 1993 Oct, 59(10), 3470 - 3
New high-toxicity mosquitocidal strains of Bacillus sphaericus lacking a 100-kilodalton-toxin gene; Liu JW et al.; Five new high-toxicity mosquitocidal strains of Bacillus sphaericus were isolated in Singapore . They all belong to phage group 8 and have binary toxin (51.4- plus 41.9-kDa) genes located on the chromosome but lack a 100-kDa-toxin gene . These strains of B . sphaericus constitute a new subgroup, as only two weakly toxic strains in phage group 8 have previously been described and all the known high-toxicity strains have both binary toxin and 100-kDa-toxin genes.

Biosci Biotechnol Biochem, 1993 Oct, 57(10), 1691 - 8
Nucleotide sequence and analysis of a gene (chiA) for a chitinase from Streptomyces lividans 66; Miyashita K et al.; A chitinase gene (chiA) from Streptomyces lividans was characterized and its nucleotides sequenced . Although the deduced amino acid sequence of chitinase A1 did not show any similarity to those of other Streptomyces chitinases that has been sequenced, the C-terminal part, containing both a putative catalytic domain and type-III-like repeating units, showed a similarity (36%) to that of chitinase D from Bacillus circulans . A site of initiation of transcription was found approximately 51 bp upstream from the GTG initiation codon . The promoter region of the chiA gene was subcloned on a 178-bp fragment into the promoter-probe vector pIJ486, resulting in the chitin stimulated expression of the neomycin resistance gene . One of the deleted subclones, which contained a 114-bp sequence upstream from the translation start codon, retained both chitin stimulated production and glucose repression . Chitin stimulated production was lost in an other deleted mutant containing the 104-bp upstream sequence.

Biosci Biotechnol Biochem, 1993 Oct, 57(10), 1632 - 7
Purification and biochemical properties of an alkaline pullulanase from alkalophilic Bacillus sp . S-1; Kim CH et al.; A novel extracellular pullulanase (PUL-E, pullulan 6-glucanohydrolase, EC 3.2.1.41) has been purified from the alkalophilic Bacillus sp . S-1 . The purified enzyme had a molecular mass of about 140 kDa on denaturated and natural conditions . The pI was 5.5 . The pullulanase, when resolved by SDS-PAGE, was negative for Schiff staining, suggesting that the enzyme is not a glycoprotein . The N-terminal amino acid sequence of the enzyme was Phe-Leu-Asn-Met-Ser-(Trp-Phe) . The enzyme displayed a temperature optimum of around 60 degrees C and a pH optimum of around pH 9.0 . The enzyme was stable to incubation from pH 4.0 to pH 11.0 at 4 degrees C for 24 h . The presence of pullulan protected the enzyme from heat inactivation, the extent depending upon the substrate concentration . The activity of the enzyme was stimulated by Mn2+ ions . Ca2+ ions and EDTA did not inhibit the enzyme activity . The enzyme hydrolyzed the alpha-1,6-linkages of amylopectin, glycogens, alpha,beta-limited dextrin, and pullulan . The enzyme had an apparent Km of 7.92 mg/ml for pullulan, a Km of 1.63 mg/ml for amylopectin, and a Km of 3.1 mg/ml for alpha,beta-limited dextrin, when measured at pH 9.0 and 50 degrees C . The enzyme caused the complete hydrolysis of pullulan to maltotriose . The activity was not inhibited by alpha, beta, or gamma-cyclodextrins . The western blotting analysis with mouse anti-serum against PUL-E showed that PUL-E is produced as a single enzyme form during bacterial cultivation.

Biotechnology (N Y), 1993 Oct, 11(10), 1151 - 5
Insect resistant rice generated by introduction of a modified delta-endotoxin gene of Bacillus thuringiensis; Fujimoto H et al.; As a first step towards development of insect resistant rice we have introduced a truncated delta-endotoxin gene, cryIA(b) of Bacillus thuringiensis (B.t.) which has specific biological activity against lepidopteran insects into a japonica rice . To highly express the cryIA(b) gene in rice the coding sequence was extensively modified based on the codon usage of rice genes . Transgenic plants efficiently expressed the modified cryIA(b) gene at both mRNA and protein levels . Bioassays using R2 generation plants with two major rice insect pests, striped stemborer (Chilo suppressalis) and leaffolder (Cnaphalocrosis medinalis), indicated that transgenic rice plants expressing the CryIA(b) protein are more resistant to these pests than untransformed control plants . Our results suggest that the B.t . endotoxin genes will be useful for the rational development of new rice varieties resistant to major insect pests.

Virology, 1993 Oct, 196(2), 619 - 28
Nucleotide sequence and genomic organization of cacao swollen shoot virus; Hagen LS et al.; Cacao swollen shoot virus is classified as a badnavirus based on its nonenveloped, bacilliform particle morphology and double-stranded DNA genome . A complete copy of the genome was cloned into a plasmid vector and the sequence was determined from 75 overlapping subclones covering both strands . The genome contains 7161 base pairs and possesses an intergenic region and five putative open reading frames (ORF) capable of coding for proteins > 10 kDa . All of the ORFs are present on the plus-strand . ORF 1 (17 kDa) and ORF 2 (14 kDa) encode proteins of unknown function . The large ORF 3 (211 kDa) encodes a polyprotein that can be divided into three regions . Based on distant homologies with viral movement proteins, region 1 may encode a protein involved in cell-to-cell spread, while region 2 encodes the viral capsid protein . Region 3 contains consensus sequences for viral aspartyl proteinase, reverse transcriptase, and ribonuclease H characteristic of pararetroviruses . The last two ORFs (13 and 14 kDa) overlap ORF 3 and are not present in the other badnaviruses described.

Biochemistry, 1993 Sep 28, 32(38), 10178 - 84
Determinants of coenzyme specificity in glyceraldehyde-3-phosphate dehydrogenase: role of the acidic residue in the fingerprint region of the nucleotide binding fold; Clermont S et al.; On the basis of the three-dimensional structure of the glycolytic NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and of sequence comparison with the photosynthetic NAD(P)-dependent GAPDH of the chloroplast, a series of mutants of GAPDH from Bacillus stearothermophilus have been constructed . The results deduced from kinetic and binding studies suggest that the absence of activity of the wild-type GAPDH with NADP as a cofactor is the consequence of at least three factors: (1) steric hindrance, (2) electrostatic repulsion between the charged carboxyl group of Asp32 and the 2'PO4, and (3) structural determinants at the subunit interface of the tetramer . The best value for kcat/KM and KD for NADP was observed for the D32A-L187A-P188S mutant . This triple mutation leads to a switch in favor of NADP specificity but with a kcat/KM ratio 50- and 80-fold less than that observed for the wild type with NAD and for the chloroplast GAPDH with NADP, respectively . Substituting the invariant chloroplastic Thr33-Gly34-Gly35 for the B . stearothermophilus Leu33-Thr34-Asp35 residues on the double mutant Ala187-Ser188 does not improve significantly the affinity for NADP while substituting Ala32 for Asp32 on the double mutant does . Clearly, other subtle adjustments in the adenosine subsite are needed to reconcile the presence of the carboxylate group of Asp32 and the 2'-phosphate of NADP . Kinetic studies indicate a change of the rate-limiting step for the mutants . This could be the consequence of an incomplete apo-holo transition.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1993 Sep 27, 331(1-2), 165 - 72
Identification of the barstar binding site of barnase by NMR spectroscopy and hydrogen-deuterium exchange; Jones DN et al.; The extracellular ribonuclease from Bacillus amyloliquifaciens, barnase, forms a tightly-bound one-to-one complex with its intracellular inhibitor barstar . The barstar binding site on barnase was characterized by comparing the differences in the chemical shift and hydrogen-deuterium exchange rates between free and bound barnase . Chemical shift assignments of barnase in the complex with barstar were determined from 3D NOESY-HMQC and TOCSY-HMQC spectra of a complex that had been prepared with uniformly 15N-labelled barnase and unlabelled barstar . Hydrogen exchange rates were obtained from an analysis of a series of {15N}HMQC spectra of a sample prepared in the same manner exchanged into D2O . The largest changes in either chemical shift or hydrogen-deuterium exchange rate are observed for residues located in the active-site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site.

J Mol Biol, 1993 Sep 20, 233(2), 293 - 304
Folding of subtilisin BPN': role of the pro-sequence; Eder J et al.; Subtilisin BPN' is an extracellular serine protease from Bacillus amyloliquefaciens that requires an N-terminal 77 amino acid pro-sequence for correct folding of the catalytic domain . We have expressed an inactive, stable pro-subtilisin variant in Escherichia coli and show that it has structural properties similar to native subtilisin in terms of its near- and far-UV circular dichroism spectra, its compactness, and its capacity to bind calcium ions stoichiometrically . Unlike subtilisin, the pro-subtilisin variant unfolds reversibly with guanidinium chloride, and unfolding occurs via a folding intermediate . This intermediate is similar to the metastable intermediate state recently found for folding of subtilisin in the absence of the pro-sequence . The intermediate state has native-like secondary but little tertiary structure, and has a compactness between that of the native and unfolded state . Pro-subtilisin folds from the intermediate to the folded state in a single co-operative transition mediated by the pro-sequence . The isolated pro-sequence does not appear from its circular dichroism and 1H-NMR spectrum to have enough intrinsic stabilizing interactions to fold autonomously . However, the difference circular dichroism spectra of the pro-subtilisin variant and native subtilisin suggest that it is folded in the context of the pro-subtilisin molecule . The inability of the pro-subtilisin variant to bind a polypeptide inhibitor supports further the hypothesis that the pro-sequence interacts with subtilisin in the region where the active site is exposed . Our results suggest that the interactions provided by the pro-sequence are important only late on the folding pathway of pro-subtilisin and stabilize the transition state for folding . Kinetic analysis of the refolding reaction in the presence and absence of the pro-sequence reveal this stabilization to be in excess of 7.5 kcal/mol; folding is accelerated more than five orders of magnitude.

Eur J Biochem, 1993 Sep 15, 216(3), 829 - 34
Determinants for the enhanced thermostability of hybrid (1-3,1-4)-beta-glucanases; Politz O et al.; Hybrid (1-3,1-4)-beta-glucanases which contain an N-terminal region derived from the Bacillus amyloliquefaciens enzyme and a C-terminal region of the closely related B . macerans enzyme may exhibit a thermostability superior to both parental enzymes . A systematic series of hybrid enzymes were constructed in order to delineate the amino acid residues that affect protein stability . Hybrid enzymes with between one and four of the N-terminal residues for the mature B . amyloliquefaciens (1-3,1-4)-beta-glucanase exhibit no significant changes in biochemical characteristics as compared with the parental B . macerans enzyme . However, significantly enhanced thermostability was observed in the hybrid enzyme containing an N-terminal segment of eight amino acid residues derived from the B . amyloliquefaciens enzyme . Site-directed mutagenesis revealed that the combined effect of Gln1, Thr2, Ser5 and Phe7 confer enhanced stability on hybrid enzymes, probably by improving the hydrogen bonding that stabilizes the interactions between the N-terminal and the centre of the folded molecule, as well as between the two termini of the polypeptide chain . Furthermore, deletion of Tyr13 in the hybrid enzyme containing the 12 N-terminal amino acids from the B . amyloliquefaciens (1-3,1-4)-beta-glucanase results in a dramatic increase in stability at 70 degrees C with the half-life of 6 min increased to around 4 h . This is twofold higher than the hitherto most stable hybrid enzyme in which the N-terminal domain consisted of 16 residues of the B . amyloliquefaciens enzyme.

Biochemistry, 1993 Sep 7, 32(35), 8994 - 9
Mutational replacements of the amino acid residues forming the hydrophobic S4 binding pocket of subtilisin 309 from Bacillus lentus; Sorensen SB et al.; The amino acid side chains of Ile107, Leu126, and Leu135 participate in the formation of the important hydrophobic S4 binding pocket of the subtilisin Savinase . Ile107 and Leu126, located on each side of the pocket, point toward each other, and Leu135 is situated at the bottom of the pocket . These amino acid residues have been substituted for other hydrophobic amino acid residues by site-directed mutagenesis, and the resulting enzymes have been characterized with respect to their P4 substrate preferences . The Leu126-->Ala or Phe substitutions reduce kcat/KM for the hydrolysis of all substrates to around 5% without altering the substrate preference . It is concluded that Leu126 is an essential structural part of the pocket which cannot be replaced without seriously affecting catalysis, consistent with the fact that Leu126 is conserved among all subtilisins . In contrast, the Ile107-->Gly, Ala, Val, Leu, or Phe and Leu135-->Ala, Val, or Phe substitutions strongly influence the P4 substrate preference, and some of the mutants exhibit large specificity changes for particular substrates when compared to wild-type Savinase . The results can be rationalized on the basis of Ile107 and Leu135 being responsible for steric repulsion of branched aliphatic and aromatic P4 side chains, respectively . Leu135 exclusively interacts with aromatic P4 side chains, and its replacement with less bulky amino acid residues alleviates steric repulsion such that the activity toward this type of substrates is enhanced . Conversely, the introduction of a more bulky amino acid residue at position 135 produces more steric repulsion and reduces the activity toward substrates with aromatic P4 side chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1993 Sep 6, 131(1), 93 - 5
Bsp24I, a new unusual restriction endonuclease; Degtyarev SK et al.; A new restriction endonuclease, Bsp24I, from Bacillus species 24, recognizing: {formula: see text} has been isolated . Its specificity and cleavage points were determined.

Gene, 1993 Sep 6, 131(1), 113 - 7
Identification and cloning of genes differentially expressed in the virulent strain of Mycobacterium tuberculosis; Kinger AK et al.; The mechanism(s) used by Mycobacterium tuberculosis to establish disease in the human host are not well understood . The virulent M . tuberculosis H37Rv strain and its avirulent derivative M . tuberculosis H37Ra provide an attractive system for the identification of virulence-specific genes of the tubercle bacillus . Differentially expressed genes in the virulent strain of M . tuberculosis (dev genes) were identified by screening a plasmid gene bank of H37Rv with a cDNA probe that was enriched in dev transcripts by subtraction of RNAs common to H37Ra . Individual dev clones coded for RNA transcripts that were differentially expressed in H37Rv in comparison to H37Ra . In contrast, mRNAs and stable RNAs that were commonly expressed in both the strains were present in equivalent amounts . The identification and cloning of dev genes marks the first step in defining bacterial gene(s) involved in the pathogenesis of M . tuberculosis.

Clin Exp Immunol, 1993 Sep, 93(3), 382 - 6
Successful primate immunization with peptides conjugated to purified protein derivative or mycobacterial heat shock proteins in the absence of adjuvants; Perraut R et al.; We have previously shown in mice that antibodies can be induced to synthetic malaria peptides conjugated to mycobacterial antigens, such as purified protein derivative (PPD) or heat shock proteins (hsp), and given in the absence of adjuvants after a previous priming with bacille Calmette-Guerin (BCG) . In the present study we investigated this model of immunization in the non-human primates, Saimiri sciureus monkeys . Monkeys primed with BCG subcutaneously and then immunized subcutaneously with the Plasmodium falciparum sporozoite (NANP)40 synthetic peptide conjugated to PPD or mycobacterial hsp of 65 or 70 kD, in the absence of adjuvants, produced antipeptide and anti-sporozoite IgG antibodies . Interestingly, the carrier effect of the hsp of 70 kD for the induction of anti-(NANP)40 antibodies was also observed in the absence of a previous priming with BCG . These data suggest that such a vaccination strategy may be applied to humans.

Chest, 1993 Sep, 104(3), 973 - 5
Bronchopulmonary bacillary angiomatosis; Foltzer MA et al.; A man with prior AIDS developed acute febrile interstitial pneumonitis, hilar and paratracheal adenopathy, and bronchial polyps . The polyps were histologically typical for bacillary angiomatosis and complete symptomatic and radiographic response to oral clarithromycin was seen . The clinical presentation of bacillary angiomatosis includes pulmonary disease and in particular bronchial polyps; clarithromycin is an effective oral antibiotic.

Scand J Immunol, 1993 Sep, 38(3), 239 - 46
Specific activation of human peripheral blood gamma/delta + lymphocytes by sonicated antigens of Mycobacterium tuberculosis: role in vitro in killing human bladder carcinoma cell lines; Wang MH et al.; Tumour regression induced in cancer patients by local instillation of Bacillus Calmette-Guerin (BCG) into the bladder has been considered to be mainly mediated by activated cellular immunity and inflammatory reactions . In the present study we investigated the cytotoxicity of T cells bearing gamma/delta T-cell receptors (gamma/delta + cells) against bladder carcinoma cells in vitro . Long-term cultured gamma/delta + T-cell lines from peripheral blood lymphocytes of healthy donors were established by stimulation with sonicated cell wall-associated antigens of Mycobacterium tuberculosis (SMA) . These gamma/delta + T cells lack the natural killer (NK) markers CD16 and CD56, as determined by flow cytometry . The SMA-specific gamma/delta + T cells exhibited profound cytotoxicity against two NK-resistant bladder tumour cell lines as well as against NK-sensitive tumour cells in a non-major histocompatibility complex-restricted manner . The pattern of tumour cells killed by gamma/delta + T cells differed significantly from those of NK cells and lymphokine-activated killer LAK cells . Furthermore, we tested the effects of recombinant human cytokines, including interleukin (IL)-1, IL-2, IL-4, IL-6, interferon (IFN)-gamma and tumour necrosis factor (TNF), on gamma/delta + T-cell-mediated cytotoxicity . It was shown that the addition of recombinant TNF in co-incubation could augment gamma/delta + T-cell-mediated killing of two bladder tumour cell lines, but not of cells of the erythroleukaemia cell line K562 . Based on these results it was concluded that mycobacterial antigens could specifically activate resting gamma/delta + T cells . The cytotoxicity of gamma/delta + T cells against bladder tumour cells and its selective enhancement by TNF may be an important mechanism involved in bladder tumour regression induced by intravesical instillation of BCG.

Insect Biochem Mol Biol, 1993 Sep, 23(6), 663 - 73
Purification and characterization of a trypsin-like digestive enzyme from spruce budworm (Choristoneura fumiferana) responsible for the activation of delta-endotoxin from Bacillus thuringiensis; Milne R et al.; A trypsin-like enzyme purified from spruce budworm (Choristoneura fumiferana) gut juice has a molecular mass of 25 kDa and its pH activity profile indicates a pKa of 8 . Sequence homology with bovine trypsin of the N-terminus and active site, and the ionization dependence for catalysis, reflect the typical trypsin-like activities measured . The action of this enzyme (designated CFT-1) is compared to the neat gut juice with regard to the proteolytic activation of the delta-endotoxin from Bacillus thuringiensis.

Cancer, 1993 Sep 1, 72(5), 1749 - 55
Intravesical 4'-epi-doxorubicin (epirubicin) versus bacillus Calmette-Guérin . A controlled prospective study on the prophylaxis of superficial bladder cancer; Melekos MD et al.; BACKGROUND . The selection of the most appropriate antineoplastic agent and optimal treatment schedule for the prophylaxis of superficial bladder cancer against tumor recurrences is the subject of continual investigations . METHODS . A controlled prospective trial involving 161 patients evaluated and compared the efficacy of intravesical epirubicin and bacillus Calmette-Guerin (BCG) administration as prophylaxis against recurrences after complete transurethral resection of superficial bladder cancer . The treatment schedule, consisting of one 6- or 8-week course of instillations (50 mg epirubicin or 150 mg BCG per instillation) followed by single maintenance doses to the responders at follow-up examinations, was modified for those of the initial responders who were at high risk for recurrence and who received an additional separate 4-week course of treatment 6 months after the start of therapy . RESULTS . Sixty percent of the patients treated with epirubicin, 68% of the patients treated with BCG, and 41% of the control subjects, who underwent resection only, remained free of recurrences for a mean follow-up of 32.9 months . The only significant difference was found between patients treated with BCG and control subjects, in favor of the former . Conversely, recurrence rate per 100 patient-months and mean interval to recurrence showed both drugs to be superior to resection alone regarding several tumor characteristics . However, a significant benefit in favor of BCG when compared with epirubicin was shown in those patients who had Stage T1 and Grade 3 tumors at presentation . CONCLUSIONS . Intravesical epirubicin and BCG were superior to transurethral resection alone in the prophylaxis of superficial bladder cancer, but with respect to superficially invasive and high-grade tumors, BCG demonstrated a remarkable advantage.

Br Vet J, 1993 Sep-Oct, 149(5), 405 - 17
HIV/AIDS and its implications for the control of animal tuberculosis; Daborn CJ et al.; The HIV/AIDS pandemic is associated with a number of opportunist mycobacterial infections, principally tuberculosis and disease due to the avian tubercle bacillus, Mycobacterium avium . Tuberculosis occurring early in the course of HIV infection is usually caused by M . tuberculosis . However some cases are due to the bovine tubercle bacillus, M . bovis, which, in turn, is transmissible from man to animals, principally by the aerogenous route although the majority of cases in man are non-pulmonary . These two mycobacterial species may be differentiated by means of a set of simple tests . The quality and quantity of information on the world-wide distribution and prevalence of bovine and human tuberculosis due to M . bovis is not uniform . There is a notable paucity of information from the tropics but available reports suggest that there are significant levels of bovine tuberculosis . If correct, this information has serious public health implications in the light of the current HIV/AIDS epidemic . Urgent investigation is required so that appropriate control measures can be instituted where indicated and possible . The avian tubercle bacillus is a very common opportunistic pathogen in the late stage of AIDS but infection leading to disease is extremely rare in healthy, HIV-negative persons . Because of its widespread environmental distribution, infection by this pathogen cannot be prevented.

Trans R Soc Trop Med Hyg, 1993 Sep-Oct, 87(5), 500 - 3
Chemotherapy of leprosy--current status and future prospects; Waters MF; The introduction of multi-drug therapy (MDT) by the World Health Organization in 1982 has proved to be the most important advance in the management and control of leprosy since the first use of the sulphone drugs 40 years earlier . For the first time, the number of registered leprosy cases has shown a decline from a peak of 5.37 million in 1985 to 3.1 million in February 1992 . The 2 standard MDT regimens have proved simple to apply in most parts of the world, are relatively cheap, generally acceptable, and have shown remarkably few toxic side-effects . Nevertheless, difficulties have arisen in distinguishing between multibacillary and paucibacillary leprosy, especially when skin smears are of poor quality . Relapses in paucibacillary leprosy have proved difficult to distinguish from late reversal reactions . In multibacillary leprosy, the duration of treatment, 2-10 years in lepromatous leprosy, is a source of difficulty, and in addition light-skinned patients dislike the skin discolouration caused by clofazimine, for fear that their diagnosis might be discovered . The discovery that 3 different groups of drugs are highly bactericidal for the leprosy bacillus, although not so rapidly bactericidal as rifampicin, raises the possibility of having simplified, shorter, or better supervised regimens in the future as second generation MDT . These drugs include the 4-fluoroquinolones, pefloxacin, ofloxacin and sparfloxacin, the tetracycline minocycline, and the macrolide clarithromycin . Finally, in low-prevalence areas it is opportune to consider chemoprophylaxis and immunoprophylaxis for child contacts of lepromatous patients.

Bioorg Khim, 1993 Sep, 19(9), 853 - 61
{Complete primary structure of Bacillus thuringiensis extracellular ribonuclease}; Dement'ev AA et al.; Complete primary structure of an extracellular low molecular mass ribonuclease of Bacillus thuringiensis was determined using Edman degradation and mass-spectrometry analysis of individual peptides obtained after hydrolysis of the protein by cyanogen bromide and staphylococcal protease . The peptides were isolated and purified by HPLC and denaturing PAGE . The enzyme consists of 109 amino acid residues (Asp 8, Asn 6, Thr 6, Ser 10, Glu 3, Gln 1, Pro 3, Gly 9, Ala 12, Val 7, Ile 7, Leu 7, Tyr 7, Phe 4, His 1, Arg 10, Trp 3 and Lys 5) and has a molecular weight of 12182 Da . A single difference was detected between primary structures of the enzyme and an extracellular ribonuclease of B . intermedius.

J Am Mosq Control Assoc, 1993 Sep, 9(3), 344 - 5
Field trial of two Bacillus thuringiensis var . israelensis formulations for control of Aedes species mosquitoes in Michigan woodlands; Wilmot TR et al.; Vectobac and Bactimos corn cob granules were evaluated for control of Aedes species mosquito larvae in woodland pools . No significant differences were seen between the 2 formulations . Both provided greater than 90% control at application rates as low as 100 mg/m2 (0.89 lb/acre) and greater than 98% control at label-specified field application rates (2.5 or 5 lb/acre).

J Am Mosq Control Assoc, 1993 Sep, 9(3), 330 - 4
Use of cellular fatty acid analysis to characterize commercial brands of Bacillus thuringiensis var . israelensis; Siegel JP et al.; The cellular fatty acid composition of Bacillus thuringiensis var . israelensis (B.t.i.) from 5 commercial brands (Vectobac, Acrobe, Skeetal, Bactimos and Teknar), as well as of the current International Standard for B.t.i . (IPS 82), was determined using a Hewlett-Packard Microbial Identification System . The original strain of B.t.i., B.t . var . kurstaki, B.t . var . thuringiensis, B.t . var . morrisoni and Bacillus sphaericus strain 2362 were used as outgroups . Acrobe, Bactimos, Teknar, Vectobac and IPS 82 consisted of the same strain . Skeetal represented a different strain than the other commercially produced B.t.i . Our results indicate that cellular fatty acid analysis can be used to distinguish among the forms of B.t.i . produced by various manufacturers.

