Eur J Biochem, 1999 Sep, 264(3), 816 - 24 Electrostatics in the active site of an alpha-amylase; Nielsen JE et al.; The importance of electrostatics in catalysis has been emphasized in the literature for a large number of enzymes . We examined this hypothesis for the Bacillus licheniformis alpha-amylase by constructing site-directed mutants that were predicted to change the pKa values of the catalytic residues and thus change the pH-activity profile of the enzyme . To change the pKa of the catalytic residues in the active site, we constructed mutations that altered the hydrogen bonding network, mutations that changed the solvent accessibility, and mutations that altered the net charge of the molecule . The results show that changing the hydrogen bonding network near an active site residue or changing the solvent accessibility of an active site residue will very likely result in an enzyme with drastically reduced activity . The differences in the pH-activity profiles for these mutants were modest . pH-activity profiles of mutants which change the net charge on the molecule were significantly different from the wild-type pH-activity profile . The differences were, however, difficult to correlate with the electrostatic field changes calculated . In several cases we observed that pH-activity profiles shifted in the opposite direction compared to the shift predicted from electrostatic calculations . This strongly suggests that electrostatic effects cannot be solely responsible for the pH-activity profile of the B . licheniformis alpha-amylase. Eur J Biochem, 1999 Sep, 264(2), 554 - 61 Structure determination of an exopolysaccharide from an alkaliphilic bacterium closely related to Bacillus spp; Corsaro MM et al.; An exopolysaccharide obtained from an alkaliphilic bacterium closely related to Bacillus spp . was found to contain D-galactopyranuronic acid (GalpA), 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose (QuipNAc4NAc), 2-acetamido-2-deoxy D-mannopyranuronic acid (ManpNAcA) and one uncommon unit of D-galactopyranuronic acid with the carboxyl group amide-linked to glycine {GalpA(Gly)} . The polysaccharide was studied by one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectroscopy both on native polysaccharide and on monosaccharides and oligosaccharides obtained from methanolysis and from anhydrous HF solvolysis . The following linear structure of the repeating unit was established: -->3)-alpha-D-GalpA(Gly)-(1-->4)-beta-D-ManpNAcA-(1-->4)-alp ha-D-Galp A-(1-->3)-alpha-D-QuipNAc4NAc-(1--> . A preliminary phylogenetic assignment for the bacterium is also reported. J Immunol, 1999 Oct 1, 163(7), 3642 - 52 Oligodeoxynucleotides containing palindrome sequences with internal 5'-CpG-3' act directly on human NK and activated T cells to induce IFN-gamma production in vitro; Iho S et al.; Previous studies have shown that the action of bacterial or synthetic oligodeoxynucleotide (oligo-DNA) on mouse NK cells to produce IFN-gamma is mediated mostly by monocytes/macrophages activated by olig-DNA . However, its action on human IFN-gamma-producing cells has not been well investigated . In the present study, we examined the effect of oligo-DNAs on highly purified human NK and T cells . Bacillus Calmette-Guerin-derived or synthetic oligo-DNAs induced NK cells to produce IFN-gamma with an increased CD69 expression, and the autocrine IFN-gamma enhanced their cytotoxicity . The response of NK cells to oligo-DNAs was enhanced when the cells were activated with IL-2, IL-12, or anti-CD16 Ab . T cells did not produce IFN-gamma in response to oligo-DNAs but did respond independently of IL-2 when they were stimulated with anti-CD3 Ab . In the action of oligo-DNAs, the palindrome sequence containing unmethylated 5'-CpG-3' motif(s) appeared to play an important role in the IFN-gamma-producing ability of NK cells . The changes of base composition inside or outside the palindrome sequence altered its activity: The homooligo-G-flanked GACGATCGTC was the most potent IFN-gamma inducer for NK cells . The CG palindrome was also important for activated NK and T cells in their IFN-gamma production, although certain nonpalindromes acted on them . Among the sequences tested, cell activation- or cell lineage-specific sequences were likely; i.e., palindrome ACCGGT and nonpalindrome AACGAT were favored by activated NK cells but not by unactivated NK cells or activated T cells . These results indicate that oligo-DNAs containing CG palindrome act directly on human NK cells and activated T cells to induce IFN-gamma production. Folia Microbiol (Praha), 1999, 44(1), 90 - 2 Alkaline phosphatase production during sporulation of Bacillus cereus; Bursik M et al.; Cell-bound alkaline phosphatase of Bacillus cereus was produced during vegetative growth and sporulation in a complex medium . Addition of glucose repressed the sporulation process and the amount of enzyme synthesized increased . The time course of alkaline phosphatase production is very similar in both sporulating and non-sporulating cells . Irrespective of sporulation, alkaline phosphatase level shows a peak of activity in the exponential phase, and another in the stationary phase of growth . This preliminary data indicates differences between B . cereus, and B . subtilis in alkaline phosphatase characteristics. Anal Chem, 1999 Sep 1, 71(17), 3846 - 52 Array biosensor for simultaneous identification of bacterial, viral, and protein analytes; Rowe CA et al.; The