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