J Am Mosq Control Assoc, 1993 Sep, 9(3), 325 - 9
Field trials of Bacillus thuringiensis H-14 and Bacillus sphaericus (strain 2362) formulations against Anopheles arabiensis in the central highlands of Madagascar; Romi R et al.; Malaria is highly endemic and unstable in the central Highlands plateau of Madagascar . The infection is seasonally transmitted by Anopheles funestus and An . arabiensis . The latter species is abundant especially in rice-growing areas . The field efficacies of commercial formulations of Bacillus thuringiensis H-14 and B . sphaericus (strain 2362) were assessed against An . arabiensis in 5 types of larval habitats . The granular formulation of B . thuringiensis (Vectobac GR) provided very good control in small ponds and rainwater ditches . In ricefields complete larval control was achieved with Vectobac GR and the flowable concentrate (Vectobac 12AS) at 2.5 kg/ha and 0.6 liter/ha, respectively . The granular formulation of B . sphaericus (ABG 6185) showed activity similar to that of Vectobac GR in small pools and ricefields . ABG 6185 was less effective in rainwater ditches where it gave satisfactory control at rates not lower than 6 kg/ha.

Immunology, 1993 Sep, 80(1), 151 - 6
Pentoxifylline: a potent inhibitor of IL-2 and IFN-gamma biosynthesis and BCG-induced cytotoxicity; Thanhauser A et al.; In the present study we investigated the influence of pentoxifylline (POF) on bacillus Calmette-Guerin (BCG)- and phytohaemagglutinin (PHA)-induced DNA synthesis and cytokine release, and BCG-induced cytotoxicity of human peripheral blood mononuclear cells (PBMC) . DNA synthesis of PBMC stimulated with either BCG or PHA was inhibited by POF . We also demonstrated that the addition of POF led to a POF dose-dependent decrease of the release of the cytokines interleukin (IL)-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) . The release of IL-6 remained unaffected . With respect to the inhibition of BCG-induced IL-2 and IFN-gamma release POF is active at the transcriptional (mRNA) level, as found by polymerase chain reaction (PCR) . However, PHA-induced mRNA expression of these lymphokines is not affected by POF . Thus, the existence of a post-transcriptional regulation of PHA-induced cytokine release by POF can be assumed . The observed inhibition of cytokine release is correlated with a potent inhibitory effect of POF on BCG-induced cytotoxicity against bladder tumour cell lines . This effect is reversible.

Zhonghua Min Guo Xiao Er Ke Yi Xue Hui Za Zhi, 1993 Sep-Oct, 34(5), 412 - 7
{Hemophilus aphrophilus meningitis: report of one case}; Wang KF et al.; Hemophilus aphrophilus, a gram negative, capnophilic slow growing bacillus, is a rarely recognized pathogen in meningitis and is most frequently seen in patients with either endocarditis or brain abscess . This article reported one case with Hemophilus aphrophilus meningitis . A 10-year-old boy presented at the emergency room with chief complaint of fever for 2 days and sudden onset of loss of consciousness . Hemophilus aphrophilus was isolated from the blood and cerebrospinal fluid . Aqueous penicillin and chloramphenicol were given for three weeks . The patient discharged without any sequelae . Three months later, fever and consciousness disturbance were noted again . No pathogen was isolated from the cerebrospinal fluid and blood culture this time, but CSF finding was consistent with bacterial meningitis . Aqueous penicillin and chloramphenicol were readministered for 30 days . The patient recovered smoothly . Because the patient had no history of CSF rhinorrhea or hypogammaglobulinemia, recurrence of the bacterial meningitis could be due to incomplete treatment during the first admission . Brain computed tomography (CT) done during the two admissions showed focal cortical enhancement in the fronto-temporo-parietal region . This is presumed to indicate infarction over these regions . The findings of brain CT are in accordance with the development of hemiplegia in the patient . It is still unknown, however, whether Hemophilus aphrophilus meningitis also causes a higher incidence of brain infarction, which was frequently noted in patients with Hemophilus influenzae meningitis.

Plasmid, 1993 Sep, 30(2), 150 - 4
A transformation vector for dictyostelium discoideum with a new selectable marker bsr; Sutoh K; A new selectable marker for transformation of Dictyostelium discoideum cells was constructed by using the bsr gene from Bacillus cereus, which confers resistance to Blasticidin S . The bsr gene was driven by Dictyostelium actin 15 promoter and Dictyostelium actin 8 terminator for expression in Dictyostelium cells . To demonstrate the feasibility of using the bsr marker, we constructed an extrachromosomal replication vector by replacing the Neor gene of pnDeI (B . Leiting and A . Noegel (1988) Plasmid 20, 241-248) with the bsr gene cassette . A mutant Dictyostelium actin 15 gene was constructed and inserted into the vector . Dictyostelium cells were transformed with the resulting vector and then transformants were selected with Blasticidin S . The selected cells showed high level expression of the mutant actin, indicating an efficient selection of transformed cells with the bsr marker.

Plasmid, 1993 Sep, 30(2), 141 - 9
IS231V and W from Bacillus thuringiensis subsp . israelensis, two distant members of the IS231 family of insertion sequences; Rezsohazy R et al.; IS231 constitutes a family of related insertion sequences (IS) from Bacillus thuringiensis . Two new IS231-related elements, IS231V and IS231W, have been isolated from the 72-MDa plasmid of B . thuringiensis subsp . israelensis . These closely related 1964-bp IS are delimited by 22-bp imperfect inverted repeats strongly similar to those of the other iso-IS231 . Although the other known IS231 harbor a single long open reading frame (ORF), IS231V and W display two slightly overlapping ORF on the same DNA strand . They show about 50% identity with the transposase of the other iso-IS231 . A frameshifting model is proposed for the synthesis of a fusion product which would constitute their active transposase.

J Invertebr Pathol, 1993 Sep, 62(2), 131 - 6
Protection from ultraviolet irradiation by melanin of mosquitocidal activity of Bacillus thuringiensis var . israelensis; Liu YT et al.; A process for production, isolation, and purification of melanin produced by the fermentation of Streptomyces lividans 66 harboring a recombinant plasmid pIJ702-bearing tyrosinase gene has been developed . The efficacy of melanin in the protection of mosquito larvacidal activity of Bacillus thuringiensis var . israelensis against uv light has been studied . Results obtained by the live cell counts and the bioassay of residual mosquitocidal activity of B . thuringiensis var . israelensis after exposure to uv radiation showed that melanin is an excellent photoprotective agent.

J Invertebr Pathol, 1993 Sep, 62(2), 121 - 30
Expression of full-length and truncated forms of crystal protein genes from Bacillus thuringiensis subsp . kurstaki in a baculovirus and pathogenicity of the recombinant viruses; Ribeiro BM et al.; Full-length and truncated forms of the crystal protein gene cryIA(b) derived from Bacillus thuringiensis subsp . kurstaki HD-1 and full-length cryIA(c) gene of B . thuringiensis subsp . kurstaki HD-73 were introduced into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus, in place of the polyhedrin gene . All gene constructs were expressed at high levels in insect cells and insects upon infection with the recombinant viruses . The protein products were shown to be biologically and immunologically similar to the natural crystal protein . The expressed proteins formed crystals (in insects) up to 10 times bigger (in length) than their bacterial counterpart . The LT50 values for recombinant viruses were not significantly shorter than wild-type virus.

An Med Interna, 1993 Sep, 10(9), 427 - 32
{An epidemiological study of tuberculosis in the health area of Santiago de Compostela during the years 1989, 1990 and 1991}; Salgueiro Rodriguez M et al.; In order to assess the incidence of tuberculous disease in our health area, we reviewed the clinical records of patients from the three hospitals of the area under study during the years 1989, 1990 and 1991, who had positive bacilloscopy, positive Lowenstein's culture in any specimen and/or compatible anatomopathologic report . After excluding 26 patients because they belonged to other health areas, 885 patients remained in the study, out of which 251 (64% men and 36% women) were from the year 1989, 270 (64% men and 36% women) from the year 1990 and 364 (62% men and 38% women) from the year 1991 . The mean age was 38.4 (SD 20.5) . Fifty-one percent of the patients were between 20 and 35 years old . The rate of new cases was 65.87 per 100.000 population in 1989, 71.05 in 1990 and 95.53 in 1991 . Seventy-four cases were HIV-positive (8%) . Tuberculous meningitis was present in 12 patients . The highest mortality was 1.79 per 100.000 population in 1990 . We conclude that tuberculosis presents a medium-high incidence in our health area.

Urologe A, 1993 Sep, 32(5), 374 - 81
{Immunotherapy of superficial bladder cancer}; Schmitz-Drager BJ et al.; Because patients with superficial bladder cancer are in a high-risk group where tumor progression is concerned, topical therapeutic strategies are necessary to prevent tumor recurrence and tumor progression . Based on experimental studies and several case reports, during the last two decades immunotherapy for superficial bladder cancer has been developed . The effects of topical instillation of bacillus Calmette-Guerin (BCG) has been carefully investigated in numerous clinical trials . Especially patients with carcinoma in situ appear to benefit from BCG therapy . Other types of local immunotherapy, e.g., instillation of interferons, interleukins, and keyhole limpet hemocyanin have been found to have fewer side effects than BDG . These new approaches are currently under clinical investigation.

East Afr Med J, 1993 Sep, 70(9), 568 - 71
BCG scar survey among primary school children in Kenya (1986-1990); Kwamanga D et al.; Bacillus Calmette Guerin (BCG) vaccination is essential in the control of tuberculosis (TB) especially in countries like Kenya where TB is still a public health problem and BCG is given to all children at birth as a matter of policy . The present survey was launched in 1986 to assess both BCG vaccination coverage and to compare its findings with the 1979/81 BCG scar survey results . Using random cluster sampling procedures, all primary school children aged 6-13 years from schools in each of 12 districts were included in the survey . A total of 46357 school children were registered . Of these, 3642 (7.9%) were excluded from the survey for a variety of reasons . Of the remaining 42715 (92.1%) children, 26781 (62.7%) had BCG scars present . Overall there was a significant upward trend of 15% in BCG vaccination coverage in the country . However, in some districts the coverage was found to have fallen quite significantlyPIP: In 1986-1990, researchers conducted a BCG scar survey in 360 randomly selected primary schools in 12 districts in Kenya to determine BCG vaccination coverage . They used primary schools because more than 70% of all school age children were enrolled in school . They compared this survey's findings with those of the 1979-1981 BCG scar house-to-house survey . The districts included Elgeyo Marakwet, Kakamega, Kilifi, Kisii, Kitui, Siagya, Kwale, Meru, Muranga, Nakuru, Nairobi, and South Nyanza . The ages of the 42,715 healthy children ranged from 6-13 years old . 26,781 (62.7%) children had a BCG scar, indicating that the National Tuberculosis Control Program had not yet reached its target of 70% BCG vaccination coverage . Nairobi had the highest BCG coverage, while Kisii district had the lowest BCG coverage (82.73% vs . 44.01%) . BCG coverage decreased as age increased (p .001) . For example, 6-year-old males and females had a BCG coverage rate of 64.43% and 62.39%, respectively, while the corresponding figures for 13-year-olds were 52.93% and 49.13% . BCG vaccination coverage increased significantly between the two surveys (an increase of 15%) (60.8% vs . 62.7%; p .01) . South Nyanza district experienced the greatest improvement in BCG coverage between the 2 surveys, while Kilifi district experienced the greatest decline in coverage . The greatest upward trend was observed in the Western and Rift Valley provinces, while the greatest downward trend was observed in the Coast and Eastern provinces .

In Vivo, 1993 Sep-Oct, 7(5), 463 - 6
Asialo-positive cells are overexpressed whereas IL-2 induced LAK activity is impaired in mice hyperimmunized with an immunomodulating mycobacterial fraction; Rashid G et al.; Previously reported studies revealed that extensive immunization with the Methanol Extraction Residue (MER) of BCG tubercle bacillus in Incomplete Freund's Adjuvant (IFA) induced marked suppression of T and B cell functions and that immunosuppression was correlated with marked decrease in splenic T-cell population and marked increase in the splenic macrophage population . The purpose of the present work was to determine if extensive immunization with MER in IFA also induced changes in splenic asialo antigen positive (NK) population and in the potential of induction of splenic LAK activity in vitro by recombinant IL-2 . By comparison with other test groups, namely mice injected with IFA only, injected once with MER in aqueous suspension and normal, untreated mice, hyperimmunization with MER resulted in marked increase in expression of asialo positive (NK) splenic cells whereas induction of splenic LAK activity by IL-2 was markedly depressed . Splenic macrophages originated from MER hyperimmunized mice had no effect on induction of LAK activity by IL-2 in a normal splenic population.

J Urol, 1993 Sep, 150(3), 1018 - 23
T-cell subsets required for intravesical BCG immunotherapy for bladder cancer; Ratliff TL et al.; Intravesical bacille Calmette-Guerin (BCG) has been shown in prospective randomized clinical trials to be the treatment of choice for superficial bladder cancer . In this investigation we evaluated the role of CD4 and CD8 lymphocytes in the antitumor response . Monoclonal antibodies to thy 1.2, CD8, CD4 and an isotype control were injected intravenously to deplete T cell populations . After depletion (verified by flow cytometry), BCG therapy was initiated . The results demonstrate that the depletion of either CD4 or CD8 T cell subsets eliminated BCG-mediated antitumor activity . Footpad delayed type hypersensitivity (DTH) was aborted only in CD4 depleted mice; it was essentially unchanged in CD8 depleted mice . However, the presence of DTH was not sufficient for induction of BCG-mediated antitumor activity . Exogenous IL-2 at levels sufficient to induce lymphokine activated killer cell activity did not substitute for CD4 cells . There was no evidence for the induction of protective immunity to the tumor after BCG therapy . These results demonstrate the requirement for T lymphocytes in BCG-mediated antitumor activity and further demonstrate that the presence of both CD4 and CD8 subsets are required . CD8 depletion experiments suggest that the presence of CD4-mediated DTH is not sufficient for the induction of antitumor activity . Furthermore, these data suggest that BCG-mediated antitumor activity is a localized phenomenon that does not induce protective immunity.

Biosci Biotechnol Biochem, 1993 Sep, 57(9), 1518 - 25
Structure of the 87-kDa beta-1,3-glucanase gene of Bacillus circulans IAM1165 and properties of the enzyme accumulated in the periplasm of Escherichia coli carrying the gene; Yamamoto M et al.; The nucleotides of a gene for the extracellular 87-kDa beta-1,3-glucanase of Bacillus circulans IAM1165 and its flanking regions were sequenced . The sequence showed an open reading frame for 877 amino acids, which corresponds to a precursor of the beta-1,3-glucanase . The coding region of 2631 bp is flanked by putative promoter and transcription terminator sequences . The signal peptide was considered to be consisted of 38 amino acids . The amino acid sequence of the mature enzyme composed of 839 amino acids showed high homology to that of the enzyme from B . circulans WL-12, although these enzymes are different in their sizes . A catalytic domain of the enzyme was estimated central region of the sequence on the basis of comparison of amono acid sequences of beta-1,3- or beta-1,3:1,4-glucanases . Properties of the periplasmic enzyme produced in Escherichia coli carrying the gene were identical with those of the extracellular enzyme produced by B . circulans IAM1165.

Biosci Biotechnol Biochem, 1993 Sep, 57(9), 1454 - 60
Direct sequencing of superoxide dismutase genes from two bacterial strains amplified by polymerase chain reaction; Lee SO et al.; The nucleotides of the Mn-superoxide dismutase (SOD) gene of Bacillus circulans and the Fe-SOD gene of Aerobacter aerogenes were sequenced by PCR . These SOD genes were specifically amplified by using oligonucleotide primers corresponding to the amino-terminal amino acid sequences and the antisense strand primer corresponding to the common amino acid sequence near the carboxyl-terminus among various Mn- and Fe-SODs thus far sequenced . The PCR products amplified from B . circulans and A . aerogenes genes contained a 486-nucleotide sequence encoding 162 amino acids and a 507-nucleotide sequence encoding 169 amino acids, respectively . Each sequence seemed to contain most of the open reading frame encoding the SOD protein when compared with other sequenced SODs . The two amino acid sequences deduced from the nucleotide sequences of PCR products had an identity of 66.1% . However, the SODs from B . circulans and A . aerogenes were immunologically distinct from each other judging from an immunoprecipitation test . The two SODs had high homologies with other bacterial Mn-SODs, especially the highest homology of 75.4% and 66.7%, respectively, with the B . stearothermophilus Mn-SOD . Genomic Southern hybridization suggested that each PCR product of the bacterial genes that were synthesized and sequenced was the product of the sole SOD gene in each bacterium.

Curr Microbiol, 1993 Sep, 27(3), 153 - 6
Low temperature protocol for efficient transformation of Mycobacterium smegmatis spheroplasts; Naser SA et al.; Spheroplasts of Mycobacterium smegmatis LM15, strain 607, were prepared by a combined treatment with glycine and lysozyme . The spheroplasts were tested for ability to take up and express purified mycobacteriophage DNA . Exposure of 1.0 x 10(8) to 1.0 x 10(9) spheroplasts to saturating DNA (1 microgram) for 15 min at 5 degrees C resulted in a transfection efficiency of approximately 0.009% . The transfer of the beta-lactamase marker with DNA purified from strain LM15 to spheroplasts of a beta-lactamase-negative mutant, strain LM144, was achieved . The DNA-treated cultures, after reversion to the bacillary form, contained 20-fold more penicillin-resistant cells than the nontreated control culture . Approximately 80% of the penicillin-resistant colonies from the DNA-treated cells were positive for beta-lactamase Cell-free extracts of penicillin-resistant transformants contained beta-lactamase activity that ranged from 0.046 to 0.134 micromol of benzylpenicillin hydrolyzed/min per mg protein . This low temperature procedure is recommended for high efficiency transformation of M . smegmatis.

FEMS Microbiol Lett, 1993 Sep 1, 112(2), 205 - 10
Phylogenetic analysis of Bacillus sphaericus and development of an oligonucleotide probe specific for mosquito-pathogenic strains; Aquino de Muro M et al.; The 16S rRNA gene sequences for six strains of Bacillus sphaericus representing five distinct DNA homology groups and one strain of Bacillus fusiformis have been determined . An-oligonucleotide probe based on the area approximately between position 186 and 198 (Escherichia coli numbering) of the 16S rRNA gene of the mosquito pathogen, strain 2362, hybridised to DNA from strains of DNA homology group IIA which contains all known mosquito pathogens and not to any of ten other Bacillus species . It can be used to identify potential mosquito-pathogenic strains of B . sphaericus in screening programmes.

Biochemistry, 1993 Aug 31, 32(34), 8836 - 41
Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C; Lewis KA et al.; A series of symmetric short-chain phosphatidylinositols (PI), including dihexanoyl-PI, diheptanoyl-PI (racemic as well as D and L forms), and 2-methoxy inositol-substituted diheptanoyl-PI, have been synthesized, characterized, and used to investigate key mechanistic questions about phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis . Key results include the following: (i) bacterial PI-PLC exhibits a 5-6-fold "interfacial activation" when its substrate is present in an interface as opposed to existing as a monomer in solution (in fact, the similarity to the activation observed with nonspecific PLC enzymes suggests a similarity in activation mechanisms); (ii) the 2-OH must be free since the enzyme cannot hydrolyze diheptanoyl-2-O-methyl-PI (this is most consistent with the formation of inositol cyclic 1,2-phosphate as a necessary step in catalysis); (iii) the inositol ring must have the D stereochemistry (the L-inositol attached to the lipid moiety is neither a substrate nor an inhibitor); and (iv) the presence of noninhibitory L-PI with the D-PI substrate relieves the diacylglycerol product inhibition detected at approximately 30% hydrolysis.

Biochemistry, 1993 Aug 24, 32(33), 8547 - 52
Sensitivity of phospholipase C (Bacillus cereus) activity to lipid packing in sonicated lipid mixtures; Rao NM et al.; We have recently demonstrated that phospholipase C (PLC) activity on membranes decreases in the presence of membrane-active peptides such as alamethicin, gramicidin S, and melittin {Rao, N . M . (1992) Biochem . Biophys . Res . Commun . 182, 682-688} . Since these peptides affect lipid packing in the membrane and induce nonbilayer phases depending on the lipid composition, we tested for the sensitivity of PLC activity to lipid packing . We monitored PLC activities on four lipid systems which demonstrate a transition from the bilayer to the nonbilayer phase as a function of one of the components . The four model systems are (1) dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE); (2) DOPE, DOPC, and cholesterol; (3) DOPE and lysophosphatidylcholine; and (4) DOPC and gramicidin D . On all four lipid systems, the PLC activity was high for lipid in the bilayer phase and decreased as the phase changed to the nonbilayer phase . The phase changes were also monitored in PLC assay conditions on the four model systems by 31P NMR to confirm the observations made with PLC . These results suggest that the lipid in bilayer and nonbilayer phases was differentially susceptible to PLC; hence, PLC activity may be used to monitor isothermal phase transitions at physiological conditions.

Biochem Biophys Res Commun, 1993 Aug 16, 194(3), 1365 - 70
The elastolytic properties of subtilisin GX from alkalophilic Bacillus sp . strain 6644 provides a means of differentiation from other subtilisins; Durham DR; A serine protease exhibiting high activity in alkaline media was purified from alkalophilic Bacillus strain GX6644 . The enzyme, subtilisin GX, has a molecular weight of 25,000 and a pI greater than 9.5 . The protease exhibited high elastolytic activity, but unlike most elastin hydrolyzing enzymes, elastin hydrolysis and binding were not inhibited by 0.1 M NaCl . The elastolytic properties of subtilisin GX together with its specificity toward amino acid phenyl esters functionally distinguishes this protease from other subtilisins . However, comparisons of the available amino-terminal sequence of subtilisin GX with subtilisins from alkalophilic and neutrophilic Bacillus species revealed extensive homology.

Cell, 1993 Aug 13, 74(3), 475 - 82
tRNA as a positive regulator of transcription antitermination in B . subtilis; Grundy FJ et al.; Most Bacillus tRNA synthetase genes are regulated by a common transcription antitermination mechanism but respond individually to limitation for the cognate amino acid . The mRNA leader regions of these genes exhibit extensive structural conservation, with a single codon specific for the appropriate amino acid at the identical position in each structure . Alteration of this sequence in the tyrS gene from UAC (tyrosine) to UUC (phenylalanine) resulted in loss of induction by tyrosine limitation and a switch to induction by phenylalanine limitation . Insertion of an extra base immediately upstream of the codon did not alter regulation, indicating a nontranslational mechanism . A nonsense codon resulted in an uninducible phenotype that was suppressible in a lysyl-tRNA nonsense suppressor mutant, indicating that tRNA acts as an effector.

FEBS Lett, 1993 Aug 9, 328(1-2), 99 - 102
The relationships between transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase of the pyruvate dehydrogenase complex; Robinson BH et al.; The amino acid sequences of four thiamine pyrophosphate-requiring enzymes were aligned with the published amino acid sequence of the transketolase of Hansenula polymorpha . Sequences of the combined alpha and beta subunits of the E1 enzyme of the pyruvate dehydrogenase complexes of Homo sapiens and Bacillus stearothermophilus aligned well with the transketolase while the E1 of the pyruvate dehydrogenase complex of Escherichia coli aligned easily provided a non-aligning segment of 77 amino acids was omitted . The non-acetylating pyruvate decarboxylase of Saccharomyces cerevisiae could only be aligned if the sequence was cut in two with the C-terminus corresponding to the N-terminus of the other TPP-dependent enzymes . Using the published 2.5 A resolution of the X-ray crystal structure of Saccharomyces cerevisiae transketolase as a template we show that a hydrophobic region of the beta-subunit of the PDH E1 alpha beta enzymes likely contains a binding site for the thiazolium ring of TPP and key motifs are retained in common by all the TPP-dependent enzymes considered, which are essential for catalysis.

FEBS Lett, 1993 Aug 2, 327(3), 351 - 4
Kinetics of CO binding to H(+)-motive oxidases of the caa3-type from Bacillus FTU and of the o-type from Escherichia coli; Muntyan MS et al.; The kinetics of CO rebinding with isolated Bacillus FTU caa3-type oxidase and with solubilized Escherichia coli membranes (GO103 strain) containing the o-type oxidase as the main O2-reducing enzyme were studied under reducing conditions by laser flash photolysis of the CO-oxidase complexes . The spectra of the optical absorbance changes upon photolysis were characteristic of CO-caa3- and CO-o-oxidase complexes in Bac . FTU and E . coli, respectively . Small quantities of d-type oxidase in E . coli GO103 membranes were detected . The kinetics of CO reassociation with reduced caa3- and o-type oxidases were monophasic with tau 25-30 ms in both cases.

FEBS Lett, 1993 Aug 2, 327(3), 347 - 50
Kinetics of CO binding to putative Na(+)-motive oxidases of the o-type from Bacillus FTU and of the d-type from Escherichia coli; Muntyan MS et al.; The kinetics of CO reassociation with isolated Bacillus FTU o-type oxidase and with solubilized membranes of Escherichia coli (GO102 strain) containing the d-type oxidase only, upon laser flash photolysis under reducing conditions, were studied . In both cases, kinetics are shown to be composed of three phases (tau 35-70 microseconds, 0.25-0.5 ms and 2-5 ms) . The spectra of the flash-induced absorbance changes of the first kinetic components proved to be characteristic of CO-o- and CO-b595 d-cytochrome complexes in Bac . FTU and E . coli, respectively . The spectra of the second and the third components appeared to be nearly the same in Bac . FTU and E . coli with peaks for the former at 436-437 and 590 nm and troughs at 419-420 and 569 nm; and for the latter with peaks at 436-437 and 558-560 nm and troughs at 419-420 and 575-578 nm . The similarity between the putative Na(+)-pumping Bac . FTU o- and E . coli d-type oxidases and their difference from the H(+)-motive Bac . FTU caa3- and E . coli o-type oxidases are discussed.