array biosensor was fabricated to analyze multiple samples simultaneously for multiple analytes . The sensor utilized a standard sandwich immunoassay format: Antigen-specific "capture" antibodies were immobilized in a patterned array on the surface of a planar waveguide and bound analyte was subsequently detected using fluorescent tracer antibodies . This study describes the analysis of 126 blind samples for the presence of three distinct classes of analytes . To address potential complications arising from using a mixture of tracer antibodies in the multianalyte assay, three single-analyte assays were run in parallel with a multianalyte assay . Mixtures of analytes were also assayed to demonstrate the sensor's ability to detect more than a single species at a time . The array sensor was capable of detecting viral, bacterial, and protein analytes using a facile 14-min assay with sensitivity levels approaching those of standard ELISA methods . Limits of detection for Bacillus globigii, MS2 bacteriophage, and staphylococcal enterotoxin B (SEB) were 10(5) cfu/mL, 10(7) pfu/mL, and 10 ng/mL, respectively . The array biosensor also analyzed multiple samples simultaneously and detected mixtures of the different types of analytes in the multianalyte format. J Clin Microbiol, 1999 Oct, 37(10), 3159 - 66 Molecular analysis of riboflavin synthesis genes in Bartonella henselae and use of the ribC gene for differentiation of Bartonella species by PCR; Bereswill S et al.; The biosynthesis pathway for riboflavin (vitamin B(2)), the precursor of the essential cofactors flavin mononucleotide and flavin adenine dinucleotide, is present in bacteria and plants but is absent in vertebrates . Due to their conservation in bacterial species and their absence in humans, the riboflavin synthesis genes should be well suited either for detection of bacterial DNA in human specimens or for the differentiation of pathogenic bacteria by molecular techniques . A DNA fragment carrying the genes ribD, ribC, and ribE, which encode homologues of riboflavin deaminase (RibD) and subunits of riboflavin synthetase (RibC and RibE), respectively, was isolated from a plasmid-based DNA library of the human pathogen Bartonella henselae by complementation of a ribC mutation in Escherichia coli . Sequence analysis of the ribC gene region in strains of B . henselae, which were previously shown to be genetically different, revealed that the ribC gene is highly conserved at the species level . PCR amplification with primers derived from the ribC locus of B . henselae was used to isolate the corresponding DNA regions in B . bacilliformis, B . clarridgeiae, and B . quintana . Sequence analysis indicated that the riboflavin synthesis genes are conserved and show the same operon-like genetic organization in all four Bartonella species . Primer oligonucleotides designed on the basis of localized differences within the ribC DNA region were successfully used to develop species-specific PCR assays for the differentiation of B . henselae, B . clarridgeiae, B . quintana, and B . bacilliformis . The results obtained indicate that the riboflavin synthesis genes are excellent targets for PCR-directed differentiation of these emerging pathogens . The PCR assays developed should increase our diagnostic potential to differentiate Bartonella species, especially B . henselae and the newly recognized species B . clarridgeiae. J Clin Microbiol, 1999 Oct, 37(10), 3097 - 101 Semiquantitative species-specific detection of Bartonella henselae and Bartonella quintana by PCR-enzyme immunoassay; Sander A et al.; Bartonella henselae is the main causative agent of cat-scratch disease, and both B . henselae and Bartonella quintana cause angioproliferative disorders such as bacillary angiomatosis . To increase the sensitivity of Bartonella detection by PCR and to improve the species differentiation, we developed a semiquantitative, species-specific PCR-based enzyme immunoassay (EIA) . The 16S rRNA gene was selected as the target sequence . Internal nucleotide sequences derived from the amplified 16S rRNA region were used to develop species-specific oligonucleotide probes for B . henselae and B . quintana . Biotin-labeled PCR products were immobilized on streptavidin-coated microtiter plates, hybridized to a digoxigenin-labeled probe, and detected with antidigoxigenin peroxidase conjugate . No cross-hybridization with other Bartonella or non-Bartonella species was observed . This EIA was as sensitive as dot blot hybridization and was 10 times more sensitive than visualization of PCR products on agarose gels . Serial dilutions of B . henselae and B . quintana suspensions demonstrated that an optical density (OD) of approximately 0.200 was equivalent to 5 CFU in the reaction mixture . By comparing the OD of the bacterial dilutions with that obtained from clinical specimens we could determine that the number of CFU in clinical samples ranged from 10(3) to 10(6) CFU/ml . The PCR-EIA developed in the present study is a rapid, sensitive, and simple method for the diagnosis of B . henselae and B . quintana infections. J Invertebr Pathol, 1999 Sep, 74(2), 127 - 36 Insecticidal activity of strains of Bacillus thuringiensis on larvae and adults of Bactrocera oleae Gmelin (Dipt . Tephritidae); Alberola TM et al.; The olive fly, Bactrocera oleae, is the key pest on olives in the Mediterranean area . The pest can destroy, in some cases, up to 70% of the olive production . Its