J Lipid Res, 1993 Aug, 34(8), 1385 - 92
Degradation of plasma membrane phosphatidylcholine appears not to affect the cellular cholesterol distribution; Porn MI et al.; To clarify the role of possible cholesterol/phosphatidylcholine interactions in cellular cholesterol distribution, we have used a phosphatidylcholine-specific phospholipase C from Bacillus cereus to degrade the cell surface phosphatidylcholine of cultured human fibroblasts . Of cellular phosphatidylcholine, approximately 15% was susceptible to degradation by the phospholipase . In spite of the dramatic redistribution of cellular cholesterol that can be observed after sphingomyelin depletion, the degradation of cell surface phosphatidylcholine did not affect the distribution of cholesterol in fibroblasts . In cholesterol-depleted cells as well as in cholesterol-loaded cells, the size of the cell surface cholesterol pool (susceptible to cholesterol oxidase) remained unchanged after phosphatidylcholine degradation . The rate of cholesterol esterification with {3H}oleic acid and the rate of {3H}cholesterol efflux from fibroblasts to high density lipoproteins also remained unchanged after degradation of plasma membrane phosphatidylcholine . An increase in the level of {3H}cholesterol efflux to high density lipoproteins was observed after degradation of plasma membrane sphingomyelin with exogenous sphingomyelinase, in-contrast to earlier reports, where no such effect was observed . The results suggest that interactions between cholesterol and phosphatidylcholine in the fibroblast plasma membranes are less important than cholesterol/sphingomyelin interactions for the asymmetric distribution of cellular cholesterol.

FEMS Microbiol Lett, 1993 Aug 1, 111(2-3), 255 - 61
Effects on toxicity of eliminating a cleavage site in a predicted interhelical loop in Bacillus thuringiensis CryIVB delta-endotoxin; Angsuthanasombat C et al.; When activated by treatment with mosquito (Aedes aegypti) gut extract, the Bacillus thuringiensis CryIVB delta-endotoxin lysed A . aegypti cells in vitro . SDS-PAGE and N-terminal sequence determination showed that in addition to removal of the C-terminal half of the molecule, the activated toxin had undergone proteolytic cleavage at two internal regions producing 47-48-kDa and 16-18-kDa polypeptides . Aligning the CryIVB protein sequence with the crystallographic structure of the CryIIIA toxin suggested that one set of cleavages occurred in a region before the start of the N-terminal helical bundle and the second cleavage site occurred in a predicted loop between helices 5 and 6 in the bundle at arginine-203 . To investigate the suggestion by Li et al . that interhelical proteolysis is important in the cytolytic mechanism of these toxins, arginine-203 was substituted by alanine . The mutated toxin now resisted proteolysis at this position and showed a marked decrease in cytolysis in vitro but an increase in larvicidal activity.

Prog Urol, 1993 Aug-Sep, 3(4), 608 - 17
{Treatment of stage Ta,T1 and Tis bladder tumors using Calmette-Guérin bacillus vaccine}; Bretheau D et al.; The authors report a series of 71 patients (sex ratio: 1F/4M, mean age: 68 years) with stage Ta (n = 20), T1a (n = 32), T1b (n = 14) and Tis (n = 5) bladder tumours treated by endoscopic resection followed by a course of intravesical BCG instillation (120 mg/week for 6 weeks) . The mean follow-up was 15 months (3-36 months) . The overall recurrence rate was 42% . A recurrence occurred in 50% of Ta (median time to recurrence: 10.1 months), 32% of T1a (median: 5.8 months), 65% of T1b (median: 7.3 months) and 20% of isolated Tis (median: 7 months) . Disease progression was observed in 9% of stage T1 tumours . The following risk factors for recurrence were identified: stage T1b (p = 0.05), multifocal tumours (p = 0.05), resistance to previous chemotherapy (mitomycin C) (p = 0.001) and association with Tis for stages T1 (p < 0.02) . The following risk factors for disease progression were identified: stage T1b (p < 0.001), grade III for stage T1 (p = 0.05) and association with Tis (p < 0.05) . Ten patients (14%) developed transient BCGitis . BCG was found to be effective in the prophylaxis of recurrence of stage Ta, T1 and Tis bladder tumours . This treatment is proposed for recurrent stage Ta grade II and III tumours and stage T1 tumours in the presence of recognised risk factors . The high risk of progression for stage T1b grade III tumours associated with Tis demands rigorous surveillance.

Clin Infect Dis, 1993 Aug, 17(2), 273 - 5
Eikenella corrodens, a rare cause of pancreatic abscess: two case reports and review; Stein A et al.; Eikenella corrodens, a slowly growing gram-negative bacillus that is a normal inhabitant of dental plaque, has been recognized as an infrequent cause of invasive disease . To date, only one case of pancreatic abscess due to E . corrodens in association with other bacteria from the oropharynx has been described . We report herein two cases of pancreatic abscess due to E . corrodens . In one case E . corrodens and Escherichia coli were found in the abscess specimens; in the other case no other pathogen was associated with E . corrodens . In addition, we review descriptions from the literature of abdominal infections caused by E . corrodens.

Clin Infect Dis, 1993 Aug, 17(2), 264 - 6
Rapid response of AIDS-related bacillary angiomatosis to azithromycin; Guerra LG et al.; A 28-year-old male with AIDS and a CD4 cell count of 100/mm3 presented with fever, hepatosplenomegaly, weight loss, and multiple, polypoid, angiomatous lesions on his face . It was determined by means of biopsy that the lesions were due to bacillary angiomatosis . The patient was treated with oral azithromycin (1 g daily as a single dose) . Rapid resolution of the skin lesions was noted . After 1 week of therapy, diminution in the size of the liver and spleen was noted . The only significant side effect noted was diarrhea, which was controlled with symptomatic therapy.

Biokhimiia, 1993 Aug, 58(8), 1258 - 65
{Two forms of extracellular low molecular weight Bacillus sp . BCF 247 ribonuclease . Isolation and characteristics of the protein}; Dement'ev AA et al.; Two homogeneous samples of low molecular mass RNAase (RNAases Bci I and Bci II) were obtained from cultural filtrates of spore-forming bacteria strain Bacillus sp . BCF 247 isolated from permafrost soils . The yields of RNAases Bci I and Bci II were 17% and 16% at the 17388- and 15376-fold degree of purification, respectively . Both enzymes have a close specific activity which is equal to approximately 4.7 x 10(-5) activity units per mg of protein . The relative molecular masses of the isolated proteins were determined and their N-terminal amino acid sequences identified . It was shown that the higher molecular mass sample of the enzyme is a pro-RNAase which, in contrast with the mature protein, contains an additional decapeptide segment in the N-terminal part of its molecule . The structure of RNAase Bci was compared with that of RNAases obtained from other Bacillus species; its ability to interact with a natural intracellular inhibitor of B . amyloliquefaciens RNAase was demonstrated.

Biokhimiia, 1993 Aug, 58(8), 1139 - 53
{New site site-specific endonuclease and methylase from Bacillus licheniformis 736}; Matvienko NN et al.; The site-specific endonuclease R Bli736I and methylase M Bli736I have been isolated from the Bacillus licheniformis strain 736 by blue-agarose, hydroxyapatite-Ultragel and heparin-Sepharose chromatography . The enzymes are free from interfering impurities . R Bli736I recognizes the 5'-GGTCTCN-3' decreases and decreases 5'-NNNNNGAGACC-3' sequences on the DNA and cleaves the DNA as indicated by arrows to form single-stranded 4-nucleotide 5'-protruding termini . This enzyme is an isoschizomer of Eco3II isolated from E . coli.

Biochem J, 1993 Aug 1, 293 ( Pt 3), 789 - 93
Developmental and biochemical characteristics of the cardiac membrane-bound arginine-specific mono-ADP-ribosyltransferase; McMahon KK et al.; ADP-ribosylation of protein in heart membrane preparations has been shown to be present in adult tissue but absent from early neonate tissue {Piron and McMahon (1990) Biochem . J . 270, 591-597} . To further this observation, the cardiac membrane-bound form of arginine-specific mono-ADP-ribosyltransferase (EC 2.4.2.31) has been characterized . Apparent Km values of 330 and 470 microM were found in heart membrane preparations from rat and quail respectively . The Vmax . value depended greatly on the species of animal studied, and was 1.1 and 48 nmol/min per mg in rat and quail preparations respectively . The specific activity of the enzyme was lowest in pig, intermediate in rat, dog and rabbit, and highest in mouse and quail cardiac membranes . In the rat, the ADP-ribosylation of protein and enzyme activity were very low in heart preparations from 1-15-day-old animals . Thereafter the ADP-ribosylation and enzyme activity increased gradually to adulthood . Bacillus cereus phosphatidylinositol-specific phospholipase C, known to hydrolyse glycosylphosphatidylinositol anchors of proteins, released the mono-ADP-ribosyltransferase from membrane preparations of both rat and quail in a dose-dependent, Zn(2+)-inhibited manner . Thus it appears that a membrane-bound form of arginine-specific mono-ADP-ribosyltransferase is present in heart membranes from a variety of species and is not species-specific . The activity of this ADP-ribosyltransferase appears to be developmentally regulated and to be bound to the cardiac membranes by a glycosylphosphatidylinositol anchor.

Kekkaku, 1993 Aug, 68(8), 521 - 6
{The appearance and enlargement of localized pulmonary granuloma with eosinophilic infiltration during tuberculosis chemotherapy}; Saitoh Y et al.; A pulmonary tumorous shadow appeared and enlarged in a 25 years-old male patient undergoing intensive chemotherapy for tuberculosis . The chest X-rays taken on admission revealed effused pleura in the right lung and nodular shadows in the upper area of the right lung . After 40 days of using isoniazid (INH), rifampicin (RFP) and streptomycin (SM), a homogeneous opacity, not previously observed, appeared in the middle area of the right lung (S5) . Microscopic examination of the tissues obtained during a transbronchial lung biopsy disclosed epithelioid cell granulomas with marked eosinophilic infiltration . The presence of eosinophilic infiltration due to the admission of antituberculosis agents was disregarded because no change was observed in the new granulomatous shadows during the drug challenge tests and the lymphocyte stimulation test to INH, RFP and SM was negative . Transient aggravation during the initial phase of chemotherapy for pulmonary tuberculosis, such as in this case, is suspected cause by some eosinophilic allergic-induced mechanisms, against bacillary components.

J Egypt Soc Parasitol, 1993 Aug, 23(2), 389 - 97
A field survey of bacteriophage contamination of mosquito breeding places, inhibiting bacterial insecticide; Ali SM et al.; Twelve geographically different mosquito breeding places were described and sampled for the detection of naturally existed bacteriophage viruses which could transduct and lysate 5 entomopathogenic bacteria . The surveyed places are classified into seepage, sewage, and irrigation breeding water . Bacterial free filtrates of the collected samples were assayed against the tested bacteria in vitro and against 3rd instar Culex pipiens larvae as well . Nine out of the twelve places could demonstrate the presence of phages . Bacillus thuringiensis H-14 was foud susceptible to phage(s) present in polluted and irrigated water of 5 location, while, B . thuringiensis Berliner was susceptible to only a specific phage of one breeding place (polluted, sewage water) . With regard to Bacillus sphaericus strains 1593 and 114, bacteriophages of sewage and irrigated water could lysate them and these phages are characterized by being of a moderate host range, except one phage which showed high specificity with strain 114 and was detected in a polluted sewage water sample collected from Dakahliya Governorate . The detected phages proved to lysate both B . thuringiensis H-14 and B . sphaericus 1593 on their larvicidal action through a series of bioassay experiment, almost all results indicate the presence of a significant inhibitory activity.

Infect Control Hosp Epidemiol, 1993 Aug, 14(8), 459 - 62
Dissemination of Bacillus cereus in an intensive care unit; Bryce EA et al.; OBJECTIVE: To report the contamination of ventilator equipment with Bacillus cereus and to outline the measures taken to trace the source of the organism . DESIGN: A prospective survey of all intensive care unit patients who were culture-positive for B cereus and obtaining of environmental cultures of the cleaning and assembly area of the respiratory services division between October 1991 and September 1992 . SETTING AND PATIENTS: Ventilated patients from a 16-bed medical and surgical intensive care unit (ICU) in a 1,000-bed adult tertiary care hospital . INTERVENTIONS AND RESULTS: From October 1991 to April 1992, B cereus colonized the ventilator circuitry of patients in the ICU . One of two washer/decontaminators in the cleaning and assembly area of the respiratory services division was found to yield the microorganism consistently from the water intake port . The design of the machine precluded easy decontamination of the port with 2% glutaraldehyde and a second outbreak occurred . Following the second outbreak, aqueous chlorhexidine in a final concentration of 0.05% was added to the first of two pasteurization cycles in an attempt to achieve sporicidal activity . This ended the outbreak . Sixty-two patients became colonized with the organism including two with nonfatal Bacillus sepsis and one death due to pneumonia associated with the organism . CONCLUSION: This experience emphasizes the importance of obtaining cultures of machine parts to identify the source of contamination and thereby direct control measures . Use of chlorhexidine gluconate at high temperatures effectively eradicated B cereus from ventilator circuitry in a practical and cost-effective manner.

Int J Biol Macromol, 1993 Aug, 15(4), 241 - 5
Studies on chitosan: 6 . Relationship between N-acetyl group distribution pattern and chitinase digestibility of partially N-acetylated chitosans; Aiba SI; The digestibility of partially N-acetylated chitosans by microbial chitinases was investigated in view of the distribution pattern of N-acetyl groups along the polysaccharide chain . Partially N-acetylated chitosans are classified into two groups; moderately N-deacetylated chitosans (MDC) with 10-30% acetyl content obtained by heterogeneous N-deacetylation of chitin and partially N-acetylated chitosans (PAC-H) with 20-70% acetyl content prepared by homogeneous N-acetylation of highly N-deacetylated chitosans (HDC) . MDC have some blocks of N-acetyl-D-glucosamine (GlcNAc) sequences but PAC-H are random-type copolymers of GlcNAc and D-glucosamine . The apparent Km values of Streptomyces griseus chitinase were 0.14 g l-1 for 30% N-acetylated MDC and 0.16 g l-1 for 30% N-acetylated PAC-H . The Km values decreased with increased N-acetylation but the values for both MDC and PAC-H with similar acetyl content were almost the same . The chitinase from S . griseus could not distinguish the difference between block and random distributions of GlcNAc . The chitinases from Bacillus sp . and Bacillus sp . PI-7S also hydrolysed MDC and PAC-H in the same manner . From these results we conclude that sequences of GlcNAc are not necessary for recognition by these chitinases in contrast to lysozyme.

Appl Environ Microbiol, 1993 Aug, 59(8), 2442 - 8
Structural stability of Bacillus thuringiensis delta-endotoxin homolog-scanning mutants determined by susceptibility to proteases; Almond BD et al.; Forty homolog-scanning (double-reciprocal-crossover) mutant proteins of two Bacillus thuringiensis delta-endotoxin genes (cryIAa and cryIAc) were examined for potential structural alterations by a series of proteolytic assays . Three groups of mutants could be identified . Group 1, consisting of 13 mutants, showed no delta-endotoxin present during overexpression conditions in Escherichia coli (48 h at 37 degrees C, with a ptac promoter) . These mutants produced full-sized delta-endotoxin detectable by polyacrylamide gel electrophoresis with Coomassie blue staining or Western immunoanalysis after 24 h of growth but not after 48 h, suggesting sensitivity to intracellular proteases . Group 2 consisted of 13 mutants that produced stable delta-endotoxins that were completely digested by 2% bovine trypsin . In contrast, native delta-endotoxin produces a 65,000-Da trypsin-resistant peptide, which is the active toxin . Group 3 mutants expressed delta-endotoxin and trypsin-stable toxins, similar to the wild type . In this study, 12 group 3 mutant toxins were compared with wild type toxins by thermolysin digestion at a range of temperatures . The two wild-type toxins exhibited significant differences in thermolysin digestion midpoints . Among the group 3 mutants, most possessed significantly different protein stabilities relative to their parental toxins . Two of the group 3 mutants were observed to have exchanged the thermolysin sensitivity properties of the parental toxins.

Proteins, 1993 Aug, 16(4), 357 - 63
Localization of hydrogen-bonds within modules in barnase; Noguti T et al.; Proteins in eukaryotes are composed of structural units, each encoded by discrete exons . The protein module is one such structural unit; it has been defined as the least extended or the most compact contiguous segment in a globular domain . To elucidate roles of modules in protein evolution and folding, we examined roles of hydrogen bonds and hydrophobic cores, as related to the stability of these modules . For this purpose we studied barnase, a bacterial RNase from Bacillus amylolique-faciens . Barnase is decomposed into at least six modules, M1-M6; the module boundaries are identified at amino acid residues 24, 52, 73, 88, and 98 . Hydrogen bonds are localized mainly within each of the modules, with only a few between them, thereby indicating that their locations are designed to primarily stabilize each individual module . To obtain support for this notion, an analysis was made of hypothetical modules defined as segments starting at a center of one module and ending at the center of the following one . We found that the hydrogen bonds did not localize in each hypothetical module and that many formed between the hypothetical modules . The native conformations of modules of barnase may be specified predominantly by interactions within the modules.

J Bacteriol, 1993 Aug, 175(16), 5276 - 80
A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis; Wu D et al.; The effect of a 20-kDa protein on cell viability and CytA crystal production in its natural host, Bacillus thuringiensis, was studied by expressing the cytA gene in the absence or presence of this protein . In the absence of the 20-kDa protein, B . thuringiensis cells either were killed during sporulation (strain cryB) or produced very small CytA crystals (strain 4Q7) . Expression of cytA in the presence of the 20-kDa protein, however, preserved cell viability, especially in strain cryB, and in both strains yielded bipyramidal crystals of the CytA protein that were larger than those of wild-type B . thuringiensis . These results suggest that the 20-kDa protein promotes crystal formation, perhaps by chaperoning CytA molecules during synthesis and crystallization, concomitantly preventing the CytA protein from interacting lethally with the bacterial host cell.

Arch Biochem Biophys, 1993 Aug 1, 304(2), 508 - 14
Purification and properties of laminaribiose phosphorylase (EC 2.4 1.31) from Euglena gracilis Z; Kitaoka M et al.; Three isoforms of laminaribiose phosphorylase, F0, F1, and F2, were purified to an electrophoretically homogeneous state from a cell free extract of Euglena gracilis Z (IAM E-6) . F1 and F2 were the major components . The three enzymes showed very similar properties except their isoelectric points . They could also use laminaribiose as the glucosyl acceptor instead of glucose . The isoforms the same molecular mass of 120 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) or 200 kDa by the gel filtration method, suggesting that they have a dimer structure . They could not be distinguished on the Ouchterlony's double diffusion test with mouse antiserum against F1 or F2 . The substrate specificities of F1 and F2 were determined to be essentially the same . Both of the reaction mechanisms of F1 and F2 were determined to be ordered bi-bi mechanisms, as in the case of cellobiose phosphorylase . A competitive substrate inhibition was observed in their synthetic reaction . Two other strains, E . gracilis var . bacillaris (IAM E-2) and E . gracilis (IAM E-3), had only one laminaribiose phosphorylase, which corresponded to F0 on native PAGE.

J Gen Virol, 1993 Aug, 74 ( Pt 8), 1611 - 6
A strong-stop DNA in rice plants infected with rice tungro bacilliform virus; Bao Y et al.; A virus-specific small nucleic acid (strong-stop DNA) was identified in rice plants infected with rice tungro bacilliform virus, but not in the virus particles . This nucleic acid was shown to consist of about 595 deoxyribonucleotides with about 70 ribonucleotides covalently linked at the 5' end . Hybridization with sequence-specific oligonucleotides showed that the ribonucleotides were from the plant cytoplasmic tRNA(iMet) sequence . PCR analysis detected hairpin structures at the 3' end of the DNA.

Clin Orthop, 1993 Aug, (293), 144 - 7
Eikenella corrodens vertebral osteomyelitis . A case report and literature review; Raab MG et al.; A debilitated 73-year-old white man was diagnosed with back pain secondary to acute hematogenous Eikenella corrodens vertebral osteomyelitis on the basis of history and physical examination, radiographs, computed tomography, magnetic resonance imaging, and open biopsy of the L3 vertebral body . A rare cause of vertebral osteomyelitis, possibly reported only once before in the world literature, E . corrodens is a facultative anaerobic gram-negative bacillus and a normal oral inhabitant . E . corrodens should be considered in the differential diagnosis of vertebral osteomyelitis and can be managed with immobilization and long-term intravenous antibiotics.

Jpn J Genet, 1993 Aug, 68(4), 243 - 55
Complementation assay of primer protein: gene expression systems of plasmid vectors support the infection of suppressor sensitive mutant phages M2 and phi 29; Kishi T et al.; Two different expression systems of genes of primer proteins (pE for phage M2, and p3 for phi 29) were constructed to study the protein primed DNA replication of Bacillus phages M2 and phi 29 . In one system, expression of the genes was under the control of the inducible spac promoter, whereas in the other system, the expression was under the control of the constitutive promoter in plasmid pUB110 . Complementation tests in vivo were performed between the primer proteins expressed by these systems and mutant phages having suppressor sensitive mutations in the genes of the primer proteins . The phages M2 susE and phi 29 sus3 were complemented by pE and p3 expressed by the systems, respectively . However, the complementation and apparent phage DNA synthesis were not detected in the combinations between susE of phage M2 and p3 of phage phi 29, and vice versa . Although pE and p3 proteins exhibited structurally and functionally similar characteristics, these proteins showed species specificity in the protein primed DNA replication of bacteriophages M2 and phi 29.

Orthop Rev, 1993 Aug, 22(8), 943 - 9
A 43-year-old woman with right hip pain; Yin Y et al.; Although tuberculous arthritis is currently uncommon in the United States, the hip joint is a common location . Tuberculous arthritis usually presents as a mild, slowly progressing process . It often involves a single joint . Radiological findings include osteopenia of the involved joint, soft tissue swelling, bone destruction without evidence of bone formation, and sequestrum formation . Several conditions should be considered in the differential diagnosis . The detailed roentgenographic appearances and many of the differential diagnoses are included in this paper . Final diagnosis is made by positive culture results or finding the bacillus histologically.

J Oral Pathol Med, 1993 Aug, 22(7), 292 - 7
Oral Kaposi's sarcoma: a 10-year retrospective histopathologic study; Regezi JA et al.; Microscopic diagnosis of early Kaposi's sarcoma continues to be a challenge to the pathologist, as does the identification of bacillary angiomatosis (BA) which may have a similar appearance . 120 oral Kaposi's sarcoma (KS) biopsies submitted to the UCSF oral pathology service from 1981-1991 were reviewed in order to describe the clinical-pathologic spectrum of these lesions and to search for unrecognized cases of BA . Also, histopathologic features of oral KS were compared to 30 oral pyogenic granulomas, and immunohistochemical stains for endothelium-associated CD34 antigen were done . The diagnosis of KS was confirmed in all biopsies and no cases of BA were found . Histologically, the KS specimens exhibited numerous features that separated them from pyogenic granulomas, and could themselves be divided into two clinical-pathologic subtypes: small, well-delineated macular lesions (31), which were characterized by inconspicuous patches of spindle cells containing ill-defined vascular spaces; and larger, infiltrative nodular lesions (89), which were characterized by spindle cells lining vascular slits and bizarre-shaped vessels . Extravasated RBCs were evident in almost all KS lesions; hemosiderin deposits and hyaline globules were seen in half of each of the small and large lesions . Nuclear atypia was minimal and mitotic activity was slight . Lymphocytes in small lesions added to the difficulty of microscopic interpretation of these incipient lesions . CD34 was expressed on all spindle cells lining vascular spaces in larger lesions and on spindle cells of small, subtle lesions . We conclude that within the spectrum of lesions that are diagnosed as oral KS, two clinical-pathologic types can be identified: macular small spindle-cell lesions and nodular infiltrative vascular lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

J Comput Aided Mol Des, 1993 Aug, 7(4), 367 - 96
Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases; Vriend G et al.; Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability . The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B . thermoproteolyticus (thermolysin) and B . cereus . Several new techniques have been developed to improve the model-building procedures . Also a 'model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model . The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes . Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches . Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully . At elevated temperatures NPs are irreversibly inactivated as a result of autolysis . It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics . From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step . Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs . However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding . In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation . Mutations that alter the stability of the NP by a large amount are located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activation) . One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B . stearothermophilus . After eliminating this weakest link a drastic increase in thermostability was observed and the search for the second-weakest link, or the second-lowest energy local unfolding pathway is now in progress . Hopefully, this approach can be used to unravel the entire early phase of unfolding.

Tuber Lung Dis, 1993 Aug, 74(4), 254 - 60
Nationwide community survey of tuberculosis epidemiology in Saudi Arabia; al-Kassimi FA et al.; In the first nationwide community-based survey of the epidemiology of tuberculosis in Saudi Arabia, 7721 subjects were screened in the 5 provinces (using an equal proportional allocation formula) for 2 parameters: (1) prevalence of positive Mantoux test in non BCG vaccinated subjects; (2) prevalence of bacillary cases on sputum culture . The prevalence of positive Mantoux reaction in children aged 5-14 years was 6% +/- 1.8; higher in urban areas (10%), and lower in rural areas (2%), thus classifying Saudi Arabia among the middle prevalence countries . These relatively good results (by Third World standards) could reflect the rise of the standard of living and wide availability of free treatment for active cases with a lowered risk of infection in the community . This view is supported by the fact that in our survey, only one subject grew Mycobacterium tuberculosis in the sputum . However, there were foci of high prevalence of Mantoux reaction in the urban communities in the Western province (20% +/- 8.7 urban; 1% +/- 1.9 rural) . The problem may be caused by the fact that the province receives every year over a million pilgrims, some of whom are known to settle illegally and escape the usual screening for tuberculosis imposed on foreign labourers . In conclusion, even in the absence of an enforceable national programme for the eradication of tuberculosis, the economic standard and wide availability of free treatment for active cases has resulted in relatively low rates of prevalence of tuberculin sensitivity in children . The foci of high prevalence in the Western Province require special screening arrangements.

Biokhimiia, 1993 Aug, 58(9), 1420 - 9
{Metalloproteinase of Bacillus mesentericus, strain V-313}; Morozova IP et al.; A homogeneous metalloproteinase has been isolated with a 28% yield from the culture fluid of Bacillus mesentericus, strain B-313 . The isolation procedure included chromatography on bacitracin-silochrome and gel filtration on Acrylex P-10 and Sephadex G-75 . The enzyme has a molecular mass of 41,000 Da; its N-terminal sequence, which appears as A-A-T-T-G-T-G-T-T-L-K-G-K-T-V-S-L-N-I, is identical with that of the B . amyloliquefaciens enzyme . Like other metalloproteinases, the enzyme is inhibited by o-phenanthroline and EDTA, has an activity maximum at 55 degrees C and pH 6.5-7.2, is stable at pH 7.0-9.5 and at temperature below 45 degrees C for several hours, and is irreversibly inactivated in acid media . As can be judged from the kcat/Km ratio dependence on pH, two ionogenic groups with pKa of 7.4 and 6.2 are involved in the catalytic act, presumably the imidazol group of histidine and the carboxylic group . Within synthetic peptides the enzyme hydrolyzes the bonds formed by the amino group of hydrophobic amino acids, mostly of leucine residues.

Int J Food Microbiol, 1993 Aug, 19(3), 207 - 16
Effects of temperature, aw and pH on the growth of Bacillus cells and spores: a response surface methodology study; Quintavalla S et al.; Effects of water activity (aw), pH and storage temperature on the growth of spores and vegetative cells of Bacillus spp . isolated from bakery products were studied in a model system and the growth was monitored spectrophotometrically in microtitre plates . Experiments, performed following a Central Composite Rotatable Design and aimed at development of response surfaces, resulted in two polynomials based mainly on first order coefficients . For both cells and spores, temperature, aw and pH acted additively and without any synergistic effect . This was also confirmed by isoresponse plots for the combinations temperature-aw, temperature-pH and pH-aw . Applications of the models to predict the shelf life of the actual food product are discussed.

Curr Opin Immunol, 1993 Aug, 5(4), 497 - 502
Immunity to mycobacteria; Orme IM; Recent progress in the field of immunity to mycobacteria has centered on T cell subset responses and the cytokines these cells secrete . In addition, there has been steady progress in identifying and characterizing several classes of major mycobacterial proteins; included amongst these are the secreted/export proteins of Mycobacterium tuberculosis, which several laboratories now believe may represent the key protective immunity-inducing antigens of the bacillus.

Ann Intern Med, 1993 Aug 1, 119(3), 185 - 93
Tuberculin and anergy testing in HIV-seropositive and HIV-seronegative persons . Pulmonary Complications of HIV Infection Study Group; Markowitz N et al.; OBJECTIVE: To determine the prevalence and predictors of reactivity to tuberculin purified protein derivative (PPD) and skin test anergy in patients with human immunodeficiency virus (HIV) infection and in HIV-seronegative controls . DESIGN: Cross-sectional analysis of baseline data from a prospective, multicenter study of pulmonary complications of HIV infection . SETTING: Community-based cohort of persons with and without HIV infection . PATIENTS: A total of 1171 HIV-seropositive patients without AIDS (841 homosexual men, 274 intravenous drug users, and 56 women with heterosexually acquired infection); 182 HIV-seronegative persons (125 homosexual men and 57 intravenous drug users) . MEASUREMENTS: Delayed-type hypersensitivity response to tuberculin PPD, trichophytin, mumps, and Candida antigens; T-lymphocyte subsets . RESULTS: The prevalence of tuberculin PPD reactivity was higher among intravenous drug users than among homosexual men, in both HIV-seronegative (19.1% compared with 6.8%, P = 0.03) and HIV-seropositive persons (15.1% compared with 2.5%, P < 0.001) . Among HIV-infected patients, the prevalence of tuberculin reactivity varied directly and that of anergy inversely with the absolute CD4 lymphocyte count . Prevalences were 1% and 72%, respectively, in patients with fewer than 200 CD4 cells/mm3, and 8.4% and 25.5%, respectively, in those with 600 CD4 cells/mm3 (P < 0.001 for both comparisons) . Patients with HIV infection and fewer than 400 CD4 lymphocytes/mm3 had a lower prevalence of PPD reactivity than HIV-seronegative controls (2.7% compared with 10.0%, P < 0.001) . The strongest predictors of tuberculin reactivity were intravenous drug use, black race, a previous positive PPD test result, and a history of Calmette-Guerin bacillus vaccination . The strongest predictor of anergy was HIV seropositivity . CONCLUSIONS: The response to delayed-type hypersensitivity antigens depends on immune status . The value of PPD and anergy testing in HIV-seropositive patients depends on the ability of such testing to predict subsequent tuberculosis, which is imprecisely known . Until more data or better methods are available, these tests should be done as early as possible in the course of HIV infection.

Biosci Biotechnol Biochem, 1993 Aug, 57(8), 1384 - 6
Efficient production of Bacillus stearothermophilus alpha-amylase in Bacillus brevis by altering its signal peptide; Yamaguchi K et al.; To investigate the role of signal peptides in extracellular production in Bacillus brevis, the Bacillus stearothermophilus alpha-amylase signal peptide was systematically altered and the effects were analyzed in B . brevis . Efficient extracellular production of the amylase in B . brevis was accomplished either by introducing point mutations at positions between -6 and -4 or by replacing the whole signal peptide with that of B . brevis middle wall protein.

Biosci Biotechnol Biochem, 1993 Aug, 57(8), 1294 - 8
Effects of moranoline, 4-O-alpha-D-glucopyranosylmoranoline and their N-substituted derivatives on thermostability of cyclodextrin glycosyltransferase, glucoamylase, and beta-amylase; Maruo S et al.; In repeated glycosylmoranolines synthetic reaction at 55 degrees C, cyclodextrin glycosyltransferase (CGT-ase, EC 2.4.1.19) from Bacillus stearothermophilus retained its activity for more than 600 days . A main stabilizing compound was found to be 4-O-alpha-D-glucopyranosylmoranoline . The thermostabilizing activities of moranoline, 4-O-alpha-D-glucopyranosylmoranoline, and their N-substituted derivatives were studied . Moranoline and its N-substituted derivatives stabilized glucoamylase . 4-O-alpha-D-Glucopyranosylmoranoline and its N-substituted derivatives stabilized CGT-ase and beta-amylase.

Appl Environ Microbiol, 1993 Aug, 59(8), 2404 - 10
Effect of promoter modification on mosquitocidal cryIVB gene expression in Synechococcus sp . strain PCC 7942; Soltes-Rak E et al.; The impact of promoter modification on the expression of the mosquitocidal Bacillus thuringiensis subsp . israelensis cryIVB gene when used to transform the cyanobacterium Synechococcus sp . strain PCC 7942 has been examined . Maximal transcript and protein abundances were achieved by the addition of the lacZ promoter upstream of the cryIVB sequence . Replacement of the endogenous corresponding Bacillus sequences with the Synechococcus petF1 promoter, ribosome binding site, and initiation codon also resulted in increased expression of the cryIVB gene relative to the expression obtained with the Bacillus promoter alone but decreased expression relative to the expression achieved with the tandem array of the Bacillus and lacZ promoters . Synechococcus cells carrying plasmids in which the expression of the cryIVB gene was regulated by either the lacZ or the petF1 promoter were readily consumed by first-instar Culex restuans larvae and proved to be toxic for these organisms.

J Exp Med, 1993 Aug 1, 178(2), 479 - 88
Dendritic cell progenitors phagocytose particulates, including bacillus Calmette-Guerin organisms, and sensitize mice to mycobacterial antigens in vivo; Inaba K et al.; Dendritic cells, while effective in sensitizing T cells to several different antigens, show little or no phagocytic activity . To the extent that endocytosis is required for antigen processing and presentation, it is not evident how dendritic cells would present particle-associated peptides . Evidence has now been obtained showing that progenitors to dendritic cells can internalize particles, including Bacillus Calmette-Guerin (BCG) mycobacteria . The particulates are applied for 20 h to bone marrow cultures that have been stimulated with granulocyte/macrophage colony-stimulating factor (GM-CSF) to induce aggregates of growing dendritic cells . Cells within these aggregates are clearly phagocytic . If the developing cultures are exposed to particles, washed, and "chased" for 2 d, the number of major histocompatibility complex class II-rich dendritic cells increases substantially and at least 50% contain internalized mycobacteria or latex particles . The mycobacteria-laden, newly developed dendritic cells are much more potent in presenting antigens to primed T cells than corresponding cultures of mature dendritic cells that are exposed to a pulse of organisms . A similar situation exists when the BCG-charged dendritic cells are injected into the footpad or blood stream of naive mice . Those dendritic cells that have phagocytosed organisms induce the strongest T cell responses to mycobacterial antigens in draining lymph node and spleen . The administration of antigens to GM-CSF-induced, developing dendritic cells (by increasing both antigen uptake and cell numbers) will facilitate the use of these antigen-presenting cells for active immunization in situ.

Biochemistry, 1993 Jul 27, 32(29), 7475 - 8
Role of tyrosine residues in Hg(II) detoxification by mercuric reductase from Bacillus sp . strain RC607; Rennex D et al.; Two tyrosine residues of mercuric reductase (MerA), Tyr-264 and Tyr-605, which were shown by the X-ray crystal structure to be involved in metal binding, were changed to phenylalanine residues by site-directed mutagenesis, both singly (Y264F, Y605F) and to form a double mutant (Y264,605F) . The effect of these mutations on Hg(II) reduction activity varied . While MerA Y605F has a similar apparent Km to the wild-type enzyme and an apparent kcat reduced by 6-fold, MerA Y264F has an apparent Km 5-fold lower than the wild type and apparent kcat 160-fold lower . The double mutant MerA Y264,605F has the same apparent Km as MerA Y264F, but its apparent kcat was reduced by a further 7-fold . These results show that the roles of the two tyrosine residues are not equivalent and that Y264 is important for catalysis, possibly by destabilizing the binding of Hg(II) to the two ligating thiolates at the active site of MerA.

Commun Dis Rep CDR Rev, 1993 Jul 16, 3(8), R107 - 10
Cat scratch disease and bacillary angiomatosis: aetiological agents and the link with AIDS; Birtles RJ et al.; The recent characterisation of a new species of the genus Rochalimaea is the culmination of a ten year search for the identity of the agent of bacillary angiomatosis, a condition that is increasingly recognised in association with AIDS . This work has also implicated Rochalimaea henselae in the aetiology of cat scratch disease contact with cats is a risk factor for both conditions . This report describes both diseases and reviews progress toward understanding their microbiology . R . quintana is also implicated as an agent of bacillary angiomatosis and was recognised much earlier as the cause of trench fever.

J Biol Chem, 1993 Jul 15, 268(20), 15277 - 84
Rat muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase . Study of the kinase domain by site-directed mutagenesis; Crepin KM et al.; Sequence alignment and modeling of the 2-kinase domain of the liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, on 6-phosphofructo-1-kinase from Bacillus stearothermophilus and Escherichia coli (Bazan, J . F., Fletterick, R . J., and Pilkis, S . J . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 9642-9646) suggested that Cys-160 of the 2-kinase would correspond to Asp-127 of the 1-kinase, which acts as a general base catalyst . We have studied the validity of this alignment by site-directed mutagenesis of residues in the 2-kinase domain of skeletal muscle 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase . Cys-160 was mutated to Asp or Ser . Two adjacent residues, Glu-157 and Asp-162, either of which could act as a general base catalyst, were mutated to Ala . Asp-162 corresponds to Asp-129 in the bacterial 1-kinase, which is also essential for catalysis and might bind Mg2+ . None of these mutations significantly decreased the Vmax of the 2-kinase, suggesting that the mutated amino acids are not essential for catalysis and therefore do not play the same role as Asp-127 and Asp-129 in the bacterial 1-kinase . Mutation of Glu-157 and Asp-162 to alanine had no effect on the kinetic parameters of the bifunctional enzyme, indicating that these two negatively charged residues are not involved in catalysis and substrate binding.

Biochem Biophys Res Commun, 1993 Jul 15, 194(1), 194 - 201
Gene structure and upstream regulatory regions of human CYP2C9 and CYP2C18; de Morais SM et al.; There is a genetic polymorphism in humans in the metabolism of S-mephenytoin which has been suggested to be mediated by either CYP2C18 or CYP2C9 . We have isolated genomic clones for CYP2C9 and CYP2C18 from the liver of an individual phenotyped in vitro as an extensive metabolizer of S-mephenytoin . Analysis of the genes reveals nine coding exons spanning approximately 55 kb . The intron-exon organization was similar to that of other members of the CYP2C subfamily . Analysis of 2200 bp of 5' upstream sequence for CYP2C9 and 1300 bp 5' upstream sequence for CYP2C18 reveals canonical TATA boxes situated 57 bp upstream from the first codon, multiple consensus sequences for glucocorticoid regulatory elements, and identification of a 15 base sequence with high homology to a 5'-flanking sequence responsible for barbiturate-inducible expression of P450BM-3 in Bacillus megaterium . The upstream region for CYP2C9 was highly homologous (75%) to that of human CYP2C8 through most of the 2200 bp sequenced, but the upstream region of CYP2C18 was similar to CYP2C8 and CYP2C9 for only the first 200 bases . The availability of the sequences of the upstream regions and intron-exon junctions of CYP2C9 and CYP2C18 will allow future analysis of these genes in humans which differ in their ability to metabolize S-mephenytoin and other drugs.

Int J Cancer, 1993 Jul 9, 54(5), 734 - 40
Identification of colon-tumor-associated antigens by T-cell lines derived from tumor-infiltrating lymphocytes and peripheral-blood lymphocytes from patients immunized with an autologous tumor-cell/bacillus Calmette-Guérin vaccine; Ransom JH et al.; Tumor immunity developing as a response to an autologous colon-tumor/bacillus Calmette-Guerin (BCG) vaccine appears to be associated with induction of CD4+ helper T cells, implied by the observation that vaccine efficacy is associated with major histocompatibility complex class-II molecule expression on the vaccine tumor cells . Therefore, in an attempt to identify colon-tumor-associated antigens responsible for conferring immunity, we examined and compared the proliferative responses of peripheral-blood lymphocytes (PBL) from patients immunized with the autologous tumor/BCG vaccine to T-cell lines cloned expanded from colon-tumor-infiltrating lymphocytes to 5 antigens isolated on the basis of their reactivity by colon-tumor-reactive human monoclonal antibodies . Enzymatically dissociated colon tumors provided a source for establishment of cloned T-cell lines, tumor cell lines propagated in vitro or in vivo as nude-mouse xenografts and EBV-transformed B-cell lines used as antigen-presenting cells . Of 104 different T-cell lines tested, only 3 proliferated in response to CTAA 28A32-46K, and I to the CTAA28A32-32K antigen . In contrast, PBL from 64% of patients immunized with the autologous colon-tumor/BCG vaccine responded to the CTAA 28A32-32K antigen . This antigen is related to a family of calcium- and phospholipid-binding placental proteins termed annexins . Since proliferative responses developed to this antigen after vaccination in 64% of individuals, this antigen may be an important common colon-tumor-associated rejection antigen.

Biochemistry, 1993 Jul 6, 32(26), 6624 - 31
Three histidine residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp . 1011: effects of the replacement on pH dependence and transition-state stabilization; Nakamura A et al.; Cyclodextrin glucanotransferase (CGTase) catalyzes the formation of cyclodextrins from amylose through an intramolecular transglycosylation reaction . On the basis of the three-dimensional structures of CGTases three histidine residues, which are conserved between CGTases and alpha-amylases, are located at the active center and are proposed to constitute the substrate binding sites . The three histidine residues (His-140, His-233, and His-327) of CGTase from alkalophilic Bacillus sp . 1011 were individually replaced by site-directed mutagenesis to probe their roles in catalysis . Asparagine-replaced CGTases (H140N-, H233N-, and H327N-CGTase) retained cyclization activity but had altered production ratios of alpha-, beta-, and gamma-cyclodextrin . Replacement of histidine by asparagine residues strongly affected the kcat for beta-cyclodextrin-forming, coupling, and hydrolyzing activities, whereas it barely affected the Km values . The activation energies for alpha-cyclodextrin hydrolysis were increased more than 12 kJ/mol by the replacement . Furthermore, the Ki values of acarbose, which is thought to be a transition-state analog of glycosidase catalysis, were 2-3 orders of magnitude larger in asparagine-replaced CGTases than that in wild-type CGTase . Therefore, the three histidine residues participate in the stabilization of the transition state, whereas they participate little in ground-state substrate binding . H327N-CGTase had decreased activity over an alkaline pH range, indicating that His-327 is important for catalysis over an alkaline pH range.

J Biol Chem, 1993 Jul 5, 268(19), 14109 - 15
Formation of 1,3-cyclic glycerophosphate by the action of phospholipase C on phosphatidylglycerol; Shinitzky M et al.; The action of phospholipase C (PLC) from Bacillus cereus on phosphatidylglycerol (PG), derived from egg yolk phosphatidylcholine (PC), was examined in an ether-water mixture . The PLC cleavage of PG and PC followed a Michaelis-Menten kinetics with apparent Vmax values per 1 microgram enzyme of 0.26 and 0.91 mumol.min-1 and Km values of 10 and 12 mM, respectively . When the same enzymic reaction was carried out in minimally buffered aqueous solution of 1% Triton X-100, the decrease in pH with respect to phospholipid cleavage was as expected with PC but much less pronounced with PG . This could be accounted for by the formation of a cyclic glycerophosphate, rather than alpha-glycerophosphate, in the PLC hydrolysis of PG . Examination of the chemical nature of the water-soluble product of PG by phosphorus nuclear magnetic resonance (31P NMR) revealed a single band at 2.31 ppm, while the bands of alpha-glycerophosphate and beta-glycerophosphate appeared at 5.12 and 4.57 ppm, respectively . Basic hydrolysis of the phospholipase cleavage product of PG (0.1 M NaOH for 1 min at 80 degrees C) followed by neutralization shifted its 31P NMR band to 5.18 ppm, which practically coincided with that of alpha-glycerophosphate . Analogous experiments were carried out with PG labeled with 3H at the carbon 2 of the glycerol headgroup ({3H}PG) . Autoradiography of thin layer chromatography (TLC) of the {3H}PG enzymic hydrolyzate displayed a single 3H-labeled compound, which could be converted to alpha-glycerophosphate by basic hydrolysis . These results strongly suggest that the phosphate headgroup of PG is cleaved off by PLC as 1,3-cyclic glycerophosphate . A series of PLC experiments with phosphatidyl dihydroxyacetone and phosphatidyl 1,3-propanediol as model substrates supported this assignment . Two-dimensional homonuclear 1H NMR correlated spectra as well as infrared spectra carried out on the isolated sodium salt of this product could further confirm such a structure . The unique structure and chemical nature of 1,3-cyclic glycerophosphate may bear a distinct physiological function.

J Urol, 1993 Jul, 150(1), 188 - 9
Psoas abscess following intravesical bacillus Calmette-Guerin for bladder cancer: a case report; Hakim S et al.; An 87-year-old man with an abdominal aortic aneurysm received intravesical bacillus Calmette-Guerin therapy for transitional cell carcinoma of the bladder . He presented 9 months later with a psoas abscess that mimicked a contained retroperitoneal abdominal aortic aneurysm rupture . The abscess cultures yielded Mycobacterium bovis . Recent transurethral resection and high voiding pressures after instillations of bacillus Calmette-Guerin may have led to distant dissemination of the drug.

J Biochem (Tokyo), 1993 Jul, 114(1), 88 - 95
The molecular features and catalytic activity of CuA-containing aco3-type cytochrome c oxidase from a facultative alkalophilic Bacillus; Yumoto I et al.; Cytochrome aco3 of Bacillus YN-2000 was purified by an improved procedure and its molecular features and catalytic activity were extensively studied . The enzyme molecule was composed of three subunits with M(r)s of 50,000, 41,000, and 22,000, and contains 1 molecule each of cytochrome a, cytochrome c, and cytochrome o3 in the minimal structural unit (M(r), 113,000) . The 41,000 subunit (subunit II) contains heme c . The EPR, optical, and resonance Raman spectra of the oxidized enzyme demonstrated the presence of CuA whose coordination environment bore close resemblance to that of the aa3-type cytochrome c oxidase . Resonance Raman studies demonstrated that the cytochrome a moiety was similar to that of an aa3-type oxidase and also that the cytochrome o3 contained a five-coordinated high-spin heme with histidine as an axial ligand . The Fe-CO stretching mode of the cytochrome o3.CO complex was observed at 520 cm-1, which is the same frequency as that of cytochrome aa3.CO . The oxygen consumption activity of cytochrome aco3 was measured using several kinds of cytochromes c as the electron mediators . The reaction between cytochrome aco3 and eucaryotic cytochromes c was completely inhibited by poly-L-lysine . In contrast, poly-L-lysine was indispensable for sufficient reaction between the oxidase and Bacillus YN-2000 cytochrome c-553, the physiological electron donor . The combined results on the structure and enzymatic properties suggest that the cytochrome aco3 is very similar to cytochrome caa3 except that the cytochrome aco3 has cytochrome o3 in place of cytochrome a3 and the cytochrome c component has a very low redox midpoint potential (95 mV).(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1993 Jul, 114(1), 69 - 75
Site-directed mutagenesis of a hexapeptide segment involved in substrate recognition of phenylalanine dehydrogenase from Thermoactinomyces intermedius; Kataoka K et al.; Phenylalanine dehydrogenase from Thermoactinomyces intermedius and leucine dehydrogenase from Bacillus stearothermophilus show a 59% sequence similarity in their substrate-binding domains, although their substrate specificities are different . We prepared a phenylalanine dehydrogenase mutant enzyme whose inherent hexapeptide segment (124F-V-H-A-A-129R) in the substrate-binding domain was replaced by the corresponding part of leucine dehydrogenase (M-D-I-I-Y-Q) in order to investigate the mechanism of substrate recognition by phenylalanine dehydrogenase . The catalytic efficiencies (kcat/Km) of the mutant enzyme with aliphatic amino acids and aliphatic keto acids as substrates were 0.5 to 2% of those of the wild-type enzyme . In contrast, the efficiencies for L-phenylalanine and phenylpyruvate decreased to 0.008 and 0.035% of those of the wild-type enzyme, respectively . These results suggest that the hexapeptide segment plays an important role in the substrate recognition by phenylalanine dehydrogenase.

Can J Microbiol, 1993 Jul, 39(7), 649 - 58
Insertion sequence elements in Bacillus thuringiensis subsp . darmstadiensis; Ryan M et al.; Two variants of insertion sequence IS231, named IS231G and H, were isolated from Bacillus thuringiensis subsp . darmstadiensis 73-E-10-2 (BTD2), an isolate toxic to dipteran insects, and characterized by DNA sequence analysis . They are encoded consecutively as direct repeats on an EcoRI fragment of 5.6 kilo base pairs . Direct tandem repeats of IS231 elements have not been previously reported . Both elements are closely related to other members of the IS231 family that have been isolated from B . thuringiensis strains toxic to lepidopteran as well as to dipteran insects . A close correlation exists between the evolutionary relationships of the IS231 sequences determined to data and the toxicity spectrum of the host cell . Probing of BTD2 DNA with a radiolabeled IS231G fragment demonstrated that IS231 elements are located on 55- and 34-MDa plasmids as well as on chromosomal DNA . Chromosomal DNA, but not plasmids, from BTD2 also hybridizes to another, unrelated insertion sequence, IS240, from B . thuringiensis subsp . israelensis, an isolate toxic to dipteran insects . BTD2, therefore, contains IS elements once thought to reside exclusively in either dipteran- or lepidopteran-specific subspecies of B . thuringiensis.