control relies mainly on chemical treatments, sometimes applied by aircraft over vast areas, with their subsequent ecological and toxicological side effects . Bacillus thuringiensis is a spore-forming soil bacterium which produces a protein crystal toxic to some insects, including the orders of Lepidoptera, Diptera, and Coleoptera and other invertebrates . The aim of this study was to search for isolates toxic to B . oleae . Several hundred B . thuringiensis isolates were obtained from olive groves and olive presses in different areas of Greece, Sardinia (Italy), and Spain and from cooperating scientists throughout the world . Some isolates were found toxic only to adults or larvae and some to both stages of the olive fly . In addition, the most toxic isolates were assayed on Opius concolor Szepl . (Hym . Braconidae), the most important parasitoid of the olive fruit fly . Only 3 isolates out of 14 gave significant mortality against this parasitoid . Several of the most toxic crystalliferous isolates may contain novel toxins since they gave no PCR products when probed with primers specified for 39 known toxin genes . Proc Natl Acad Sci U S A, 1999 Sep 14, 96(19), 10608 - 13 Molecular cloning of an apoptosis-inducing protein, pierisin, from cabbage butterfly: possible involvement of ADP-ribosylation in its activity; Watanabe M et al.; We have previously reported that the cabbage butterfly, Pieris rapae, contains a 98-kDa protein, named pierisin, that induces apoptosis in a variety of human cancer cell lines . In the present study, sequencing and cloning of a cDNA encoding pierisin was accomplished . PCR-direct sequencing showed that the gene encodes an 850-amino acid protein with a calculated molecular weight of 98,081 . An intact clone at the amino acid level encompassing the entire coding region was obtained by recombination of two independent clones, and the molecular mass of its in vitro expressed protein was about 100 kDa on SDS/PAGE, the same as that of purified native pierisin . The expressed protein induced apoptosis in human gastric carcinoma TMK-1 and cervical carcinoma HeLa cells, like the native protein, indicating functional activity . The deduced amino acid sequence of pierisin showed 32% homology with a 100-kDa mosquitocidal toxin from Bacillus sphaericus SSII-1 . In addition, pierisin showed regional sequence similarities with ADP-ribosylating toxins, such as the A subunit of cholera toxin . A glutamic acid residue at the putative NAD-binding site, conserved in all ADP-ribosylating toxins, was also found in pierisin . Substitution of another amino acid for glutamic acid 165 resulted in a great decrease in cytotoxicity and induction of apoptosis . Moreover, inhibitors of ADP-ribosylating enzymes reduced pierisin-induced apoptosis . These results suggest that the apoptosis-inducing protein pierisin might possess ADP-ribosylation activity that leads to apoptosis of the cells. Biophys Chem, 1999 Aug 30, 80(3), 153 - 63 The electric dipole moment of DNA-binding HU protein calculated by the use of an NMR database; Takashima S et al.; Electric birefringence measurements indicated the presence of a large permanent dipole moment in HU protein-DNA complex . In order to substantiate this observation, numerical computation of the dipole moment of HU protein homodimer was carried out by using NMR protein databases . The dipole moments of globular proteins have hitherto been calculated with X-ray databases and NMR data have never been used before . The advantages of NMR databases are: (a) NMR data are obtained, unlike X-ray databases, using protein solutions . Accordingly, this method eliminates the bothersome question as to the possible alteration of the protein structure due to the transition from the crystalline state to the solution state . This question is particularly important for proteins such as HU protein which has some degree of internal flexibility; (b) the three-dimensional coordinates of hydrogen atoms in protein molecules can be determined with a sufficient resolution and this enables the N-H as well as C = O bond moments to be calculated . Since the NMR database of HU protein from Bacillus stearothermophilus consists of 25 models, the surface charge as well as the core dipole moments were computed for each of these structures . The results of these calculations show that the net permanent dipole moments of HU protein homodimer is approximately 500-530 D (1 D = 3.33 x 10(-30) Cm) at pH 7.5 and 600-630 D at the isoelectric point (pH 10.5) . These permanent dipole moments are unusually large for a small protein of the size of 19.5 kDa . Nevertheless, the result of numerical calculations is compatible with the electro-optical observation, confirming a very large dipole moment in this protein. Public Health, 1999 Jul, 113(4), 177 - 9 Route of administration of BCG in a school population: outcome of an audit of clinical practice in North Bedfordshire; Chisholm CJ et al.; Following findings of variations in policies and practices of BCG (Bacillus Calmette-Guerin) administration in different parts of England and Wales, recommendations were made for BCG vaccination to be administered intradermally using disposable equipment . Intradermal methods were used for BCG in Bedfordshire until 1981 but it was changed in 1982 to percutaneous method using the reusable modified heaf gun . Suggestion to revert from percutaneous to intradermal method caused anxiety amongst health professionals