J Bacteriol, 1993 Jul, 175(14), 4414 - 26
Purification and characterization of protocatechuate 2,3-dioxygenase from Bacillus macerans: a new extradiol catecholic dioxygenase; Wolgel SA et al.; Protocatechuate 2,3-dioxygenase (2,3-PCD) from Bacillus macerans JJ1b has been purified to homogeneity for the first time . The enzyme catalyzes proximal extradiol ring cleavage of protocatechuate (PCA) with the attendant incorporation of both atoms of oxygen from O2 . The holoenzyme has a mass of 143 +/- 7 kDa as determined by ultracentrifugation and other techniques . It is composed of four apparently identical subunits with M(r)s of 35,500, each containing one iron atom . Mossbauer spectroscopy of 57Fe-enriched enzyme showed that the irons are indistinguishable and are high spin (S = 2) Fe2+ in both the uncomplexed and substrate-bound enzyme . However, the quadrupole splitting, delta EQ, and isomer shift, delta, of the Mossbauer spectrum changed from delta EQ = 2.57 mm/s and delta = 1.29 mm/s to delta EQ = 2.73 mm/s and delta = 1.19 mm/s upon PCA binding to the enzyme, showing that the iron environment is altered when substrate is present . The enzyme was also found to bind variable and substoichiometric amounts of Mn2+, but this metal could be removed without loss of activity or stability . The inherently electron paramagnetic resonance (EPR)-silent Fe2+ of the enzyme reversibly bound nitric oxide to produce an EPR-active species (g = 4.11, 3.95; S = 3/2) . The specific activity of the enzyme was found to be correlated with the amount of the S = 3/2 species formed, showing that activity is dependent on Fe2+ . Anaerobic addition of substrates to the enzyme-nitric oxide complex significantly altered the EPR spectrum, suggesting that substrates bind to or near the iron . The enzyme was inactivated by reagents that oxidize the Fe2+, such as H2O2 and K3FE(CN)6; full activity was restored after reduction of the iron by ascorbate . Steady-state kinetic data were found to be consistent with an ordered bi-uni mechanism in which the organic substrate must add to 2,3-PCD before O2 . The enzyme has the broadest substrate range of any of the well-studied catecholic dioxygenases . All substrates have vicinal hydroxyl groups on the aromatic ring except 4-NH2-3-hydroxybenzoate . This is the first substrate lacking vicinal hydroxyl groups reported for catecholic extradiol dioxygenases . 2,3-PCD is the final member of the PCA dioxygenase family to be purified . It is compared with other members of this family as well as other catecholic dioxygenases.

J Gen Microbiol, 1993 Jul, 139 ( Pt 7), 1523 - 30
Identification of the L-tartrate dehydratase genes (ttdA and ttdB) of Escherichia coli and evolutionary relationship with the class I fumarase genes; Reaney SK et al.; The genes encoding an oxygen-labile stereospecific L-tartrate dehydratase (L-Ttd, EC 4.2.1.32) have been identified as the orfZ1 and orfZ2 genes located upstream of the rpsU-dnaG-rpoD operon at 67 min in the Escherichia coli linkage map . They were previously cloned and sequenced by M . Nesin and others (Gene 51, 149-161, 1987) and have now been independently cloned, partially resequenced, and designated as an operon (ttdAB) containing two translationally coupled genes . The enzyme behaves as a tetramer (M(r) 105,000) containing two pairs of non-identical subunits, TtdA (M(r) 32589) and TtdB (M(r) 22641), which otherwise resembles the homodimeric iron-sulphur-containing Class I fumarases of E . coli and Bacillus stearothermophilus . The amino acid sequences of the TtdA-TtdB subunits are colinearly related to a single fumarase subunit, indicating a common evolutionary ancestry . E . coli can use L-, D- and meso-tartrates as aerobic growth substrates and as reducible substrates for supporting anaerobic growth on glycerol . L-Ttd was induced during anaerobic growth on glycerol plus L- and meso-tartrates, and a stereospecific D-tartrate dehydratase was induced by all three stereoisomers under comparable conditions . No meso-tartrate dehydratase was detected, nor were any dehydratases detected after aerobic growth on tartrate minimal media suggesting that different catabolic routes operate under aerobic conditions.

Biophys J, 1993 Jul, 65(1), 215 - 26
Time-resolved fluorescence of the single tryptophan of Bacillus stearothermophilus phosphofructokinase; Kim SJ et al.; The fluorescence of the single tryptophan in Bacillus stearothermophilus phosphofructokinase was characterized by steady-state and time-resolved techniques . The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition . Time-resolved emission spectral data were fitted to discrete and distributed lifetime models . The fluorescence decay is a double exponential with lifetimes of 1.6 and 4.4 ns and relative amplitudes of 40 and 60% . The emission spectra of both components are identical with maxima at 327 nm . The quantum yield is 0.31 +/- 0.01 . The shorter lifetime is independent of temperature; the longer lifetime has weak temperature dependence with activation energy of 1 kcal/mol . The fluorescence intensity and decay are the same in H2O and D2O solutions, indicating that the indole ring is not accessible to bulk aqueous solution . The fluorescence is not quenched significantly by iodide, but it is quenched by acrylamide with bimolecular rate constant of 5 x 10(8) M-1 s-1 . Static and dynamic light scattering measurements show that the enzyme is a tetramer in solution with hydrodynamic radius of 40 A . Steady-state and time-resolved fluorescence anisotropies indicate that the tryptophan is immobile . The allosteric transition has little effect on the fluorescence properties . The fluorescence results are related to the x-ray structure.

Ann Pharmacother, 1993 Jul-Aug, 27(7-8), 870 - 3
Intrathecal administration of amikacin for treatment of meningitis secondary to cephalosporin-resistant Escherichia coli; Preston SL et al.; OBJECTIVE: To report a case of gram-negative bacillary meningitis (GNBM) secondary to cephalosporin-resistant Escherichia coli that was treated with intrathecal and intravenous amikacin and intravenous imipenem/cilastatin (I/C) . CASE SUMMARY: A patient who had undergone two recent neurosurgical procedures developed GNBM and bacteremia . He was treated empirically with ceftazidime . Both bloodstream and cerebrospinal fluid isolates were identified as E . coli, resistant to third-generation cephalosporins, penicillins, tobramycin, and gentamicin . The patient was subsequently treated with intravenous and intrathecal amikacin plus intravenous I/C . He experienced subjective and objective improvement on days 2-4 of antimicrobial therapy; two generalized tonic-clonic seizures occurred on days 7 and 12 . Intrathecal amikacin was discontinued after 6 days, and intravenous amikacin and I/C were discontinued after 23 and 27 days, respectively . The patient's mental status did not completely return to premeningitis baseline . DISCUSSION: Third-generation cephalosporins are the treatment of choice for GNBM . In the case reported herein, bacterial resistance to these agents prompted the use of a therapy that has not been well studied and is also considered to be less safe and perhaps less efficacious . Treatment of GNBM with an intrathecally administered aminoglycoside or with intravenous I/C plus an aminoglycoside is reviewed . CONCLUSIONS: Patients with GNBM secondary to third-generation cephalosporin-resistant organisms may require therapies that may be less effective and more toxic . Further study of alternative agents is warranted.

Biofizika, 1993 Jul-Aug, 38(4), 672 - 7
{Change in the permeability of liposome phospholipid membranes under the effect of Bacillus thuringiensis delta-endotoxins}; Iarkov SP et al.; We studied release of the fluorescent calcein marker from egg-lecithin liposomes under action of intact delta-endotoxin from Bacillus thuringiensis var . israilensis, and hydrolysed preparation of delta-endotoxin from Bacillus thuringiensis var . kurstaki together with the motional parameters of 5- and 12-doxyl stearic radical probes in the liposomal membrane . The yield of the non-penetrating fluorescent marker was shown to be a sensitive indicator of the disturbance of the membrane barrier function.

Mol Biol (Mosk), 1993 Jul-Aug, 27(4), 952 - 9
{Creation of a hybrid protein gene based on Bacillus thuringiensis delta endotoxins CryIIIA and CrIA(a) and expression of its derivatives in Escherichia coli}; Shadenkov AA et al.; The 5'-terminal fragment (containing 1-565th codons) of Bacillus thuringiensis var . tenebrionis gene for the Coleoptera-specific delta-endotoxin CryIIIA was cloned . This sequence was extended with either only a homologous fragment of CryIA(a) or that one together with in-frame NPTII or GUS coding sequences . The obtained gene derivatives were expressed in E . coli . The analysis of hybrid polypeptides confirmed the enzymatic activities of bifunctional proteins and showed toxic properties of "insectotoxin-NPTII" against Colorado potato beetle (Leptinotarsa decemlineata).

Kekkaku, 1993 Jul, 68(7), 469 - 78
{Retreatment of pulmonary tuberculosis--duration of chemotherapy}; Wada M et al.; Although standard chemotherapy for initial treatment of pulmonary tuberculosis has been established, regimens for retreatment of tuberculosis have not yet been established . One hundred fifty nine retreatment pulmonary tuberculosis cases admitted to Fukujuji Hospital were retrospectively analyzed . Regardless of the age at the start of retreatment, majority of cases were treated previously between 1955 and 1960 . Bacillary negative conversion rate, duration of chemotherapy, follow-up period and bacteriological relapse rate were compared according to resistance against isoniazid and/or rifampicin . Sixty four cases were sensitive to both INH and RFP . For this group the average duration of chemotherapy was 14.6 months, mean follow up period was 47.3 months and relapse rate was 3.1% . This rate was similar to that of initial treatment cases . Sixty one (94%), were treated with more than two sensitive drugs containing INH and RFP . The 22 INH-resistant and RFP-susceptible cases were treated for 18.6 months and followed up for 55.2 months . The relapse rate of this group was 13.6% . Thirteen cases were treated with more than 2 sensitive drugs containing RFP . Eleven cases were resistant to both INH and RFP . Five of them were surgically operated of which 3 cases were converted to negative and among the nonsurgical cases in this group only one remained sputum positive . All of these retreatment regimens did not contain pyrazinamide and ofloxacin . Although bacillary positive rate of INH and RFP susceptible cases was 13.0% at 6 months after treatment and 5.3% at 12 months after treatment, that of INH resistant and RFP susceptible cases were 25.0% and 12.5%, respectively . A certain rule of retreatment could be obtained from the result of this study.

Appl Environ Microbiol, 1993 Jul, 59(7), 2007 - 13
Purification and preliminary characterization of permethrinase from a pyrethroid-transforming strain of Bacillus cereus; Maloney SE et al.; Bacillus cereus SM3 was isolated on a mineral salts medium with Tween 80 as the primary carbon source . It was able to hydrolyze second- and third-generation pyrethroids, thereby generating noninsecticidal products . The enzyme responsible for this hydrolytic reaction was named permethrinase for this study . This is the first instance in which pyrethroid detoxification has been achieved with a cell-free microbial enzyme system . Permethrinase was purified by ion-exchange chromatography and gel filtration chromatography . The molecular mass of native permethrinase was 61 +/- 3 kDa, as estimated by Sephadex G-100 gel filtration . This novel microbial esterase seems to be a carboxylesterase . Permethrinase activity had an optimum pH of 7.5 and a temperature optimum of 37 degrees C . No cofactors or coenzymes were required for permethrinase activity . The enzyme may be a serine esterase, as it seems to be sensitive to the organophosphorus compound tetraethylpyrophosphate at concentrations in the micromolar range . Addition of dithiothreitol afforded permethrinase protection against the inhibitory effects of the sulfydryl agents p-chloromercuribenzoate and N-ethylmaleimide . The enzyme was stable over a range of temperatures . Cell extracts of strain SM3 also contained another esterase, which was active towards beta-naphthylacetate, but this enzyme was distinct from permethrinase.

Infect Control Hosp Epidemiol, 1993 Jul, 14(7), 390 - 4
Evaluation of a rapid readout biological indicator for flash sterilization with three biological indicators and three chemical indicators; Rutala WA et al.; OBJECTIVE: Flash sterilization is most commonly used for emergency sterilization of unwrapped items in a gravity displacement sterilizer for three minutes . Sterilization quality assurance is monitored by biological indicators that require a 24-hour incubation prior to reading . In this study, we compared a new biological indicator that provides results within 60 minutes with three conventional, 24-hour biological indicators for monitoring flash sterilization and three chemical indicators . DESIGN: Conventional biological indicators tested included the conventional Attest 1261, Proof Flash and Assert, while the rapid readout indicator tested was Attest 1291 . Attest Rapid Readout detects the presence of a Bacillus stearothermophilus enzyme by reading a fluorescent product that is produced by the enzymatic break-down of a nonfluorescent substrate . Chemical indicators tested included Comply, Incheque, and Thermalog S . Survival at 132 degrees C in a gravity displacement sterilizer was measured by media color change after incubation for 24 hours at 56 degrees C for the three conventional biological indicators, fluorescence at 60 minutes for the Attest Rapid Readout biological indicator, and color change for the chemical indicators . Each exposure time was replicated four times with 10 of each biological and chemical indicator per run . RESULTS: The conventional biological indicators (Attest, Proof Flash, and Assert) had 90%, 48%, and 40% spore survival at two minutes exposure; 23%, 3%, and 0% at three minutes exposure; and 3%, 0%, and 0% at four minutes exposure respectively . The Attest Rapid Readout biological indicator had 88%, 33%, and 0% enzyme activity detectable at 2, 3, and 4 minutes exposure . The chemical indicators Comply, Incheque, and Thermalog S revealed sterilization failure rates of 100%, 100%, and 100% at 0 minutes exposure; 100%, 100%, and 45% at one minute; 0%, 0%, and 28% at two minutes exposure; 0%, 0%, and 18% at three minutes exposure; and 0%, 0%, and 0% at four minutes exposure, respectively . CONCLUSION: The sensitivity of the Attest Rapid Readout parallels the conventional biological indicators . These data suggest that a 60-minute rapid readout biological indicator is equivalent to the 24-hour biological indicators . If further studies demonstrate that a four-minute flash sterilization cycle provides a needed safety margin to ensure sterilization, then consideration should be given to requiring a four-minute flash sterilization cycle . Chemical indicators were too sensitive to the processing conditions (eg, steam) and are inadequate to ensure adequate sterilization.

Eur J Biochem, 1993 Jul 1, 215(1), 167 - 70
Increase of specificity of RNase from Bacillus amyloliquefaciens (barnase) by substitution of Glu for Ser57 using site-directed mutagenesis; Yakovlev GI et al.; Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus intermedius show the specificity towards the nature of a nucleoside at the O3' end of the phosphodiester bond to be split in the preference order G > A >> U > C in the cleavage reactions of polynucleotides . It follows from the X-ray data that the substrate guanosine base is bound at the active site of these RNases in the same manner as for high-specificity guanylic RNases . We supposed that the difference in specificity for the two types of RNases is due to the additional hydrogen bond between the protein and a purine base in the case of bacterial guanyl-preferring RNases in contrast to the high-specificity guanylic RNases . To examine this hypothesis we prepared the Ser57 --> Glu mutant of B . amyloliquefaciens, in which this hydrogen bond is eliminated . Kinetic studies demonstrate that the specificity of this mutant towards guanylic substrates is 35-times greater than that of the wild-type RNases from B . amyloliquefaciens and close to that of RNases T1.

Oftalmologia, 1993 Jul-Sep, 37(3), 215 - 20
{Tuberculous sclerokeratitis and cutaneous tuberculids}; Preoteasa D; Sixteen years old patient presents bilateral tuberculous sclerokeratitis and papulonecrotic cutaneous tuberculids-type acnitis . The patient is a bearer of a latent tuberculous focere following a prolonged contact with a cavitar bacillary . The positive diagnosis was much facilitated by the association with acneiform dermic tuberculids at face, intense and positive local reaction to PPD and a favourable therapeutic response to tuberculostatics . The particularity of the case consists in a concomitant, cutaneous and ocular affection since the first episode, the affection evolving as a typical bilateral tuberculous sclerokeratitis.

J Bacteriol, 1993 Jul, 175(14), 4545 - 9
The lonD gene is homologous to the lon gene encoding an ATP-dependent protease and is essential for the development of Myxococcus xanthus; Tojo N et al.; Myxococcus xanthus contains two genes (lonV and lonD) homologous to the Escherichia coli lon gene for an ATP-dependent protease . We found that the lonD gene encodes a 90-kDa protein consisting of 827 amino acid residues . The lonD gene product shows 49, 48, and 52% sequence identity to the products of the M . xanthus lonV, E . coli lon, and Bacillus brevis lon genes, respectively . When a lonD-lacZ fusion was used, lonD was expressed during both vegetative growth and development . However, while lonD-disrupted strains were able to grow normally vegetatively, the development of M . xanthus was found to be arrested at an early stage in these strains . The mutant strains were able to form neither fruiting bodies nor myxospores.

J Bacteriol, 1993 Jul, 175(14), 4538 - 44
Myxococcus xanthus encodes an ATP-dependent protease which is required for developmental gene transcription and intercellular signaling; Gill RE et al.; The bsgA gene of Myxococcus xanthus plays an essential role in the regulation of early gene expression during fruiting body formation and sporulation . bsgA mutants behave as though unable to initiate a required cell-cell interaction and consequently fail to transcribe normal levels of many developmentally induced genes . We determined the nucleotide sequence of bsgA, which predicts a single gene encoding a 90.4-kDa protein . The deduced BsgA protein shares 45 and 48% amino acid identity with the lon genes of Escherichia coli and Bacillus brevis, respectively . The cloned bsgA gene was expressed in E . coli, and the BsgA protein was partially purified and found, like its E . coli homolog, to be an ATP-dependent protease . Thus, the basis for the phenotype of bsgA mutants is likely to be a defect in intracellular proteolysis.

Urology, 1993 Jul, 42(1), 26 - 30
Upper tract transitional cell carcinoma following treatment of superficial bladder cancer with BCG; Miller EB et al.; Eighty-two consecutive patients treated with intravesical bacillus Calmette-Guerin (BCG) for recurrent superficial bladder tumors were evaluated for development of metachronous upper tract tumors (UTT) . All patients had normal upper tract studies within three months of starting BCG treatment . With a median follow-up of sixty-two months (range 25 to 124), 11 patients (13.4%) were found to have UTT . The median interval between initiation of BCG therapy and diagnosis of the UTT occurrence was thirty-eight months (range 7 to 110) . All patients were asymptomatic when the UTT was diagnosed . An abnormal surveillance intravenous or retrograde pyelogram was the method of diagnosis in 8 patients . Positive cytology alone directed diagnoses in 2 patients, and 1 patient was diagnosed in the workup of hematuria . Overall upper tract cytology was positive in 7 of 11 patients . Nephroureterectomy was performed in 9 patients and 2 had ureteroscopic biopsy and fulguration . Median follow-up after treatment of UTT was thirty-two months (range 3 to 80) . UTT pathologic stage was Pa in 2 patients, P1 in 1 patient, and P2 or higher in 8 patients . Distant metastasis developed in 7 patients, 2 patients have recurrent superficial bladder tumors, and 2 patients are free of disease . The reported incidence in the literature for UTT tumors in patients with previous superficial or muscle invasive tumors ranges from 1.6 percent to 8.5 percent . The 13.4 percent incidence of UTT in the present study demonstrates the increased risk for patients in this series who were selected for BCG therapy . These risk factors include high tumor grade, associated carcinoma in situ (CIS), multiple tumors, T1 tumors, and failure of prior intravesical therapy . The fact that all patients were asymptomatic at the time of diagnosis of UTT emphasizes the importance of long-term periodic surveillance with radiographic and cytologic studies of the upper tracts for patients with similar risk factors.

J Bacteriol, 1993 Jul, 175(13), 4176 - 85
Sequence, transcriptional, and functional analyses of the valine (branched-chain amino acid) dehydrogenase gene of Streptomyces coelicolor; Tang L et al.; The gene encoding the valine (branched-chain amino acid) dehydrogenase (Vdh) from Streptomyces coelicolor has been characterized as follows . The vdh gene was identified by hybridization to a specific oligodeoxynucleotide that was synthesized on the basis of the N-terminal amino acid sequence of purified Vdh . Nucleotide sequence analysis predicts that the vdh gene contains a 364-amino-acid open reading frame that should produce a 38,305-M(r) protein . The deduced amino acid sequence of the Vdh protein is significantly similar to those of several other amino acid dehydrogenases, especially the leucine and phenylalanine dehydrogenases from Bacillus spp . The vdh gene is apparently transcribed from a single major transcriptional start point, separated by only 8 bp from the 5' end of a divergent transcript and located 63 bp upstream from the vdh translational start point . Mutants with a disrupted vdh gene have no detectable Vdh activity and have lost the ability to grow on valine, leucine, or isoleucine as the sole nitrogen source . This vdh mutation does not significantly affect growth or actinorhodin production in a minimal medium, yet the addition of 0.2% L-valine to the medium provokes approximately 32 and 80% increases in actinorhodin production in vdh+ and vdh strains, respectively.

Cancer Immunol Immunother, 1993 Jul, 37(2), 105 - 11
Induction of bacillus-Calmette-Guérin-activated killer cells from human peripheral blood mononuclear cells against human bladder carcinoma cell lines in vitro; Thanhauser A et al.; Cytotoxicity against two human bladder carcinoma cell lines (BT-A and BT-B) was investigated using human peripheral blood mononuclear cells (PBMC) stimulated with viable bacillus Calmette-Guerin (BCG) or sonicated BCG (s-BCG) . We applied a cytotoxicity assay based on radioactive labelling of tumour cells by incorporation of L{3H}methionine . The results were compared with the cytotoxicity exerted by lymphokine-activated killer (LAK) cells generated by interleukin-2 (IL-2) and interferon gamma (IFN gamma) . BCG-stimulated PBMC showed a cytotoxic potential against BT-A and BT-B comparable to that of IFN gamma-generated LAK cells, but this did not reach the level of IL-2-generated LAK cells . We termed these cytotoxic effectors BCG-activated killer (BAK) cells . In contrast to their cytotoxicity against bladder tumour cells, BAK cells did not differ from unstimulated PBMC in the killing of K562 cells . Only viable but not sonicated BCG was able to induce cytotoxicity against BT-A and BT-B . We could demonstrate the presence of the cytokines IFN gamma, IL-2, tumour necrosis factor alpha (TNF alpha) and TNF beta in the supernatants harvested during the generation of BAK cells . Monoclonal antibodies neutralizing IFN gamma were able to inhibit BCG-mediated cytotoxicity, giving evidence of the involvement of IFN gamma in the induction of BAK cells . Furthermore, we performed experiments to investigate the cytotoxic potential of distinct cell populations . The cells effective in BCG-activated killing of bladder tumour cells could be localized within the CD8+/CD56+ lymphocyte subset . CD4+ cells and macrophages did not exhibit cytolytic activity . Our findings imply that the activation by BCG of CD8+/CD56+ killer cells might be an important antitumoral mechanism during BCG therapy against superficial urothelial bladder cancer.

J Exp Med, 1993 Jul 1, 178(1), 197 - 209
Protective immunity elicited by recombinant bacille Calmette-Guerin (BCG) expressing outer surface protein A (OspA) lipoprotein: a candidate Lyme disease vaccine; Stover CK et al.; The current vaccine against tuberculosis, Mycobacterium bovis strain bacille Calmette-Guerin (BCG), offers potential advantages as a live, innately immunogenic vaccine vehicle for the expression and delivery of protective recombinant antigens (Stover, C.K., V.F . de la Cruz, T.R . Fuerst, J.E . Burlein, L.A . Benson, L.T . Bennett, G.P . Bansal, J.F . Young, M.H . Lee, G.F . Hatfull et al . 1991 . Nature {Lond} . 351:456; Jacobs, W.R., Jr., S.B . Snapper, L . Lugosi and B.R . Bloom . 1990 . Curr . Top . Microbiol . Immunol . 155:153; Jacobs, W.R., M . Tuckman, and B.R . Bloom . 1987 . Nature {Lond.} . 327:532); but as an attenuated intracellular bacterium residing in macrophages, BCG would seem to be best suited for eliciting cellular responses and not humoral responses . Since bacterial lipoproteins are often among the most immunogenic of bacterial antigens, we tested whether BCG expression of a target antigen as a membrane-associated lipoprotein could enhance the potential for a recombinant BCG vaccine to elicit high-titered protective antibody responses to target antigens . Immunization of mice with recombinant BCG vaccines expressing the outer surface protein A (OspA) antigen of Borrelia burgdorferi as a membrane-associated lipoprotein resulted in protective antibody responses that were 100-1,000-fold higher than responses elicited by immunization with recombinant BCG expressing OspA cytoplasmically or as a secreted fusion protein . Furthermore, these improved antibody responses were observed in heterogeneous mouse strains that vary in their immune responsiveness to OspA and sensitivity to BCG growth . Thus, expression of protective antigens as chimeric membrane-associated lipoproteins on recombinant BCG may result in the generation of new candidate vaccines against Lyme borreliosis and other human or veterinary diseases where humoral immunity is the protective response.

Plant J, 1993 Jul, 4(1), 71 - 9
Specificity of a promoter from the rice tungro bacilliform virus for expression in phloem tissues; Bhattacharyya-Pakrasi M et al.; The major promoter region for the transcription of the genome of rice tungro bacilliform virus (RTBV), a newly described badnavirus, has been identified . Fragments of the RTBV genome upstream of the site of transcription initiation were isolated and tested for promoter activity using a beta-glucuronidase receptor gene (gusA) . Assays of transient gusA expression were performed following introduction of the chimeric gene into protoplasts via electroporation . The chimeric RTBV-promoter: gusA gene was more active in rice protoplasts than in maize or tobacco protoplasts, but was weaker than gusA controlled by an enhanced 35S promoter from cauliflower mosaic virus . Analysis of gusA gene expression following introduction of chimeric reporter genes into intact leaves via micro-projectile bombardment indicated that the GUS activity is present primarily in vascular tissues . Transgenic rice plants carrying the chimeric gusA gene had GUS activity only in the phloem of the vascular bundles in the leaf . Tissue printing studies demonstrated that RTBV accumulates in the vascular bundles of infected rice leaves . The results of our study indicate that phloem-specific expression from the RTBV promoter is an intrinsic property of the viral promoter.