with anecdotal reports of scar formations and keloids causing disfigurement at injection site . An audit was therefore carried out to assess the outcome of intradermal BCG administration initially by doctors and then by nurses . The outcome did not justify initial anxieties. J Lab Clin Med, 1999 Sep, 134(3), 244 - 52 A double-blind, placebo-controlled study of Mycobacterium-specific human immune responses induced by intradermal bacille Calmette-Guérin vaccination; Hoft DF et al.; Recent studies have indicated that type 1 T cell responses (potent interferon-gamma and cytolytic responses, with absence of interleukin-4 production) are important for protective immunity against mycobacteria . These observations suggest that assays of type 1 T cell responses may be useful as surrogate markers of protective immunity in the evaluation of new tuberculosis vaccines . To be useful as surrogate markers, immunologic assays must distinguish between vaccine recipients and control subjects in clinical trials . Previous studies have shown that bacille Calmette-Guerin (BCG) vaccination can induce human type 1 T cell responses, but randomized trials have not been done to determine whether measurement of these responses can distinguish between BCG recipients and control subjects . We have conducted a double-blind, placebo-controlled trial of intradermal vaccination with two different BCG strains . We compared the mean lymphoproliferative, cytotoxic, Th1 and Th2 cytokine, and antibody responses detected in BCG and placebo recipients . These studies demonstrated that significant increases in Mycobacterium-specific T cell proliferative responses and type 1 cytokine responses were induced by BCG when compared with results with a placebo . In addition, BCG induced significant increases in Mycobacterium-specific antibody responses with an isotype profile characteristic of a type 1 cytokine bias . T cell and antibody assays involving the use of mycobacterial whole cell lysates or live BCG were able to discriminate between BCG and placebo recipients better than were assays using mycobacterial culture filtrates . These studies provide important information for the development of immunologic assays that might be useful as surrogate markers of protective immunity in future trials of new tuberculosis vaccines. J Med Microbiol, 1999 Sep, 48(9), 849 - 56 Seroprevalence of Bartonella henselae in cats in Germany; Haimerl M et al.; Bartonella henselae and B . quintana infections in man are associated with various clinical manifestations including cat-scratch disease, bacillary angiomatosis and bacteraemia . While cats are the natural reservoir for B . henselae, the source of B . quintana is unclear . In this study, the sera of 713 cats from Germany were examined for the presence of antibodies against B . henselae, B . quintana or Afipia felis by an indirect immunofluorescence assay (IFA) . Bartonella-specific antibody titres of > or =50 were found in 15.0% of the cats . There was substantial cross-reactivity among the various Bartonella antigens, although single sera showed high titres against B . henselae but not against B . quintana and vice versa . Antibodies against A . felis were not detected in any of these cats . Statistical analysis indicated that there is no correlation between Bartonella infections and the sex, age or breed of the cat or its hunting behavior . There was also no correlation between bartonella and toxoplasma infections in cats . However, whereas 16.8% of cats from northern Germany had B . quintana-specific antibodies, only 8.0% of cats from southern Germany were seropositive for B . quintana . No statistically significant difference was found for B . henselae . IFA-positive and IFA-negative sera were used for immunoblot analysis including B . henselae and B . quintana . Marked reactivity was observed with protein bands at 80, 76, 73, 65, 37, 33 and 15 kDa . The results of this study suggest that B . henselae, and possibly a B . quintana-related pathogen, but not A . felis, are common in cats in Germany, and that there are differences in the geographic distribution of bartonella infections in cats. Semin Oncol, 1999 Aug, 26(4), 439 - 47 Immunologic approaches to the treatment of prostate cancer; Harris DT et al.; The presence of several organ-specific molecules that could serve as immunogens or targets of an immune attack, the nonessential nature of the prostate gland, the substantial failure rate after treatment of the primary tumor, and the lack of effective chemotherapy for metastatic disease make prostate cancer an ideal candidate for immunotherapy . This report reviews the current status of two novel approaches to the treatment of prostate cancer . The first is an effort to induce antitumor immunity by enriching the cytokine environment within the primary cancer by intraprostatic injection of Leukocyte Interleukin (Cel-Sci Corp, Vienna, VA), a mixture of natural cytokines that includes interleukin-1 beta (IL-1beta), IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) . The second approach uses OncoVax-P (Jenner Biotherapies, Inc, San Ramon, CA), a vaccine consisting of liposome-encapsulated recombinant prostate-specific antigen (PSA) and lipid A . When administered as an emulsion or in association with bacillus Calmette-Guerin (BCG)/cyclophosphamide or GM-CSF with or without IL-2/cyclophosphamide, immunologic tolerance is broken as evidenced by the generation of humoral and cellular