J Clin Microbiol, 1993 Jul, 31(7), 1730 - 4
Polymerase chain reaction-based restriction fragment length polymorphism analysis of a fragment of the ribosomal operon from Rochalimaea species for subtyping; Matar GM et al.; Restriction endonuclease analysis of a polymerase chain reaction-amplified DNA fragment which included the spacer region between the genes coding for 16S and 23S rRNAs and a portion of the gene coding for 23S rRNA (spacer + 23S) was done on 10 previously characterized clinical isolates of Rochalimaea henselae, one clinical isolate of Rochalimaea quintana, and the type strains of R . henselae, R . quintana, Rochalimaea vinsonii, and Bartonella bacilliformis . Brucella abortus DNA was not amplified by the primer set used . The clinical isolates of Rochalimaea were obtained from blood or tissue from patients with and without preexisting disease . The amplicon from each strain was digested with five endonucleases (AluI, HaeIII, TaqI, HinfI, and MseI) . AluI and HaeIII were useful in species differentiation and subtyping of R . henselae . R . henselae isolates showed six different restriction patterns with AluI and four patterns with HaeIII . TaqI, HinfI, and MseI were useful only in species differentiation . These observations indicate that PCR amplification of the spacer + 23S region of the ribosomal DNA of Rochalimaea spp., along with restriction endonuclease analysis, allows differentiation of Rochalimaea spp . from closely related genera, differentiation among the species within Rochalimaea, and differentiation of strains within R . henselae . The subtyping potential of this method may be useful for further clinical and epidemiologic studies of the spectrum of diseases caused by R . henselae.

J Clin Microbiol, 1993 Jul, 31(7), 1677 - 82
Molecular strain typing of Mycobacterium tuberculosis to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures; Small PM et al.; Molecular strain typing by restriction fragment length polymorphism analysis was used to demonstrate that two clusters of Mycobacterium tuberculosis cultures involving six patients resulted from cross-contamination in the mycobacteriology laboratory . Contaminated cultures were processed by the decontamination procedure and were read on the BACTEC instrument following acid-fast bacillus smear-positive specimens from patients with active tuberculosis . Investigation of these episodes suggested opportunities for modification of laboratory procedures to minimize cross-contamination and confirmed the adverse medical and public health consequences of false-positive cultures . Strain-typing results were used in decisions regarding patient care, including the curtailment of unnecessary treatment in one patient . Molecular strain typing appears to be a valuable means of identifying false-positive cultures of M . tuberculosis in selected settings.

Biotechnol Prog, 1993 Jul-Aug, 9(4), 411 - 20
Evaluation of column flotation in the downstream processing of fermentation products: recovery of a genetically engineered alpha-amylase; Miranda EA et al.; Flotation is a simple, inexpensive, and versatile unit operation with a largely unexplored potential in biotechnology . There is a general lack of research concerning biotechnological applications in this area, especially in the recovery of fermentation products . Moreover, the few reports in the literature do not consider the modern concept of column flotation as practiced in the mineral industry . We report herein the application of column flotation for the recovery of a Bacillus stearothermophilus alpha-amylase expressed in Escherichia coli by the use of a food-grade polymer, (hydroxypropyl)methylcellulose (HPMC), and ammonium sulfate . First, the enzyme was removed from the liquid phase by partition to a salted-out HPMC phase . The enzyme-containing polymer flocs were then floated from the liquid . Recovery of active enzyme was as high as 90%, with throughput as high as 94 m3/(h.m2) . The floatability of the enzyme from a periplasmic extract was higher than extracellular enzyme in the broth due to the presence of depressors of molecular weight lower than 10,000 in the broth.

J Exp Med, 1993 Jul 1, 178(1), 101 - 11
Leishmania major-specific, CD4+, major histocompatibility complex class II-restricted T cells derived in vitro from lymphoid tissues of naive mice; Shankar AH et al.; Several studies indicate that the outcome of experimental murine cutaneous leishmaniasis caused by Leishmania major (Lm) is determined by immunological events occurring shortly after infection . These events lead to outgrowth of either protective CD4+ T cells in the C57BL/6 mouse, which cures, or exacerbative cells in the BALB/c mouse, which succumbs to disease . Potential factors influencing the outgrowth of protective or exacerbative T cells include antigen-presenting cells (APC), cytokines, and parasite antigens . An in vitro system, in which one could precisely control the factors shaping early events in the T cell response to Lm, would be very useful . To this end, we have examined the in vitro response of naive lymphocytes to Lm promastigotes . The data presented here show that Lm-specific CD4+ T cell receptor alpha/beta + T cells can be generated in vitro from spleen and lymph node cell populations of naive mice . Furthermore, they can be obtained from the CD44low (unprimed) population of T lymphocytes, indicating that in vitro priming occurs . The ability to generate these T cells is dependent on the presence of live parasites and is not due to a parasite-derived nonspecific T cell mitogen . Restimulation, as assayed by proliferation, requires APC bearing syngeneic I-A . Optimal restimulation of the in vitro derived T cells is achieved only when live promastigotes are used . The T cells do not proliferate in response to a frozen-and-thawed lysate of promastigotes, yet they exhibit mild reactivity to lysates prepared from heat-shocked promastigotes . Furthermore, they do not recognize two predominant antigens on the promastigote surface, lipophosphoglycan and gp63 . T cells derived in vitro with Lm show crossreactivity with live L . donovani, less crossreactivity with live L . mexicana, and no crossreactivity with live Bacillus-Calmette-Guerin or live Brugia malayi microfilariae . Finally, these early T cells, whether derived from healing C57BL/6 or nonhealing BALB/c mice, produce interleukin 2 (IL-2), IL-4, and interferon gamma.

Mikrobiologiia, 1993 Jul-Aug, 62(4), 633 - 8
{Tyrosol--the autoregulatory d1 factor of the yeast Saccharomyces cerevisiae.}; Batrakov SG et al.; The autoregulatory d1 factor of the yeast, Saccharomyces cerevisiae, that induces the transition of vegetative cells into refractory resting forms, has been isolated from the cell-free culture medium as an individual crystalline compound . It has been shown to be 2-(4-hydroxyphenyl)ethane-1-ol which is also known as tyrosol . When added to the producer culture at 5-15 microM concentration, tyrosol stimulated the endogenous respiration of cells, but inhibited at 20-80 microM concentration . At 200-800 microM concentration, it induced the occurrence of resting forms . The action of tyrosol was not specific, for it also inhibited the cell respiration of the bacteria, Escherichia coli and Bacillus cereus, at 64-86 microM concentration.

Carbohydr Res, 1993 Jun 21, 244(2), 315 - 23
A convenient synthesis of beta-D-galactosyl disaccharide derivatives using the beta-D-galactosidase from Bacillus circulans; Usui T et al.; beta-D-Gal-(1-->4)-beta-D-GlcNAc-OC6H4NO2-p (p-nitrophenyl N-acetyl-beta-lactosaminide) and beta-D-Gal-(1-->6)-beta-D-GlcNAc-OC6H4NO2-p (p-nitrophenyl N-acetyl-beta-isolactosaminide) were regioselectively synthesized from lactose and p-nitrophenyl 2-acetamido-2-deoxy-glucopyranoside, employing transglycosylation by the beta-D-galactosidase from Bacillus circulans and by controlling the concentration of organic solvent in the reaction system . The (1-->4)-linked disaccharide was formed exclusively when the concentration of organic solvent was high, whereas the (1-->6)-linked isomer was produced with a low concentration . Further utilization of the transglycosylation by the enzyme led to the regioselective formation of beta-D-Gal-(1-->4)-D-GalNAc and beta-D-Gal-(1-->4)-beta-D-GalNAc-OC6H4NO2-p . With the enzyme, beta-D-galactosyl transfer occurred preferentially at the O-4 position of GlcNAc and GalNAc, regardless of the configuration of the hydroxyl group.

MMWR Morb Mortal Wkly Rep, 1993 Jun 18, 42(23), 456 - 9
Pseudomonas cepacia at summer camps for persons with cystic fibrosis; Structural features of lipoarabinomannan from Mycobacterium bovis BCG . Determination of molecular mass by laser desorption mass spectrometry; Centre National de la Recherche Scientifique, Departement Glycoconjugues et Biomembranes, Toulouse, FranceIt was recently shown that mycobacterial lipoarabinomannan (LAM) can be classified into two types (Chatterjee, D., Lowell, K., Rivoire B., McNeil M . R., and Brennan, P . J . (1992) J . Biol . Chem . 267, 6234-6239) according to the presence or absence of mannosyl residues (Manp) located at the nonreducing end of the oligoarabinosyl side chains . These two types of LAM were found in a pathogenic Mycobacterium tuberculosis strain and in an avirulent M . tuberculosis strain, respectively, suggesting that LAM with Manp characterizes virulent and "disease-inducing strains." We now report the structure of the LAM from Mycobacterium bovis Bacille Calmette-Guerin (BCG) strain Pasteur, largely used throughout the world as vaccine against tuberculosis . Using an up-to-date analytical approach, we found that the LAM of M . bovis BCG belongs to the class of LAMs capped with Manp . By means of two-dimensional homonuclear and heteronuclear scalar coupling NMR analysis and methylation data, the sugar spin system assignments were partially established, revealing that the LAM contained two types of terminal Manp and 2-O-linked Manp . From the following four-step process: (i) partial hydrolysis of deacylated LAM (dLAM), (ii) oligosaccharide derivatization with aminobenzoic ethyl ester, (iii) HPLC purification, (iv) FAB/MS-MS analysis; it was shown that the dimannosyl unit alpha-D-Manp-(1-->2)-alpha-D-Manp is the major residue capping the termini of the arabinan of the LAM . In this report, LAM molecular mass determination was established using matrix-assisted UV-laser desorption/ionization mass spectrometry which reveals that the LAM molecular mass is around 17.4 kDa . The similarity of the LAM structures between M . bovis BCG and M . tuberculosis H37Rv is discussed in regard to their function in the immunopathology of mycobacterial infection.

J Biol Chem, 1993 Jun 15, 268(17), 12334 - 40
A specific binding protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp . berliner; Vadlamudi RK et al.; Biopesticides based on the bacterium Bacillus thuringiensis have attracted wide attention as safe alternatives to chemical pesticides . In this paper, we report, for the first time, the identification and purification of a single binding protein from a lepidopteran insect, Manduca sexta, that is specific for a cryIA toxin of B . thuringiensis . The purified protein appeared as a single band of 210 kDa on a two-dimensional gel, had a pI of approximately 5.5, and stained with Schiff's reagent . The band material was sensitive to proteolytic digestion and was rich with acidic amino acids, indicating its protein nature . Radiolabeled toxin bound to the protein with a Kd value of 708 pM and could be specifically blocked by unlabeled toxin but not by toxins from other subspecies of B . thuringiensis . This study lays the groundwork to clone the toxin binding protein and to determine the molecular mechanism(s) of toxin action.

Biochemistry, 1993 Jun 15, 32(23), 5978 - 84
Directed mutagenesis and barnase-barstar recognition; Hartley RW; Directed mutagenesis has been applied to the cloned genes of barnase and barstar, the extracellular ribonuclease of Bacillus amyloliquefaciens and its intracellular inhibitor, to locate residues involved in the mutual recognition of these two proteins . Arg59 and His102 of barnase and Asp35 and Asp39 of barstar have been so identified . With both Cys40 and Cys82 mutated to alanines, barstar is still produced in high yield and is functional both in vitro and in vivo . Methods devised for determining relative and absolute dissociation coefficients for various combinations of mutant and wild-type proteins have allowed us to determine a dissociation coefficient for the complex of wild-type barnase and barstar of about 10(-13) M, with off and on rate constants of 10(-5) s-1 and 10(8) M-1 s-1, respectively.

Eur J Biochem, 1993 Jun 15, 214(3), 771 - 8
An analysis of Bacillus thuringiensis delta-endotoxin action on insect-midgut-membrane permeability using a light-scattering assay; Carroll J et al.; Changes in the membrane permeability of Manduca sexta midgut brush-border-membrane vesicles (BBMV) after addition of Bacillus thuringiensis delta-endotoxins were studied using osmotic swelling experiments, volume changes being monitored as the change in 90 degrees light scattering . Typically, control BBMV exhibited limited permeability for sucrose and salts (KCl), while being permeable for urea and glucose . The action of delta-endotoxin was examined using proteolytically activated Cry-IA(c) and CryIB toxins . CryIA(c) produced a marked change in the solute permeability of M . sexta BBMV, significant effects being observed at 3.75 pmol/mg BBMV . The permeability change was relatively non-selective with cations, anions and neutral solutes all traversing the membrane to an increased extent in the presence of CryIA(c) . In contrast, the CryIB toxin had no effect on BBMV permeability.

Eur J Biochem, 1993 Jun 15, 214(3), 659 - 69
Characteristics of an exochitinase from Streptomyces olivaceoviridis, its corresponding gene, putative protein domains and relationship to other chitinases; Blaak H et al.; Streptomyces olivaceoviridis efficiently degrades chitin . Shotgun cloning of partially Sau3A-cleaved DNA using the multicopy vector pIJ702 and Streptomyces lividans 66 as host resulted in the identification of the plasmid pCHI O1 which harbours an insert of 4.6 kb . In the presence of chitin as sole carbon source, transformants of S . lividans 66 carrying pCHI O1 or its derivatives with smaller inserts overproduced an exochitinase which was purified to homogeneity . The chitin-inducible enzyme with an isoelectric point of 4.0 shows optimal activity at pH 7.3 and 55 degrees C, has an apparent molecular mass of 47 kDa and is competitively inhibited by the pseudosugar allosamidin . The enzyme was identified as an exochitinase since it generates exclusively chitobiose from chitotetraose, chitohexaose, and colloidal high-molecular mass chitin . Sequence analysis of a reading frame of 1794 base pairs and comparison of the deduced amino-acid sequence allowed the identification of the putative catalytic domain, one region with significant similarity to the type-III module of fibronectin and one domain of unknown function . Multiple sequence alignment and hydrophobic-cluster analysis of 25 chitinolytic enzymes from bacteria, fungi and plants allowed the identification of their characteristic domains . The exochitinase from S . olivaceoviridis shares highest similarity with the chitinase D from Bacillus circulans.

Med Clin (Barc), 1993 Jun 12, 101(3), 102 - 4
{Bacillary (epithelioid) angiomatosis with a nodular presentation and self-limited evolution}; Teira R et al.; Bacillary angiomatosis (epithelioid) is a recently described clinicopathologic syndrome, principally associated to infection by the human immunodeficiency virus . The case of a patient who was seen for fever and the appearance of four painful, erythematous and indurated subcutaneous nodules on the anteroexternal face of the right lower extremity 15 days previously is presented . No microorganisms were observed by microbiologic and histologic techniques however the latter showed a vascular proliferation with prominent endothelium of epithelioid morphology and notable interstitial inflammatory reaction according to the pattern described as characteristic of epithelioid angiomatosis . The fever and the nodules disappeared spontaneously . The clinical and histopathologic characteristics of this disease as well as the recent contributions with respect to the identification of the possible causative bacillus are discussed.

J Mol Biol, 1993 Jun 5, 231(3), 870 - 6
The crystal structure of tris-inhibited phospholipase C from Bacillus cereus at 1.9 A resolution . The nature of the metal ion in site 2; Hansen S et al.; We report here the crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and the widely used biochemical buffer tris (hydroxymethyl)-methylamine (Tris) . The structure has been determined at 1.9 A resolution and refined to R = 20.3% . Tris has metal-binding properties, especially to Zn2+, and has been reported to reduce the activity of PLC . The amine nitrogen atom in Tris is co-ordinated to one of the three Zn2+ ions in the active site of the enzyme, thus confirming its chelating properties and the involvement of the metal ions in the catalytic process . The occupancy of the Zn2+ ion in site 2 in native PLC is 0.6 which could imply the presence of Ca2+ rather than Zn2+ . The fact that Tris binds to this metal ion, the nature of the site 2 co-ordination shell and comparison with several homologous Zn-metalloenzymes indicate that PLC is a 3-Zn metalloenzyme . This study is one of a series which explores the active site of PLC by complexing the enzyme with inhibitors and substrate analogues.

J Chromatogr, 1993 Jun 4, 639(1), 75 - 80
Preparation of DNA polymerase from Bacillus caldotenax; Burrows JA et al.; A procedure with four chromatography steps was developed for the purification of DNA polymerase from Bacillus caldotenax by using fast protein liquid chromatography . The procedure was suitable for use with process-scale media . Elution profiles obtained from ion-exchange chromatography and triazine-dye affinity chromatography with fast protein liquid chromatography and process-scale media were similar . The enzyme showed stronger interaction, however, with phenyl-Sepharose FF in the scaled-up process than with the phenyl-Superose used in fast protein liquid chromatography . The surprising binding of the DNA polymerase to sulphonated ion-exchange media at pH 7.5 may be explained by the structure of the enzyme.

Protein Expr Purif, 1993 Jun, 4(3), 223 - 31
High-level production of the mouse epidermal growth factor in a Bacillus brevis expression system; Wang B et al.; In order to determine the three-dimensional structure, the folding pathways, and the residues which are critical for biological functions of the mouse epidermal growth factor (mEGF), large amounts of wild-type and site-specific mutants are needed for biological and physiochemical studies . Genes coding for mEGF and its mutants were expressed in a Bacillus brevis system in which the expressed foreign proteins were secreted into the culture medium . However, proteases were also secreted that resulted in partial degradation of the desired foreign proteins . One mutant with much less protease secretion was isolated by applying nitrosoguanidine mutagenesis on a B . brevis strain . A new vector to facilitate DNA mutagenesis and sequencing was also constructed for this system . Methodologies for the transformation of B . brevis and the purification of expressed mEGF were developed . Under the optimized conditions, the production of the mEGF and its mutants was about 50 mg per liter, and yielded up to 10 mg of purified mEGF . Several mEGF mutants have been produced and their receptor binding abilities have been measured.

Am J Pathol, 1993 Jun, 142(6), 1959 - 66
Granuloma formation in severe combined immunodeficient (SCID) mice in response to progressive BCG infection . Tendency not to form granulomas in the lung is associated with faster bacterial growth in this organ; North RJ et al.; Intravenous inoculation of Mycobacterium bovis, strain bacillus Calmette-Guerin caused infection in the lungs, livers, and spleens of severe combined immunodeficient (SCID) mice and in the same organs in immunocompetent co-isogenic C.B17 mice . However, whereas infection in the latter mice was stabilized and partly resolved, it was progressive in SCID mice and eventually lethal, with the most rapid bacterial growth occurring in the lungs . Histological examination of infected organs showed that well-developed, compact epithelioid granulomas formed at sites of bacterial multiplication in livers, spleens, and lungs of C.B17 mice . Granulomas also formed in the livers and spleens of SCID mice, despite their inability to generate immunity . However, in the lungs of SCID mice, bacillus Calmette-Guerin was regionally distributed mostly in isolated alveolar macrophages and in aggregates of macrophages resembling small granulomas . The possibility that this tendency not to form granulomas in the lung is the reason for the more rapid growth of bacillus Calmette-Guerin in this organ is discussed.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 5322 - 6
Isolation of a proline-rich mycobacterial protein eliciting delayed-type hypersensitivity reactions only in guinea pigs immunized with living mycobacteria; Romain F et al.; Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced only by a previous immunization with living attenuated mycobacteria, usually bacillus Calmette-Guerin (BCG) . Living and killed bacteria share a number of common antigens . To identify and to purify molecules that are dominant antigens during immunization with living bacteria, a two-step selection procedure was used . Quantitative delayed-type hypersensitivity (DTH) reactions elicited in guinea pigs immunized either with living or with killed BCG were used to select or counterselect antigens present in BCG culture filtrates . Each major fraction eluted from a series of HPLC columns (gel filtration, DEAE, reverse-phase chromatography) was assayed and titrated on guinea pigs of each group . A protein with an unusual amino acid composition (40% proline, 12% threonine) was purified and N-terminally sequenced . To our knowledge, the sequence Thr-Pro-Pro-Xaa-Glu-Xaa-Pro-Pro-Pro-Pro-Gln-Xaa-Val-Xaa-Leu has not been previously reported . The protein was 100-fold more potent on guinea pigs immunized with living bacteria than on guinea pigs immunized with dead bacteria to elicit a DTH reaction.

Arch Pathol Lab Med, 1993 Jun, 117(6), 578 - 83
Probe technology for the clinical microbiology laboratory; Hall GS; Molecular-based techniques, such as use of genetic probes, can greatly reduce turnaround time of the identification of some isolates in the clinical microbiology laboratory . For example, culture confirmation of an acid-fast bacillus such as Mycobacterium tuberculosis or Mycobacterium avium-intracellulare by a gene probe can be performed within hours of the isolation of the organism . Conventional methods might require more than 2 to 4 weeks for this identification . Identification of a mold, such as Histoplasma capsulatum, with a gene probe, can be done in less than 24 hours, with minimal growth available; conventional fungal identification takes 1 to 2 weeks or more . Whether a probe is used in the clinical microbiology laboratory depends on a number of factors including accuracy of the probe, cost, commercial availability, and ease of performance . In addition, the relevance of a more rapid result needs to be considered . A gene probe may be more specific than conventional methodologies, and this may increase its advantage . Direct specimen testing with probes is presently hampered by inadequate sensitivities . Prior amplification may decrease this limitation . It is hoped that methods will be available in the near future that provide amplification and probing of clinical specimens for laboratory diagnosis to be made within a short time following specimen collection.

J Infect Dis, 1993 Jun, 167(6), 1452 - 5
A large outbreak of gastroenteritis caused by diarrheal toxin-producing Bacillus cereus; Luby S et al.; An outbreak of diarrhea occurred after a university field day . Of 643 attendees who returned mailed questionnaires, 139 (22%) reported illness . Persons who ate barbecued pork, which was unrefrigerated for 18 h after cooking, were five times more likely to become ill than those who did not eat pork (26% vs . 5%; relative risk, 5.4; 95% confidence interval, 1.4-20.9) . A leftover pork sample grew a Bacillus cereus isolate, > 10(5) cfu/g, that produced diarrheal toxin . Thirty-four percent of ill persons noted onset of illness outside the 6- to 24-h incubation period traditionally ascribed to B . cereus-mediated diarrhea, and an unusually high percentage (23%) noted fever . B . cereus may cause a wider spectrum of disease than previously described.

J Exp Med, 1993 Jun 1, 177(6), 1723 - 33
Mycobacterial virulence . Virulent strains of Mycobacteria tuberculosis have faster in vivo doubling times and are better equipped to resist growth-inhibiting functions of macrophages in the presence and absence of specific immunity; North RJ et al.; The kinetics of growth of two virulent strains of mycobacteria (M . tuberculosis Erdman and M . tuberculosis H37Rv) and two attenuated strains (M . tuberculosis H37Ra and M . bovis Bacillus Calmette-Guerin {BCG}) were studied in the lungs, livers, spleens, and kidneys of severe combined immunodeficient (SCID) mice and of their coisogenic CB-17 immunocompetent counterparts . It was found, in keeping with the findings of earlier investigators (Pierce, C . H., R . J . Dubos, and W . B . Schaefer . 1953 . J . Exp . Med . 97:189.), that in immunocompetent mice, virulent organisms grew progressively only in the lungs, whereas the growth of attenuated organisms was controlled in all organs . In SCID mice, in contrast, virulent mycobacteria grew rapidly and progressively in all organs, as did BCG, although at a slower rate . However, H37Ra failed to grow progressively in any organs of SCID mice, unless the mice were treated with hydrocortisone . In fact, hydrocortisone treatment enabled virulent, as well as attenuated, organisms to grow strikingly more rapidly in all organs of SCID mice and in all organs of CB-17 mice . A histological study showed that in SCID mice, multiplication of mycobacteria in the liver occurs in the cytoplasm of macrophages in granulomas and presumably in macrophages in other organs . It is suggested, therefore, that the macrophages of SCID mice possess a glucocorticoid-sensitive mycobacterial mechanism that prevents virulent and avirulent mycobacteria from expressing their true minimal doubling times . In the absence of this mechanism in the lungs of hydrocortisone-treated SCID mice, the doubling times of Erdman, H37Rv, BCG, and H37Ra were 17.7, 17.4, 44.6, and 98.6 h, respectively . The possible importance of a rapid multiplication rate for mycobacterial virulence is discussed.

Indian J Malariol, 1993 Jun, 30(2), 81 - 9
Laboratory and field evaluation of Spherix, a formulation of Bacillus sphaericus (B-101), to control breeding of Anopheles stephensi and Culex quinquefasciatus; Mittal PK et al.; Spherix, a powder formulation of Bacillus sphaericus strain B-101, serotype H5a 5b, was evaluated against larvae of Anopheles stephensi and Culex quinquefasciatus in both the laboratory and field . In laboratory tests the formulation @ 0.1 g/sq m produced 100% mortality against larvae of both mosquito species at room temperature (28-32 degrees C) . The larvicidal activity of Spherix against An . stephensi @ 0.5 g/sq m persisted for over 12 weeks under laboratory conditions . Field evaluation of Spherix @ 0.25-2.0 g/sq m produced 95-100% reduction in the larval density of both An . stephensi and Culex quinquefasciatus within 48 h in different habitats, and the larvicidal activity persisted for 2-4 weeks in water habitats.

Int J Lepr Other Mycobact Dis, 1993 Jun, 61(2), 250 - 4
Therapeutic efficacy of some new quinolones and a combination of ofloxacin with rifampin against Mycobacterium leprae infection induced in athymic nude mice; Tomioka H et al.; Ofloxacin (OFLX), having superior antileprosy activity among the various quinolones, was studied for its combined therapeutic efficacy with rifampin (RMP) against Mycobacterium leprae infection induced in nude mice . When OFLX (3 mg/mouse) was given to infected mice in combination with RMP (0.01 mg/mouse) by gavage once daily six times per week, from day 31 to day 80 postinfection, a significant combined effect was observed . This study demonstrates the possibility of using OFLX in multidrug regimens for the clinical control of bacilliferous leprosy patients.