immunity . Both of these approaches have been shown to be feasible and safe, and are now being tested in patients with less advanced disease to determine if manipulation of the immune system can favorably influence clinical outcome. J Chromatogr A, 1999 Aug 13, 852(2), 407 - 16 Isolation and characterization of cyclic alpha-(1-->4)-glucans having degrees of polymerization 9-31 and their quantitative analysis by high-performance anion-exchange chromatography with pulsed amperometric detection; Koizumi K et al.; Cyclic alpha-(1-->4)-glucans with degrees of polymerization (DPs) 9-31 were isolated from a mixture of cyclization products formed in the early stage of the action of cyclodextrin glucanotransferase (CGTase) on synthetic amylose, and characterized by matrix-assisted laser desorption ionization time-of-flight MS, 13C-NMR and HPLC of their partial acid hydrolyzates . High-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection for an accurate estimation of cyclic alpha-(1-->4)-glucans was developed using those isolate glucans as quantification standards, and by HPAEC, the time course of the cyclization reaction of CGTase from an alkalophilic Bacillus sp . A2-5a on synthetic amylose was determined. Nihon Hansenbyo Gakkai Zasshi, 1999 Jul, 68(2), 109 - 16 Brief survey of leprosy situation in Congo: sero-epidemiologic profile in correlation with some emerging viral infections; Milanga M et al.; The Democratic Republic of Congo (DRC, former Zaire) in central Africa remains the foremost country for leprosy in Africa, with a total of 4877 registered cases, of which 4573 are new cases since 1997 . These numbers are well above the regional average . About 94% of these patients are under multidrug therapy (MDT) coverage in the Congo, which ranks 8th in coverage rate among the surrounding nine nations . Available data on anatomo-clinical profile and bacillarity are provided, with reservations on the use of these data drwn due to relatively small sample sizes . The seroprofile of the disease was reviewed with regard to the association of other immunity impairing infections like HBV infection and the recently highly incident retroviral epidemics (HIV-1, HTLV-1, and HTLV-2) . The leading role of non-governmental organizations is cited for improving leprosy patient conditions and also for future prospects, where the necessity of coordinated strategies with the government is emphasized . Recommendations for new trends and steps relevant to improving existing and future leprosy control strategies are put into perspective. FEBS Lett, 1999 Sep 17, 458(2), 175 - 9 Amino acid substitution in alpha-helix 7 of Cry1Ac delta-endotoxin of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner; Chandra A et al.; Insecticidal proteins or delta-endotoxins of Bacillus thuringiensis are highly toxic to a wide range of agronomically important pests . The toxins are formed of three structural domains . The N-terminal domain is a bundle of eight alpha-helices and is implicated in pore formation in insect midgut epithelial membranes . All the delta-endotoxins share a common hydrophobic motif of eight amino acids in alpha-helix 7 . A similar motif is also present in fragment B of diphtheria toxin (DT) . Site-directed mutagenesis of Cry1Ac delta-endotoxin of B . thuringiensis was carried out to substitute its hydrophobic motif with that of DT fragment B . The mutant toxin was shown to be more toxic to the larvae of Helicoverpa armigera (cotton bollworm) than the wild-type toxin . Voltage clamp analysis with planar lipid bilayers revealed that the mutant toxin opens larger ion channels and induces higher levels of conductance than the wild-type toxin. Exp Hematol, 1999 Sep, 27(9), 1368 - 74 Listeria monocytogenes and recurrent mycobacterial infections in a child with complete interferon-gamma-receptor (IFNgammaR1) deficiency: mutational analysis and evaluation of therapeutic options; Roesler J et al.; We describe the history of a girl with interferon-gamma-receptor (IFNgammaR1) deficiency and studies performed to identify the molecular and clinical characteristics of this recently discovered disorder . This is the first report of a child from Northern Europe with IFNgammaR1 deficiency . The patient, now 7 years old, first presented with swelling and reddening at the Bacille Calmette-Guerin (BCG) vaccination site, swelling of lymph nodes, hepatomegaly, and an unusually severe varicella rash at the age of 4 months . At that time, she was diagnosed with BCG histiocytosis without typical granuloma formation and was treated with antituberculous agents . During the clinical course of her illness, several different types of atypical mycobacteria and (for the first time in an IFNgammaR1-deficient patient) Listeria monocytogenes were detected . Flow cytometric analysis showed that the patient's monocytes could not bind a monoclonal antibody specific for the IFNgamma-receptor . Our analysis of mRNA derived from the alpha-chain (IFNgammaR1) gene of this receptor revealed deletions of 173 bp and 4 bp in cDNA sequences originating from individual alleles . The 173 bp deletion was located between nucleotide positions 200 and 372, exactly matching those of exon 3, and the 4 bp deletion was located between nucleotide positions 561 and 564 of the coding region of the cDNA . Analysis of genomic DNA revealed the presence of a G to T transition at the 5'end of the splice consensus sequence of intron 3, which explains the absence of exon 