Mol Gen Genet, 1993 Jun, 239(3), 416 - 24
Overproduction by gene amplification of the multifunctional arom protein confers glyphosate tolerance to a plastid-free mutant of Euglena gracilis; Reinbothe S et al.; Cells of the plastid-free mutant line of Euglena gracilis var . bacillaris, W10BSmL, can be adapted to glyphosate {N-(phosphonomethyl)glycine} by gradually increasing the concentration of the herbicide in the culture medium . The molecular basis of glyphosate tolerance is the selective ca . ten-fold overproduction of the multifunctional arom protein catalyzing steps 2-6 in the pre-chorismate pathway . Determination of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (E.C.2.5.1.19), shikimate:NADP+ oxidoreductase (E.C.1.1.1.25) and shikimate kinase (E.C.2.7.1.71) activities after non-denaturing gel electrophoresis, in combination with two-dimensional separations, revealed an increase in all three enzyme activities associated with overproduction of a 165 kDa protein in cells adapted to 6 mM glyphosate . Further evidence for an involvement of the multifunctional arom protein in aromatic amino acid synthesis in the plastid-free W10BSmL cells was obtained by Northern hybridization with ARO1-, aroA-, aroL- and aroE-specific Saccharomyces cerevisiae gene probes encoding the entire arom protein or parts of the EPSP synthase, shikimate:NADP+ oxidoreductase and shikimate kinase domains, respectively . Overproduction in adapted relative to control cells of a 5.3 kb transcript that cross-hybridized with all of the different probes could be demonstrated . The elevated content of the arom transcript correlated with a selective amplification of two out of five genomic sequences that hybridized with the S . cerevisiae ARO1 gene probe in Southern blots . One of the amplified genomic fragments is assumed to encode the previously identified monofunctional 59 kDa EPSP synthase, which is thought to be an organellar protein, that accumulates to a certain extent in its enzymatically active precursor form of 64.5 kDa in the plastid-free W10BSmL cells.

Biochemistry, 1993 Jun 1, 32(21), 5644 - 9
Side-chain mobility of the beta-lactamase A state probed by electron spin resonance spectroscopy; Calciano LJ et al.; beta-Lactamase from Bacillus licheniformis forms a stable compact intermediate state at low pH and moderate salt concentration (the A state), with properties consistent with a molten globule . A single cysteine residue was introduced into this class A beta-lactamase by site-directed mutagenesis at position 166 . A spin label was attached to the thiol of this cysteine residue via a disulfide bond as a probe of the side-chain mobility . The mutant protein and the spin-labeled derivative exhibited similar conformational properties to the wild-type enzyme at acidic pH . The A state induced by chloride or trichloroacetate (TCA) anions was characterized by circular dichroism and esr . The A state at pH 0.5 (0.32 M HCl), or at pH 2 in the presence of 8 mM TCA or 0.4 M Cl-, had comparable amounts of secondary structure to the native state but lacked significant tertiary structure, as judged by the lack of near-UV circular dichroism . Analysis of the esr spectral line widths showed that the mobility of the spin label in the A state was similar to that in the native state and much less mobile than in the unfolded state, indicating significant constraints on the side-chain mobility in this region of the molecule in the A state . The implications of this finding to the structure of the A state are discussed.

J Chemother, 1993 Jun, 5(3), 207 - 11
Current views on intravesical treatment and chemoprophylaxis of superficial bladder cancer . The present role of epirubicin and doxorubicin; Pavone-Macaluso M et al.; Since 1972, a large number of studies have shown that intravesical treatment with doxorubicin (adriamycin) is effective against carcinoma in situ and multiple papillary tumors . Furthermore, it significantly reduces the recurrence rate after transurethral resection . Its efficacy has been compared with that of Bacillus Calmette-Guerin (BCG), which is the only treatment accepted by the US Food and Drug Administration for therapy of carcinoma in situ (Tis) . In more recent years, a few studies have been performed using intravesical epirubicin in the hope that different properties of the molecule might enhance the activity of the anthracyclines, but produce fewer and milder side-effects . After weekly instillations of epirubicin (50 mg in 50 ml of sterile water) a complete response is achieved in 47% of patients with a histologically proven papillary marker lesion . The prophylactic efficacy of even a single instillation of epirubicin within 6 hours after transurethral resection (TUR) was proved in a randomized study (30863) of the EORTC (European Organization for Research on Therapy of Cancer) Urological Group . A randomized Italian trial (Blinst 4) of chemoprophylaxis after TUR investigated the efficacy of different intravesical administration schedules of epirubicin (50 mg in 50 ml of sterile water) . All treatment regimens were more effective than no treatment . The sequential intravesical combination of epirubicin and interferon-alpha-2b has shown, in our personal experience, encouraging clinical results and our laboratory data suggest the synergic activation of the local immune response.

J Biochem (Tokyo), 1993 Jun, 113(6), 646 - 9
Polypeptide folding of Bacillus cereus ATCC7064 oligo-1,6-glucosidase revealed by 3.0 A resolution X-ray analysis; Kizaki H et al.; The crystal structure of an oligo-1,6-glucosidase from Bacillus cereus ATCC7064 was determined by the X-ray diffraction method at 3.0 A resolution . The structure was solved by the multiple isomorphous replacement method and refined to a crystallographic R-factor of 0.208, using the molecular dynamics refinement program, X-PLOR . The electron density map revealed the folding of a polypeptide chain consisting of 558 amino acid residues . The molecule can be subdivided into three domains (N-terminal domain, subdomain, and C-terminal domain) . The N-terminal domain has an (alpha/beta)8-barrel structure called the TIM-barrel structure . The C-terminal domain is characterized by a beta-barrel structure composed of eight antiparallel beta-strands, while the subdomain has a loop-rich structure with a small alpha-helix and a beta-sheet . The enzyme has a large cleft between the N-terminal domain and the subdomain . The cleft leads from the molecular surface to the molecular center . The bottom of the cleft is at the C-terminal end of the parallel beta-strands of the (alpha/beta)8-barrel . These structural features closely resemble those of alpha-amylases from Aspergillus oryzae and pig pancreas.

J Clin Pharmacol, 1993 Jun, 33(6), 562 - 7
Determination of gentamicin pharmacokinetics by bioelectrical impedance in critically ill adults; Zarowitz BJ et al.; This investigation compares the accuracy of calculating gentamicin pharmacokinetic parameters by a noninvasive body composition technique (bioelectrical impedance analysis; BIA) with an empiric method, against the two-point method as the criterion standard . A prospective concurrent open label design was used . The 32 medical and surgical intensive care unit beds at Henry Ford Hospital, a not-for-profit, university-affiliated teaching hospital, served as the setting . Twenty critical ill adults, Therapeutic Index Scoring System (TISS) = 4, who required gentamicin as part of their normal course of therapy for gram-negative bacillary infections, were evaluated . Gentamicin Vd and k were calculated by three methods . After measurement of body composition parameters by BIA, previously derived gentamicin dosing equations were used to predict gentamicin volume of distribution (Vd) and elimination rate constant (k) (BIA method) . Empiric estimates of these parameters (Vd = 0.3L/kg and k derived from creatinine clearance) were compared with the BIA parameters against a criterion standard Vd and k determined from a two-point sampling of gentamicin serum concentrations . Measurements of BIA parameters and gentamicin serum concentrations were made in duplicate with coefficients of variation, < or = 2% and < or = 3%, respectively . The BIA and empiric methods produced resultant pharmacokinetic parameters (Vd and k) not different than those measured by the two-point method . There were no statistically significant differences in mean error (bias), or mean squared error (precision) for both Vd and k assessed by the empiric or BIA methods.(ABSTRACT TRUNCATED AT 250 WORDS)

Biokhimiia, 1993 Jun, 58(6), 913 - 20
{Renaturation of bacterial metalloproteinases}; Vaganova TI et al.; After denaturation by phenol or acid ethanol bacterial metalloproteinases secreted by Bacillus thermoproteolyticus and Bacillus megaterium cells can be renatured by dissolution in 99.7% formic acid with subsequent dilution of the solution and its neutralization with an appropriate amount of an alkali . Renaturation is optimal at pH 9.0 in the presence of 30% glycerol as stabilizer, Ca2+ and Zn2+ ions needed for the formation of a native structure and reconstitution of the enzyme catalytic center . The active enzyme yield is 60-80% . Reactivated metalloproteinases retain their enzymatic properties, amino acid composition and molecular mass . Under these conditions autolysis of metalloproteinases does not significantly influence their renaturation.

Biokhimiia, 1993 Jun, 58(6), 896 - 907
{Isolation and characteristics of Bacillus megaterium metalloproteinase}; Morozova IP et al.; Stepwise application of affinity chromatography on bacitracin-silochrome, gel filtration on Acrylex P-10, rechromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-15, a homogeneous metalloproteinase (M(r) = 35,000 Da) has been isolated from the cultural filtrate of B . megaterium strain 599 . The amino acid composition and N-terminal sequence (20 amino acids) of the enzyme have been determined . The proteinase is not inhibited by diisopropyl-fluorophosphate, is inhibited by o-phenanthroline, EDTA, and Zn2+, and is activated by Co2+ . The enzyme has a peak activity at 60-65 degrees C . The maximum of the enzymatic activity after hydrolysis of synthetic substrates is at pH 6.5-7.0 . The enzyme is stable at pH 7.0-9.0 and retains its stability at 45-60 C for several hours . In acid media the enzyme undergoes irreversible inactivation . The dependence of kcat/Km on pH points to the involvement of an ionogenic group with pKa 7.5 in the catalytic act, most probably of the imidazole group of histidine . The metalloproteinase hydrolyzes synthetic peptide substrates at the bonds formed by the amino groups of hydrophobic amino acids-Phe, Leu, Ile and Val.

Eur J Clin Microbiol Infect Dis, 1993 Jun, 12(6), 456 - 8
Infection caused by the nonfermentative gram-negative bacillus CDC group IV c-2: case report and literature review; Ramos JM et al.; A 10-year-old girl with acute lymphocytic leukemia developed nosocomial septicemia caused by the gram-negative bacterium CDC group IV c-2 . Recovery of the patient followed appropriate treatment with ceftriaxone, to which the organism was susceptible in vitro . Four other reported cases of infection caused by this organism are reviewed.

Lipids, 1993 Jun, 28(6), 479 - 81
Protein kinase C-dependent stimulation of phospholipase D in phospholipase C-treated fibroblasts; Kiss Z et al.; Treatment of {14C}choline- or {14C}ethanolamine-labeled NIH 3T3 fibroblasts with Bacillus cereus phosphatidyl-choline-specific phospholipase C (PLC) enhanced phospholipase D (PLD)-mediated hydrolysis of the respective 14C-labeled phospholipids . PLD activity was stimulated by 1.5 U/mL of PLC and by 100 nM of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) to similar extents . Treatment of {14C}palmitic acid-labeled fibroblasts with PLC in the presence of ethanol also enhanced PLD-mediated formation of phosphatidylethanol; the effects of PLC and PMA were nonadditive . PLC had no effect on PLD activity in fibroblasts in which PKC was down-regulated by prolonged (24 h) treatment with 300 nM PMA . These data indicate that treatment of fibroblasts with exogenous PLC results in PKC-dependent activation of PLD.

J Helminthol, 1993 Jun, 67(2), 115 - 22
The prevalence and intensity of infection with helminth parasites in Mus spretus from the Setubal Peninsula of Portugal; Behnke JM et al.; The results of a 5 year study of helminth parasites of Mus spretus, are reported . Six nematode and 5 cestode species were identified but no helminth showed 100% prevalence in M . spretus, the most commonly encountered nematode and cestode species being Syphacia obvelata (46.6%) and Taenia taeniaeformis (22.4%) . Among the more unusual helminth species identified was Eucoleus bacillatus, a capillariid nematode inhabiting the stomach musculature . This species was identified in 3 of the 5 years of the study . The results are discussed in the broader context of previous studies and the epidemiology of rodent helminth infections in general.

Med J Malaysia, 1993 Jun, 48(2), 229 - 31
Actinomycosis of the accessory breast treated with cotrimoxazole; Mohammed KN; Actinomycosis is a chronic suppurative granulomatous disease caused by the filamentous bacteria, Actinomyces israelii, which was once thought to be a fungus . It is a Gram-positive, aerobic or microaerophillic, non acid-fast hyphal organism which fragments into coccoid or bacillary forms and, unlike the fungus, does not form conidia . Accessory breast tissue usually occurs along the milk lines, frequently in the axilla and rarely in the thighs . Actinomycosis of the breast is very uncommon and we report the case of a multiparous woman who had a painful lump in the axilla which, on histopathologic examination, showed actinomycosis within the accessory breast tissue.

J Am Mosq Control Assoc, 1993 Jun, 9(2), 221 - 4
Investigations on possible resistance in Aedes vexans field populations after a 10-year application of Bacillus thuringiensis israelensis; Becker N et al.; In the Upper Rhine Valley (Germany), Bacillus thuringiensis var . israelensis has been widely used against floodwater mosquitoes over an area of approximately 500 km2 for more than 10 years . The susceptibility of larvae of Aedes vexans field populations in 3 untreated (Lake Constance) and 3 treated areas (Upper Rhine Valley) was assessed by means of bioassays with B.t.i . (Bactimos WP, 6,000 AAU/mg), following WHO guidelines . Log-probit analyses and statistical evaluations of the data showed that the LC50 values as well as slopes of bioassays of the larvae deriving from the different areas showed no significant differences . Two populations in the treated area were even more susceptible than populations from the untreated areas . These results have been confirmed by resistance ratios, which were less than one in all tests carried out.

J Am Mosq Control Assoc, 1993 Jun, 9(2), 150 - 4
Compatibility of cyclopoid copepods with mosquito insecticides; Marten GG et al.; Larvivorous copepods (Macrocyclops, Mesocyclops and Acanthocyclops) were tested for their sensitivities to commonly used mosquito larvicides and adulticides . The cyclopoids were not harmed by Bacillus thuringiensis (H-14) (B.t.i.) or larviciding oil . Control of mosquito larvae in field trials was accelerated by applying B.t.i . at the same time cyclopoids were introduced to a breeding site . Among adulticides tested, the cyclopoids were least sensitive to permethrin . Field trials demonstrated that permethrin does not harm cyclopoids when applied at label specifications.

Clin Pediatr (Phila), 1993 Jun, 32(6), 372 - 3
Cerebrospinal fluid pleocytosis in children with pneumonia but lacking evidence of meningitis; Nussinovitch M et al.; Headache, nuchal rigidity, positive Kernig's sign, and even convulsions may be observed during severe bacterial infections such as pneumonia, pyelonephritis, typhoid fever, and bacillary dysentery . In such cases, meningitis can be excluded only by documentation of normal cerebrospinal fluid (CSF) . The authors describe four children with lobar pneumonia in whom the clinical signs of meningeal irritation were associated with a mild increase in the white blood cell count in the CSF (pleocytosis) although there was no other evidence of meningeal infection.

Lepr Rev, 1993 Jun, 64(2), 104 - 9
In leprosy the presence of mycobacteria in the nerve is an essential factor in the cycle and spectrum of Mycobacterium leprae infection; Negesse Y et al.; A total of 220 untreated leprosy patients who underwent parallel skin and nerve biopsies are included in this study, which is intended to evaluate the extent of previously reported differences in bacillary load between skin and nerve lesions in leprosy and to describe the response of peripheral blood lymphocytes to Mycobacterium leprae antigens in such patients . In 161 patients out of the 220, the skin and nerve biopsies were diagnostic for leprosy . When patients were grouped according their skin and nerve lesions, the 3 groups observed were (1) paucibacillary skin and nerve lesions; (2) multibacillary skin and nerve lesions, and (3) paucibacillary skin and multibacillary nerve lesions . There was no observation of a group of patients with multibacillary skin and paucibacillary nerve lesions . In all patients with multibacillary nerve lesions, regardless of the type of skin lesions, a low response of peripheral blood lymphocytes to M . leprae was consistently noted . These results suggest that the bacillary load in the nerve is certainly one of the factors determining the immunological spectrum observed in leprosy.

Kekkaku, 1993 Jun, 68(6), 435 - 44
{HIV-infection, AIDS and BCG vaccination}; Toida I; Bacille Calmette-Guerin (BCG) has been widely used as a safe and effective vaccine for the protection of tuberculosis, but recent epidemic of human immunodeficiency virus (HIV) infection evoked serious concerns about the safety of BCG when vaccinated to HIV-infected persons: that is, because BCG is a live, though avirulent, bacterial vaccine, it might grow in immuncompromised host and might cause dissemination and/or exacerbated local adverse reactions . In fact, during the decade since the first report on AIDS in 1981, several reports were published on the adverse reactions, systemic or local, induced by BCG in HIV-infected persons . In this paper, the present author attempted to review such reports as comprehensively as possible . From critical examinations of the literatures, it was concluded that: 1) None of the reports dealing with dissemination of BCG provided satisfactorily enough evidence to identify the isolated mycobacteria as Mycobacterium bovis BCG . In some cases, infection with wild strain of M . bovis, instead of BCG, should be considered as more plausible pathogen . Especially, two reports, which suggested the late reactivation and dissemination of BCG vaccinated 30 years ago, could not be accepted without more detailed description of the procedures and results of the identification tests . In some cases, application of BCG were considered to be inappropriate . According to the present author's judgement, when BCG was applied appropriately as an anti-tuberculosis vaccine, generalized infections were most plausively induced by BCG only in 4 cases during this decade . 2) As for the local adverse reactions, many reported cases of outbreak of local adverse reactions, such as local ulceration and supprative lymphadenitis, were not related to HIV-infection at all, but were due to the usage of an inferior vaccine produced by a specified manufacturer (Pasteur Institute, Paris) . Conclusion was that BCG could be safely vaccinated to children born from HIV-seropositive mothers, even if children themselves were also infected with HIV, so long as BCG vaccine of good quality was used . 3) Positive conversion rate of post-vaccination tuberculin skin-test seemed to be lower in HIV-infected children than in children born from HIV-seronegative mothers . But, about 30% of the HIV-infected children converted to tuberculin-positive after BCG vaccination suggesting the effectiveness of the vaccination for the considerable fraction of the babies at the highest risk of tuberculosis infection . Positive conversion rate was much higher in HIV-noninfected children born from HIV-seropositive mothers.(ABSTRACT TRUNCATED AT 250 WORDS)

Rev Clin Esp, 1993 Jun, 193(1), 12 - 6
{Tuberculosis and HIV infection . Evaluation of 132 cases}; Carcaba V et al.; Tuberculosis is currently one the more frequent opportunistic infections in patients infected by Human Immunodeficiency Virus (HIV) in our setting . Its extrapulmonary localization is considered as diagnostic of the Acquired Immunodeficiency Syndrome (AIDS) . We have evaluated the epidemiological, clinical, microbiological, histological and immunological characteristics of 120 patients in the Asturias region who had a tuberculosis diagnosed in any localization, during the period between 1984 and 1991, belonging to a series of 570 patients infected by HIV . Pulmonary types were comparatively analyzed to the extrapulmonary and disseminated ones . Tuberculosis was pulmonary only in 44 occasions (PT), in 36 it was extrapulmonary (EPT) and in 52 disseminated (DT) . The more frequent risk factor for the HIV infection was the parenteral consumption of drugs (78.8%) . The final diagnosis was microbiologic in 81% of the cases, while bacilloscopia was positive in 62% of the cases . The histologic study showed the presence of granulomas in 86% of the tissues studied and necrosis in 81% . EPT and DT were related with a worse immune situation, bigger mortality rates attributed to tuberculosis and worse survival (p 0.069) . Tuberculosis in patients infected by HIV appears mainly in CDVP, being its symptoms the normal ones; but extrapulmonary forms are clearly predominant and within this group those with a ganglionar localization . Normal diagnostic procedures yield a good result . EPT and DT are significantly related to a more severe immunodeficiency in comparison with PT . Survival and prognosis are better in the PT group.

Heredity, 1993 Jun, 70 ( Pt 6), 597 - 603
X chromosome variations of Bacillus thuringiensis supernatant resistance and ribosomal DNA content in Drosophila melanogaster wild-type Oregon R lines; Paumard-Rigal S et al.; We have used the supernatant of Bacillus thuringiensis cultures to follow variations of the ribosomal DNA content in a wild-type Oregon R line of Drosophila melanogaster . These variations are revealed by differences in the degree of resistance to the lethal effect of the supernatant added in the culture medium . Different X chromosomes, all originated from the same X chromosome, confer different degrees of resistance . Increases and reductions in the number of the X ribosomal DNA transcriptional units have been found and are correlated with variations in the degree of supernatant resistance . However, no significant variations in X rDNA content were observed between the initial line and this line tested 20 generations later . These results show that the X rDNA is subjected to frequent modifications that must be essentially complementary and thus originate from reciprocal molecular events . Variations of resistance have been observed in the absence of recombination between homologous chromosomes; this is in agreement with the hypothesis that unequal recombinations between sister chromatids are largely involved in the variations of the X ribosomal DNA in Oregon R wild-type strains.

Hautarzt, 1993 Jun, 44(6), 361 - 4
{Bacillary angiomatosis . A pseudoneoplastic infection in AIDS patients}; Puppin D Jr et al.; Bacillary angiomatosis is a newly recognized pseudoneoplastic vascular disease seen in the setting of human immunodeficiency virus (HIV) infection . The disease is characterized by a cutaneous infection with reddish papules or nodules that are similar to pyogenic granulomas or Kaposi's sarcoma in clinical appearance . It is caused by the mildly gram-negative bacillus Rochalimaea henselae, which can be identified in tissue sections by means of Warthin-Starry stain . The historical, clinical, histopathological, and microbiological aspects and the differential diagnosis and therapy of bacillary angiomatosis are reviewed . It is important to be aware of these characteristics, because these lesions can easily be treated with antibiotic therapy.

J Pharm Sci, 1993 Jun, 82(6), 555 - 62
Pharmaceutical characterization of Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine used for the treatment of superficial bladder cancer; Groves MJ; Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine, developed in the 1920s as a treatment and prophylactic for tuberculosis, has proved to be a nonspecific stimulant of the immune system and is now the major form of clinical immunotherapy approved for the treatment of superficial bladder cancer in the United States . However, methods for the production and physical characterization of the vaccine have not been significantly developed since Calmette and Guerin first devised their process for attenuating the organism in 1908 . When reconstituted with sterile water immediately before use, the vaccine consists of a suspension of cellular fragments and aggregates and a mixture of dead and living cells . The dose is determined by the number of colony-forming units that develop when the vaccine is allowed to grow in a nutrient medium . This measurement of dose and viability is misleading because each cellular aggregate may consist of several hundred individual cells, but only one need be living to give rise to a single visible colony . Viability should therefore be measured on the basis of residual ATP levels . In this report, the mode of action of BCG vaccine against bladder cancer is reviewed, and attention is drawn to some factors that may need to be controlled during manufacturing and subsequent quality assurance procedures . The morphology of the various parts of the complex pleomorphic life cycle of this Mycobacterium species has been investigated, and the vaccine has been physically evaluated to provide a characterization by contemporary methodologies, including measurement of ATP content and particle size distribution of the dispersed mycobacterial aggregates.

Appl Environ Microbiol, 1993 Jun, 59(6), 1725 - 30
Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6; Khasin A et al.; Bacillus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp at pH 9 and 65 degrees C . The enzyme was purified and concentrated in a single adsorption step onto a cation exchanger and is made of a single polypeptide with an apparent M(r) of 43,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) . Xylanase T-6 is an endoxylanase that completely degrades xylan to xylose and xylobiose . The pIs of the purified protein were 9 and 7 under native and denaturing conditions, respectively . The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10 . At 65 degrees C and pH 7, the enzyme was stable for more than 10 h; at 65 degrees C and pH 9, the half-life of the enzyme was approximately 6 h . Kinetic experiments at 55 degrees C gave Vmax and Km values of 288 U/mg and 1.63 mg/ml, respectively . The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by Zn2+, Cd2+, and Hg2+ . Xylan completely protected the protein from inactivation by N-bromosuccinimide . The N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region of xylanase A from the alkalophilic Bacillus sp . strain C-125.

FEMS Microbiol Lett, 1993 Jun 1, 110(1), 97 - 100
Sphingomyelinase is part of the 'enterotoxin complex' produced by Bacillus cereus; Granum PE et al.; The three components of the 'enterotoxin complex' have been purified and the sequence of the first 14-15 amino acids of the proteins determined . Limited homology was found in the N-terminal sequence of the three proteins . The molecular mass of the proteins was determined to be 48, 40 and 34 kDa, respectively . Only the 40-kDa protein was toxic to Vero cells, whilst the 34-kDa protein was found to be hemolytic . The sequence of the first 14 N-terminal amino acids of this protein was identical to the sequence of the sphingomyelinase residues 28-41 (the N-terminal after loss of the signal sequence), except for a change from Gln to Glu in position 33 of the sphingomyelinase sequence.

FEMS Microbiol Lett, 1993 Jun 1, 110(1), 127 - 31
Characterization of the membrane sensor PenJ for beta-lactam antibiotics from Bacillus licheniformis by amino acid substitution; Takagi M et al.; The PenJ protein of the penicillinase gene (penP) expression system from Bacillus licheniformis is an antirepressor and membrane receptor for beta-lactam antibiotics . A putative beta-lactam antibiotic binding site including Ser402 and Lys405, which are homologous to the conserved sequence for the beta-lactam binding site (Ser-X-X-Lys) is present . An amino acid substitution was introduced at Ser402 to Ala, removing the hydroxyl group of the serine . The mutant PenJ, S402A, was still functional . However, two other mutants, S402T (Ser402-->Thr) and K405A (Lys405-->Ala), were not functional . Thus, the hydroxyl group of Ser402 does not appear to be important for penicillin binding . Amino acid substitutions (K539R, D591N and K539R.G541V) were introduced in PenJ in the region of the putative phosphoryl binding domain . None of these mutant PenJ proteins was a functional antirepressor . These results suggested that the putative phosphoryl binding domain might be an important region for signal transduction.