3 . The other allele carried the 4-base-pair deletion (ACTC) at nucleotide positions 15-18 of exon 5 . Twelve months after an allo inverted question markgeneic bone marrow transplantation, the patient had clinically improved. Biol Pharm Bull, 1999 Aug, 22(8), 841 - 5 Electrophysiological studies on the mechanism for enhanced intestinal transport of water-soluble compounds by antibiotic peptide bacitracin; Quan YS et al.; Bacitracin is an antibacterial cyclic dodecapeptide produced by Bacillus licheniformin . Besides antibacterial activity, it is reported to have a protease inhibitory activity and an absorption enhancing action . Here we determined the effects of bacitracin on transport of water-soluble dye fluoresceinisothiocyanate (FITC)-dextran across the rat intestinal mucosal membrane using an electrophysiological technique . Bacitracin enhanced the intestinal mucosal-to-serosal transport of FITC-dextran in concentration-dependent and pH-dependent manners . In particular, the addition of bacitracin to the mucosal side led to a remarkable enhancement of FITC-dextran transport across the colonic membrane . Furthermore, its exhibition of transport enhancement required the existence of metal divalent cations, Ca2+ and Mg2+, in the mucosal compartment . Electrophysiological study using voltage-clamp technique revealed that a relatively lower concentration of bacitracin (5 mM) enhanced the transport of 6-carboxyfluorescein via a paracellular pathway in the colonic membrane and higher concentration of bacitracin (20 mM) affects both transcellular and paracellular routes, resulting in significant enhancement of 6-carboxyfluorescein across the colonic membrane . These findings might provide the useful information for enhancing the intestinal transport of poorly absorbable drugs by bacitracin which has multiple functions. J Am Mosq Control Assoc, 1999 Sep, 15(3), 371 - 9 Residual activity of Bacillus thuringiensis serovars medellin and jegathesan on Culex pipiens and Aedes aegypti larvae; Thiery I et al.; Bacillus thuringiensis serovar medellin strain 163-131 and Bacillus thuringiensis serovar jegathesan (B.t.jeg.) strain 367 are very toxic to mosquito larvae . However, they are 10 times less toxic than Bacillus thuringiensis var . israelensis (B.t.i.) to mosquito larvae under laboratory conditions . Lyophilized powders were produced from these strains and their toxicities were compared to that of powder produced from the B.t.i . strain . Larvicidal activity was titrated using Aedes aegypti (Bora-Bora strain) larvae, with IPS82 powder as the standard . The efficacy of these powders in the field was determined using Culex pipiens (Montpellier strain) in Paris, France, and Ae . aegypti larvae (French Guiana strain) in Cayenne, French Guiana, in standardized conditions . Residual activity was also assessed in the laboratory, using Cx . pipiens (SLAB strain), in Montpellier, France . Any negative effect of direct sunlight, soil, or polluted water on the residual activity of the 3 powders was recorded . Increasing bacterial concentration by a factor of 8 had little effect on the duration of larvicidal activity, except in the presence of polluted water and when substrate was added . All powders had similar initial efficacies against both types of mosquito larvae, in all conditions except water rich in organic matter . Bacillus thuringiensis serovar medellin had the lowest residual activity, both in the laboratory and in the field, whereas B.t.jeg . remained toxic for as long as B.t.i. J Am Mosq Control Assoc, 1999 Sep, 15(3), 366 - 70 Duration of activity of the microbial larvicide VectoLex CG (Bacillus sphaericus) in Illinois catch basins and waste tires; Siegel JP et al.; The duration of activity of a formulation of Bacillus sphaericus, VectoLex CG, for control of Culex species was evaluated in 338 catch basins in Urbana, IL, and compared to Altosid in 346 catch basins in Champaign, IL . The activity of VectoLex in car and truck waste tires was evaluated in a tire dump located in Pembroke Township, IL . In catch basins, 1 g of VectoLex per catch basin gave the same control as one Altosid briquet . Both larvicides were effective against Culex sp . in catch basins for 1 month, and the duration of control with VectoLex lasted 44 days in one catch basin . VectoLex was considerably cheaper to apply than Altosid briquets, at 0.64 cents per catch basin compared to 90.75 cents, respectively . However, the Altosid briquets were judged to be easier to apply from a vehicle than VectoLex granules . VectoLex (22.6 kg) was used to treat approximately 6,000 car and truck tires; some of the tires were in direct sunlight whereas others were shaded . Aedes triseriatus was the dominant species in these tires . Tires treated with VectoLex contained significantly fewer mosquitoes than control tires, and even 65 days after application, control tires were 16.7 times more likely to contain larvae . We conclude that VectoLex was effective when used in Illinois catch basins and tire dumps, and emphasize that it is more appropriate to base tire treatment rates on the total number of tires present than on a kilogram per hectare basis. J Am Mosq Control Assoc, 1999 Sep, 15(3), 356 - 65 Field evaluation of new water-dispersible granular formulations of Bacillus thuringiensis ssp . israelensis and Bacillus sphaericus against Culex mosquitoes in microcosms; Su T et al.; A variety of formulations of Bacillus thuringiensis var . israelensis de Barjac (B.t.i.) and Bacillus sphaericus Neide (B.s.) have been studied for mosquito control under laboratory and field conditions . High efficacy, specificity, low risk of development of resistance, long shelf-life, and transportability, as well as the safety to nontarget organisms of these 2 microbial agents have been well documented . Some of the currently available formulations of B.t.i . and B.s . have low potency per unit mass . Research and development efforts are focusing on commercializing formulations with high potency and low minimum effective dosage that are suitable for long-distance shipment . To achieve this goal, new water-dispersible granule (WDG) formulations of both microbial agents were prepared and made available by Abbott Laboratories for evaluation . The newly developed WDGs of B.t.i . and B.s . with high potency dispersed readily in water with gentle agitation . These WDGs were evaluated and the minimum effective dosages were determined in microcosms against natural populations of Culex mosquitoes . The minimum effective dosage for B.t.i . WDGs with 4,000 International Toxic Units (ITU)/mg was 0.27-0.53 lb/acre which yielded significant control for up to 7-12 days . The minimum effective dosage for B.s . WDGs with 350-630 ITU/mg was 0.05-0.10 lb/acre, which yielded significant control of immature mosquitoes for up to 14-20 days. J Infect Dis, 1999 Oct, 180(4), 1386 - 9 Absence of Kaposi's sarcoma-associated herpesvirus DNA in bacillary angiomatosis-peliosis lesions; Relman DA et al.; Bartonella henselae and B . quintana induce an unusual vascular proliferative tissue response known as bacillary angiomatosis (BA) and bacillary peliosis (BP) in some human hosts . The mechanisms of Bartonella-associated vascular proliferation remain unclear . Although host factors probably play a role, microbial coinfection has not been ruled out . Because of the vascular proliferative characteristics noted in both Kaposi's sarcoma (KS) and BA and occasional colocalization of KS and BA, the possibility was explored that KS-associated herpesvirus (KSHV) might be associated with BA lesions . Tissues with BA and positive and negative control tissues were tested for the presence of KSHV DNA by a sensitive polymerase chain reaction assay . Only 1 of 10 BA tissues, a splenic biopsy, was positive in this assay; this tissue was from a patient with concomitant KS of the skin . Thus, KSHV is probably not involved in the vascular proliferative response seen in BA-BP. Biochim Biophys Acta, 1999 Aug 25, 1440(1), 107 - 17 Binding of phosphatidylinositol-specific phospholipase C to phospholipid interfaces, determined by fluorescence resonance energy transfer; Hendrickson HS et al.; Dissociation constants for binding of phosphatidylinositol-specific phospholipase C from Bacillus cereus (bcPI-PLC) and the mammalian phosphatidylinositol-specific phospholipase C-delta(1) to lipid interfaces containing phosphoinositol, phosphocholine, and phosphomethanol head groups were determined by fluorescence resonance energy transfer . Dansyl-labeled lipid probes were used as acceptors, with intrinsic tryptophan of the enzyme as the donor . Titration of protein into lipid provided data from which K(d) and N, the limiting number of lipid molecules per protein bound, were calculated by non-linear regression analysis of exact binding equations . These results were compared with apparent K(m) values from kinetic data . K(d) values in the low microM range in terms of lipid monomers or low nM range in terms of binding sites were calculated with good fits of experimental data to theoretical binding curves . bcPI-PLC binds with high affinity to PI interfaces, slightly lower affinity to PC interfaces, and much lower affinity to PM interfaces . The mammalian enzyme also binds with high affinity to PI interfaces, but shows little or no binding with PC interfaces under similar concentration conditions . These K(d) values correlate reasonably with apparent K(m) values from kinetic experiments. J Immunol, 1999 Sep 15, 163(6), 3226 - 31 In vivo immunomodulation following intradermal injection with DNA encoding IL-18; Kremer L et al.; IL-18, a recently identified cytokine synthesized by different cell types, including Kupffer cells, activated macrophages, and keratinocytes, induces IFN-gamma production by T cells and NK cells . The cDNA encoding IL-18 with its natural signal peptide was cloned under control of the CMV promoter and injected into the skin of mice . A single intradermal injection of this construction led to efficient in vivo expression of IL-18 in cutaneous dermal cells and induced IFN-gamma mRNA production, indicating that it was produced in a biologically active form . In addition, a massive cellular infiltrate was observed in the skin 2 days after injection . When the mice were subsequently infected with Mycobacterium bovis bacillus Calmette-Guerin (BCG), they produced lower levels of anti-BCG Abs than control animals . However, in contrast to their lowered humoral immune response, the mice produced higher amounts of Ag-specific IFN-gamma after in vitro restimulation, as compared with the controls . Therefore, injection of DNA encoding IL-18 into the skin modulates both Ag-specific humoral and T cell responses upon mycobacterial infection . It increases the Th1 type response, which may be particularly useful for the development of new immunotherapeutic or immunoprotective approaches against