Southeast Asian J Trop Med Public Health, 1993 Jun, 24(2), 363 - 8
Field evaluation of a microgel droplet formulation of Bacillus sphaericus 1593M (Biocide-S) against Anopheles culicifacies and Anopheles subpictus in south India; Sundararaj R et al.; A microgel droplet formulation of Bacillus sphaericus 1593M (Biocide-S) was field tested at two different doses viz, 0.22 and 0.43 g/m2 in casuarina pits against a malarial vector Anopheles (Cellia) culicifacies Giles, and An . (Cel.) subpictus Grassi in coconut garden pits in the coastal areas of South India . Both doses significantly reduced the abundance of late instars and pupae of An . culicifacies for 3 and 5 weeks respectively . A significant reduction was recorded in the abundance of late instars and pupae of An . subpictus for 2 and 3 weeks respectively.

Res Microbiol, 1993 Jun, 144(5), 411 - 6
Inclusion bodies and crystals of Bacillus sphaericus mosquitocidal proteins expressed in various bacterial hosts; Charles JF et al.; Mosquitocidal proteins of 42 and 51 kDa from Bacillus sphaericus were produced in acrystallogenic B . sphaericus and B . subtilis strains . In both species, transformants containing each protein expressed individually were non-toxic for mosquito larvae and produced small inclusions which did not have a crystalline ultrastructure . When both components of the binary toxin were expressed together, toxicity was restored: oval and round amorphous inclusions were produced in B . subtilis, and typical native-type crystals were synthesized in B . sphaericus . In B . subtilis, native-type crystals were produced only when the two components of the binary toxin were synthesized as a 93-kDa fusion protein.

Wei Sheng Wu Xue Bao, 1993 Jun, 33(3), 210 - 3
{Morphogenesis of Bacillus sphaericus Ts-1 and formation of toxic proteins to mosquito larva}; Zhang B et al.; The close relationships between morphogenesis of Bacillus sphaericus Ts-1 and formation of toxic proteins were studied systemically and comparatively by Enzyme-Linked Immunosorbent Assay, FITC-IgG Location and SDS-PAGE etc . This study showed that the toxic proteins of Ts-1 were produced in 8h culture at 30 degrees C . The toxicity increased steadily along with the sporification and then declined as the free spores increased greatly without collection immediately . It is suggested that substances homologous with crystal toxic proteins were situated on the cell walls.

Bull Acad Natl Med, 1993 Jun, 177(6), 893 - 910; discussion 910-6
{The campaign against tuberculosis in 1993 . Imperatives and current perceptions}; Chretien J; The continuing presence of tuberculosis in the world and its reappearance in industrialized countries again emphasizes the need to maintain and reinforce tuberculosis control . Despite the existence of effective drugs, there are still, deplorably, nearly 20 million cases of active tuberculosis throughout the world, with at least 3 million deaths annually . Almost 1.7 billion people are infected with the Koch bacillus . These figures are approximate and probably underestimate the real situation, as precise epidemiological data are not available . The rules of tuberculosis control are reviewed in this article, with distinctions drawn between low prevalence countries, where the objective is to eradicate the disease, and high prevalence countries, where it must be contained and reduced as quickly as possible the interruption of the claim of transmission of tuberculosis and the necessary collective measures are described in turn . The role of new techniques, particularly those based on molecular biology, is discussed . The importance of case finding and treatment in groups at risk in France is underlined, as are the role of BCG and the importance of protective measures, particularly in hospitals and in vulnerable subjects . The association between tuberculosis and HIV is well-known, but the HIV epidemic does not seem to be the only reason for the current resurgence of the tuberculosis problem in France in particular.

Scanning Microsc, 1993 Jun, 7(2), 577 - 83; discussion 583-4
BCG cell imaging using scanning probe microscopy; Garcia AA et al.; Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) were used to obtain images of the surface of whole, intact BCG (bacille Calmette Guerin, a mycobacterium) cells in air and under solution by immobilizing the cells onto glass slides (AFM only) or highly oriented pyrolytic graphite . The technique used for AFM imaging involved depositing a submonolayer of cells under a centrifugal force followed by fixation/dehydration using polar organic solvents . AFM images agree well with images from light and electron microscopy and showed large numbers of BCG cells in their distinctive cord arrangement . The AFM also proved useful for identifying extracellular microgranules which cannot be seen with light microscopy . For STM imaging, the hydrophobicity of BCG enabled strong adhesion from aqueous solution onto graphite . STM images of BCG could only be obtained while scanning in aqueous solution, and the cells showed a large variation in contrast when different samples were imaged . The STM provided greater detail of surface features than the AFM and was able to produce images of periodic layers corroborating observations made by transmission electron microscopy.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 5287 - 91
Molecular and active-site structure of a Bacillus 1,3-1,4-beta-glucanase; Keitel T et al.; The three-dimensional structure of the hybrid Bacillus 1,3-1,4-beta-glucanase (beta-glucanase; 1,3-1,4-beta-D-glucan 4-glucanohydrolase, lichenase, EC 3.2.1.73) designated H(A16-M) was determined by x-ray crystallography at a resolution of 2.0 A and refined to an R value of 16.4% using stereochemical restraints . The protein molecule consists mainly of two seven-stranded antiparallel beta-pleated sheets arranged atop each other to form a compact, sandwich-like structure . A channel crossing one side of the protein molecule accommodates an inhibitor, 3,4-epoxybutyl beta-D-cellobioside, which binds covalently to the side chain of Glu-105, as seen in a crystal structure analysis at 2.8-A resolution of the protein-inhibitor complex (R = 16.8%) . That Glu-105 may be indispensible for enzyme catalysis by H(A16-M) is suggested by site-directed mutagenesis of this residue, which inevitably leads to an inactive enzyme.

Afr J Med Med Sci, 1993 Jun, 22(2), 13 - 7
Drug resistance and plasmids of Bacillus isolated from locally fermented foods; Ebigwe SI et al.; In an epidemiological survey of antibiotic resistance in this environment, Bacillus bacteria were isolated from commonly consumed fermented foods . The fifty strains isolated were tested for susceptibility to antibiotics and screened for the presence of plasmids . Majority (72%) were resistant to Cloxacillin and Penicillin (70%) while 40% harbour plasmids ranging from 3.0kb-36.3kb in molecular weight . A 4.6kb plasmid was found to be common to all plasmid-bearing strains . The implication for the epidemiology of antibiotic resistance is discussed.

Biosci Biotechnol Biochem, 1993 Jun, 57(6), 922 - 5
Inhibition of angiotensin I-converting enzyme by Bacillus licheniformis alkaline protease hydrolyzates derived from sardine muscle; Matsui T et al.; Hydrolyzates which inhibit the angiotensin I-converting enzyme (ACE) were prepared from sardine muscle by Bacillus licheniformis alkaline protease . Considering the practical application of preparations as a functional food material, the best proteolytic conditions with respect to taste, solubility and ACE inhibitory activity were a 0.3 wt% addition of the enzyme and 17-h proteolysis at 50 degrees C and pH 9.0 . The preparations under these conditions had potent activity (IC50 = 0.26 mg protein/ml) . Fractionation of the preparations on an ODS column with ethanol resulted in the production of more potent inhibitors . The most potent activity was obtained when eluting with 10% ethanol (IC50 = 0.015 mg protein/ml) . This fraction was apparently rich in acidic amino acids, poor in hydrophobic ones, and effective for use as a physiologically functional food material by virtue of little bitterness, a fish odor and powerful ACE inhibitory activity.

Biol Pharm Bull, 1993 Jun, 16(6), 616 - 8
Analysis of a common antigenic epitope in Bacillus cereus flagellar fraction; Murakami T et al.; The strain-specific antigenicity between Bacillus cereus decreased but the common antigenicity did not change, when the flagellar antigen was treated with pH 2 . Flagellar antigen of B . cereus H.1 was separated into two fractions by gel chromatography, one with a specific antigenicity (P1 fraction), and the other with a common antigenicity (P2 fraction) . It was revealed that a common antigenic epitope of the P2 fraction was detected on a 61 kDa protein by further purification using a Mono Q column . This common antigenicity of flagella was increased by treatment with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, suggesting that a common antigenic epitope exists inside the flagellar component.

Zentralbl Bakteriol, 1993 Jun, 278(4), 469 - 78
Suprageneric classification of thermoactinomyces vulgaris by nucleotide sequencing of 5S ribosomal RNA; Park YH et al.; The 5S rRNA nucleotide sequence of Thermoactinomyces vulgaris was determined and compared with published sequences of representative Gram-positive bacteria . The primary and secondary structure of the sequence is of the type characteristic of Gram-positive bacteria that have DNA with a low proportion of guanine plus cytosine . It was evident from the phylogenetic trees that T . vulgaris has little in common with actinomycetes but is related to the genus Bacillus, showing a moderately high relationship with B . stearothermophilus . The taxonomic implications of these relationships are discussed and an emended description of the family Bacillaceae is given.

Pediatr Infect Dis J, 1993 Jun, 12(6), 499 - 504
Seroprevalence of human immunodeficiency virus type 1 infection in Zambian children with tuberculosis; Chintu C et al.; Descriptions in the medical literature of human immunodeficiency virus type 1 (HIV-1) in children with tuberculosis (TB) are scanty . This study determined the seroprevalence of HIV-1 in 237 hospitalized children between the ages of 1 month and 14 years with a clinical diagnosis of TB (125 males and 112 females) and in 242 control children (149 males and 93 females) . The overall HIV-1 seroprevalence rate in patients with TB was 37% (88 of 237) compared with 10.7% (26 of 242) among the control group (P < 0.00001: odds ratio 5.37, 95% confidence interval = 3.21 < 5.37 < 9.47) . HIV-1 seropositivity in children with TB ranged from 53% (31 of 58) in the 12- to 18-month age group to 14% (9 of 61) in the 10- to 14-year-olds . The risk of TB attributable to HIV infection was 29% . The predominant clinical presentation in both seronegative (84.6%) and seropositive (89.7%) groups was that of pulmonary TB and there were no significant differences in clinical presentation between the two groups of patients . Only 54.8% of the patients attended follow-up clinics regularly whereas 32% were lost to follow-up within 3 months . Bacillus Calmette-Guerin vaccination coverage was 87.3% among TB patients and 90.5% in the controls . No significant differences in B . Calmette-Guerin vaccination rates between the seronegative and seropositive children were seen . Coinfection with HIV and TB in children is now one of the major public health problems in Zambian children.

J Exp Med, 1993 Jun 1, 177(6), 1691 - 8
Recognition and destruction of Bacillus Calmette-Guerin-infected human monocytes; Molloy A et al.; We have established a long-term culture system to study macrophages chronically infected with mycobacteria . Monocytes are infected with Bacillus Calmette-Guerin (BCG) and support exponential intracellular replication without profound perturbation of normal host cell function . We have used this system to investigate lymphokine-activated killer (LAK)-mediated cytolysis . We have found that interleukin 2 stimulation of peripheral blood lymphocytes generates a cytotoxic activity against human monocytes . A CD56- subpopulation of LAK cells specifically recognizes and lyses BCG-infected cells . Lysis of the host cell has no effect on parasite viability and results in the liberation of bacteria capable of infecting more cells.

Biochemistry, 1993 May 25, 32(20), 5321 - 6
Use of binding energy in catalysis: optimization of rate in a multistep reaction; Avis JM et al.; The role of binding energy in optimizing the overall rate of catalysis by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been investigated by measuring the rate constants for transfer of tyrosine from engineered mutants to tRNA . The residues chosen for mutation are those that were previously identified as binding tyrosyl adenylate and contributing to the rate constant for its formation . It was previously found that tighter binding of the tyrosyl adenylate was accompanied by an increase in the rate constant for its formation . A new linear free energy relationship is presented that links the two . We now find that the rate constant for transfer of Tyr from Tyr-AMP to tRNA decreases with increasing stability of the E.Tyr-AMP complex on mutation of Thr-51 . Position 51 is the one that is found to be the most variable of the binding-site residues among the enzymes from different species . The tightness of binding of the intermediate is thus a compromise, since stabilizing the intermediate speeds up the first step but slows down the second . The rate constants for activation and transfer by wild-type enzymes are very similar, which is optimal for the rate of the overall reaction . Those for the mutants diverge so that the rate of overall catalysis is lower.

Biochemistry, 1993 May 25, 32(20), 5312 - 20
Reaction of modified and unmodified tRNA(Tyr) substrates with tyrosyl-tRNA synthetase (Bacillus stearothermophilus); Avis JM et al.; Three species of tRNA(Tyr) have been examined as substrates for the transfer reaction of the tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus: Escherichia coli tRNA(Tyr), B . stearothermophilus tRNA(Tyr) expressed in E . coli, and B . stearothermophilus tRNA(Tyr) that has been transcribed in vitro . The binding of the first two substrates to TyrRS may be readily monitored by stopped-flow studies of tryptophan fluorescence to give the rate and equilibrium constants . The in vitro-transcribed tRNA(Tyr), which lacks the modified bases queuosine and 2-(methylthio)-N6-isopentenyladenosine in the anticodon loop, does not cause a significant change in tryptophan fluorescence upon binding . The three tRNA(Tyr) substrates exhibit very similar steady-state kinetics in the charging reaction . Pre-steady-state kinetics of the transfer reaction, monitored by stopped-flow measurements of the change in protein fluorescence on the addition of tRNA(Tyr) to the E.Tyr-AMP complex, show two exponential changes for the modified tRNA(Tyr) substrates . The first is that due to substrate binding . The second has an identical rate to the single change observed for the reaction with the in vitro-transcribed tRNA(Tyr) and to that monitored by quenched-flow measurements on the formation of Tyr-tRNA(Tyr) . Hence, the transfer reaction can be observed by stopped-flow . The dissociation constants (KtRNA) of tRNA from the enzyme and rates of tyrosine transfer (k4) show that all three tRNA molecules are kinetically equivalent substrates for TyrRS . The value of k4 is also similar to that found for authentic tRNA(Tyr) from B . stearothermophilus.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1993 May 25, 268(15), 11296 - 303
Cloning and characterization of a putative Ca2+/H+ antiporter gene from Escherichia coli upon functional complementation of Na+/H+ antiporter-deficient strains by the overexpressed gene; Ivey DM et al.; DNA libraries from alkaliphilic Bacillus firmus OF4 had been screened earlier (Ivey, D.M., Guffanti, A.A., Bossewitch, J . S., Padan, E., and Krulwich, T . A . (1991) J . Biol . Chem . 266, 23483-23489) for clones that would functionally complement a strain of Escherichia coli (NM81) with a deletion in one of its Na+/H+ antiporter genes . During those studies, an alkaliphile antiporter gene was hypothesized to have been incorporated into the chromosome of strain NM81, producing Na(+)-resistant NM8191 . After introduction of a deletion in the second known E . coli Na+/H+ antiporter gene, libraries were prepared from NM8191 and screened for complementation of Na+/H+ antiporter-deficient mutants of E . coli . Instead of retrieving an alkaliphile gene, an unexpected E . coli gene was identified on the basis of its ability to restore Na+ resistance and membrane Na+/H+ antiporter activity to such mutant strains . The active open reading frame in the clone maps at 27 min on the E . coli chromosome and is identical in sequence to a wild type counterpart . It would be predicted to encode an extremely hydrophobic protein with multiple membrane-spanning regions and a molecular weight of 39,200 . A region in one of the predicted hydrophilic loops in the gene product structure possesses striking sequence similarity to calsequestrin . The Ca2+/H+ antiporter activity of membranes from an E . coli transformant with a clone possessing only this open reading frame was indeed found to have enhanced pH-independent Ca2+/H+ antiporter activity . The Ca2+/H+ and Na+/H+ antiporter activities conferred by the clone were both inhibited by Mg2+ . The gene was designated chaA and is proposed to be the structural gene for a Ca2+/H+ antiporter whose overexpression leads to resistance to growth inhibition by both calcium and sodium.

Biochemistry, 1993 May 18, 32(19), 5145 - 50
Interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering; Schreiber G et al.; Barnase, an extracellular ribonuclease of Bacillus amyloliquefaciens, forms a very tight complex with its intracellular polypeptide inhibitor barstar . At pH 8, the values for the rate constants k1 (association) and k-1 (dissociation) are 6.0 x 10(8) s-1 M-1 and 8.0 x 10(-6) s-1, respectively . The value of Ki, the dissociation constant of barstar and barnase, calculated from the ratio k-1/k1 is 1.3 x 10(-14) M, which corresponds to a delta G of -18.9 kcal/mol at 25 degrees C . The dissociation constant increases with decreasing pH according to the ionization of an acid in free barnase of pKa 6.4, with very weak, if any, binding to the protonated form . This pH dependence for dissociation of the complex can be attributed almost entirely to residue His102 in barnase, as determined by a His102-->Ala mutation . Analysis of the pH dependence of the kinetic constants indicates that binding is, at least, a two-step process . The first, and rate-determining, step is association at close to the diffusion-controlled rate . There is then the precise docking of the complex . The value of Ki increases to 2.4 x 10(-11) M in the presence of 500 mM NaCl, and to 1.6 x 10(-11) M at pH 5 (100 mM NaCl) . The binding site of barstar on barnase was mapped by measuring the values of Ki for a broad range of site-specific mutants of barnase . Mutagenesis of residues Lys27, Arg59, Arg87, and His102 to Ala increases the values of Ki by a factor of 10(4).(ABSTRACT TRUNCATED AT 250 WORDS)

Harefuah, 1993 May 16, 124(10), 607 - 10, 668
{Actinobacillus actinomycetemcomitans endocarditis}; Avigdor A et al.; Bacterial endocarditis caused by Actinobacillus actinomycetemcomitans (AA) is an extremely rare disease . AA, a gram negative cocco-bacillus, normally resides in the oral cavity . It is involved mostly in local oral cavity infections, but severe systemic infections caused by it have been reported . We describe a 63-year-old man with endocarditis caused by AA which probably originated from a dental abscess . The course of the disease was complicated by acute myocardial ischemia, apparently caused by coronary artery embolism . To enable growth of this bacterium, blood cultures should be maintained for 2-3 weeks in a CO2-rich atmosphere.

J Biol Chem, 1993 May 15, 268(14), 10534 - 9
Differential translocation of protein kinase C isozymes by thrombin and platelet-derived growth factor . A possible function for phosphatidylcholine-derived diacylglycerol; Ha KS et al.; The translocation of protein kinase C (PKC) from the cytosolic to the particulate fraction in IIC9 fibroblasts has been studied to define the functions of 1,2-diacylglycerol (DAG) derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylcholine (PC) . alpha-Thrombin caused a biphasic change in DAG, with two peaks at 15-60 s and 5-15 min, derived from PIP2 and PC, respectively, while platelet-derived growth factor (PDGF) induced a monophasic DAG increase from PC at 5-15 min . alpha-Thrombin also induced a rapid, but transient, increase of inositol 1,4,5-trisphosphate and cytosolic Ca2+, whereas PDGF did not . Three PKC isozymes, alpha, epsilon, and zeta, were identified by Western blotting in IIC9 cells and were mainly localized in the cytosol . A fraction of cytosolic PKC alpha was rapidly translocated by alpha-thrombin at 15 s, but its membrane association was lost within 1 min . PKC epsilon was also rapidly translocated; however, its membrane association was sustained for almost 60 min . PKC zeta was not translocated by alpha-thrombin or phorbol 12-myristate 13-acetate . PDGF translocated PKC epsilon at 5 min but had little effect at 15 s and did not translocate PKC alpha or zeta . Incubation with Bacillus cereus PC- or phosphatidylinositol-specific phospholipase C, which increased DAG but not phosphatidic acid, stimulated translocation of PKC epsilon, but not PKC alpha or zeta . Addition of chelators to inhibit the rise in intracellular Ca2+ largely blocked PKC alpha translocation induced by alpha-thrombin but had no effect on PKC epsilon translocation . Addition of ionomycin allowed alpha-thrombin to induce PKC alpha translocation at 5 min . PKC alpha translocation was mimicked by 1,2-dioctanoylglycerol plus ionomycin, but not by either alone . On the other hand, PKC epsilon was translocated by the DAG alone . These results support the conclusion that PIP2 hydrolysis activates both PKC alpha and epsilon at 15 s, whereas PC hydrolysis activates only PKC epsilon at 5 min . The differential activation at 5 min can be attributed to the failure of PC hydrolysis to increase Ca2+ and not to a difference in the molecular species of DAG derived from the phospholipids.

Gene, 1993 May 15, 127(1), 79 - 85
Identification of a putative infC-rpmI-rplT operon flanked by long inverted repeats in Mycoplasma fermentans (incognitus strain); Hu WS et al.; A specific 1542-bp DNA fragment was amplified from Mycoplasma fermentans (incognitus strain) using a unique 23-nucleotide (nt) synthetic deoxyribonucleotide (oligo) (5'-TCCAAAAAGTCCGGAATTTGGGG) as the primer pair in the polymerase chain reaction (PCR) . The 23-nt sequence is part of the 29-bp terminal inverted repeat (IR) which forms the left potential stem-and-loop (s&l) structure of the previously identified M . fermentans insertion-sequence(IS)-like genetic element {Hu et al., Gene 93 (1990) 67-72} . The amplified DNA was cloned and sequenced . A pair of 27-bp IR containing the 23-nt synthetic oligo was identified at both termini . Between the IR, there are four potential open reading frames (ORFs) which are arranged adjacent to each other in the order, ORF-1, ORF-2, ORF-3 and ORF-4, with parts of ORF-1 and ORF-2 overlapping . The deduced amino acid (aa) sequences of ORF-2, ORF-3 and ORF-4 are 34 to 60% identical to the translation initiation factor IF3 (encoded by the infC gene), ribosomal proteins L35 (rpmI gene) and L20 (rplT gene) of Escherichia coli and Bacillus stearothermophilus, respectively . In bacteria, the infC-rpmI-rplT genes are organized to function as an operon . There are multiple sites with promoter-like sequences identified upstream from the putative infC gene in the mycoplasma closely resembling the gene arrangement in the bacterial operon . All three genes of ORF-2, ORF-3 and ORF-4 are preceded individually by a strong appropriately spaced (7 and 10 bp) putative Shine-Dalgarno sequence (5'-AAGGA).(ABSTRACT TRUNCATED AT 250 WORDS)

J Theor Biol, 1993 May 7, 162(1), 61 - 74
HIV-1 proteinase as structural model of intercellular transport proteins of plant viruses; Melcher U; Intracellular movement of viral infections of plants requires a virus-encoded protein . Alignment of amino acid sequences of central conserved regions of such proteins produced a sequence profile that resembled that of lentiviral proteinases . The known three-dimensional structure of the proteinase encoded by the human immunodeficiency virus-1 (HIV-1) may serve as a model for the three-dimensional structure of the central region of the plant viral proteins . Secondary structures predicted for the plant viral proteins from their amino acid sequences correlate well with those predicted from a proteinase model . In addition, the positions of temperature-sensitive and resistance-breaking mutations in the intercellular transport protein of tobacco mosaic virus are consistent with a structural similarity between the plant viral proteins and the lentiviral proteinases . In a suggested model, the dimeric proteinase-similar domain serves as tether for the attachment of N- and C-terminal domains . The C-terminal domain may be an RNA-binding domain . The similarity was used to assign intercellular transport function to a previously unidentified coding region of the genomes of bacilliform DNA viruses.

Biochim Biophys Acta, 1993 May 6, 1142(3), 321 - 6
The role of protonic and sodium potentials in the motility of E . coli and Bacillus FTU; Bogachev AV et al.; The motility of Escherichia coli and of alkalo- and halotolerant Bacillus FTU has been studied . It is found that Bac . FTU motility (i) requires Na+, (ii) is resistant to the protonophorous uncoupler pentachlorophenol (PCP) if cells grow at high pH, and is sensitive to the uncouplers at neutral pH, (iii) is sensitized to the uncouplers with the addition of monensin, (iv) sensitive to amiloride and (v) can be supported by an artificially imposed Na+ gradient in the presence of uncoupler, cyanide and arsenate . On the other hand, E . coli motility (a) does not require Na+, (b) is always uncoupler-sensitive, (c) is amiloride-resistant, and (d) can be supported by an artificially-imposed gradient of H+, not Na+ . It is concluded that the motilities of Bac . FTU and E . coli are due to the operation of the Na+ and the H+ motors, respectively . In Bac . FTU growing at alkaline pH, the Na+ motors are assumed to be energized by delta mu Na+ produced by the Na(+)-motive respiratory chain, and therefore delta mu H+ is not involved in the motility process . As to Bac . FTU growing in a neutral medium, delta mu Na+ is produced secondarily, via the Na+/H(+)-antiporter, i.e., at the expense of delta mu H+ formed by the H(+)-motive respiratory chain.

Appl Environ Microbiol, 1993 May, 59(5), 1683 - 7
Screening by polymerase chain reaction of Bacillus thuringiensis serotypes for the presence of cryV-like insecticidal protein genes and characterization of a cryV gene cloned from B . thuringiensis subsp . kurstaki; Gleave AP et al.; Polymerase chain reaction screening using cryV-specific oligonucleotides, designed to amplify the 5' half of cryV-type genes, revealed the presence of such genes in 7 of 21 Bacillus thuringiensis serotypes examined . Restriction analysis and hybridization studies indicated that these putative genes fall into at least three subclasses . The nucleotide sequence of the cryV-type gene cloned from B . thuringiensis subsp . kurstaki DSIR732 revealed an open reading frame coding for a protein of 719 amino acids, and lysates of Escherichia coli cells expressing the 81.2-kDa CryV732 protein were toxic to Epiphyas postvittana (Lepidoptera: Tortricidae).

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