infections by intracellular parasites, such as mycobacteria. J Appl Microbiol, 1999 Aug, 87(2), 241 - 5 Characterization of the exosporium of Bacillus cereus; Charlton S et al.; Exosporium components from endospores of Bacillus cereus ATCC 10876 were purified and separated by gel electrophoresis . Several of the proteins for which N-terminal sequences were recovered were found to have homologues in protein databases which have been demonstrated to have enzymic activity in other organisms . Amongst these is a zinc metalloprotease, immune inhibitor A, already described in B . thuringiensis . This has been shown to be present in an active 73 kDa form on the exosporium of B . cereus . Other proteins associated with the exosporium include the molecular chaperone GroEL and a homologue of RocA (1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12)) of B . subtilis . Although these are unlikely to represent integral structural proteins of the exosporium, the observation that they are selectively present in the spore surface layer suggests that this layer may have functional significance. In Vitro Cell Dev Biol Anim, 1999 May, 35(5), 299 - 303 Activity spectra of Bacillus thuringiensis delta-endotoxins against eight insect cell lines; Gringorten JL et al.; Eight continuous insect cell lines were tested for susceptibility to the delta-endotoxins of several lepidopteran-active strains and cloned-gene products of Bacillus thuringiensis . The assays were performed on cells suspended in agarose gel, which allowed the toxins activated at pH 10.5 to be applied directly in a high-pH buffer without causing solvent toxicity to the cells . The responses of the cell lines to the various toxins produced activity spectra that were used to identify functionally similar and dissimilar toxin proteins . IPRI-CF-1 and FPMI-MS-5, derived from neonate larvae of Choristoneura fumiferana and Manduca sexta, respectively, exhibited the greatest sensitivity to the toxins tested, whereas B . thuringiensis subsp . entomocidus had the broadest in vitro host range . Analysis of activity spectra led to the identification of the particular Cry protein that was responsible for the broad toxicity of this subspecies and demonstrated a distinct difference in toxin composition between two strains of subsp . sotto . The identical spectra observed for subsp . kurstaki HD-1 and NRD-12 is consistent with insect bioassay data obtained previously by other workers and supports the conclusion that there is virtually no difference in activity between these two strains . The in vitro assay system, referred to as the "lawn assay" and used to test B . thuringiensis activated toxins against insect cell lines, is particularly useful in mode-of-action studies and as a rapid, preliminary test for the presence of specific cytolytic proteins, rather than as a method for screening toxins of wild-type strains for insecticidal activity . The response of cells in vitro to B . thuringiensis toxins is often very different from that of the insect from which the cells were derived. FEMS Microbiol Lett, 1999 Aug 15, 177(2), 237 - 42 Disruption of the mycobacterial cell entry gene of Mycobacterium bovis BCG results in a mutant that exhibits a reduced invasiveness for epithelial cells; Flesselles B et al.; Mycobacteria belonging to the Mycobacterium tuberculosis complex have the ability to invade and replicate in non-phagocytic cells, an event that requires the presence of bacterial surface components capable of triggering a cell response and the subsequent internalization of the microorganism . In this study, we report the sequencing of the mycobacterial cell entry gene (mce) of Mycobacterium bovis bacillus Calmette-Guerin (BCG) and the generation and characterization of a mutant BCG strain with an inactivated mce gene, by homologous recombination with double cross-over . This mutant strain does not express the mycobacterial cell entry protein (Mce) and exhibits a reduced ability to invade the non-phagocytic epithelial cell line HeLa as compared to wild-type BCG. FEMS Microbiol Lett, 1999 Aug 15, 177(2), 225 - 9 The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus; Granum PE et al.; The non-haemolytic enterotoxin from Bacillus cereus has been sequenced . It is composed of three components, non-haemolytic enterotoxin A, B and C of 41.0, 39.8 and 36.5 kDa, respectively . Transcription of the operon seems to be positively regulated by plcR, a gene that also regulates phospholipase C expression . There is substantial similarity between the three proteins of non-haemolytic enterotoxin and between the non-haemolytic enterotoxin and haemolytic enterotoxin proteins. Appl Environ Microbiol, 1999 Sep, 65(9), 4032 - 9 Development and field performance of a broad-spectrum nonviable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance; Sanchis V et al.; The main problems with Bacillus thuringiensis products for pest control are their often narrow activity spectrum, high sensitivity to UV degradation, and low cost effectiveness (high potency required) . We constructed a sporulation-deficient SigK(-) B . thuringiensis strain that expressed a chimeric cry1C/Ab gene, the product of which had high activity against various lepidopteran pests, including Spodoptera littoralis (Egyptian cotton leaf worm) and Spodoptera exigua (lesser {beet} armyworm), which are not readily controlled by other Cry delta-endotoxins . The SigK(-) host strain carried the cry1Ac gene, the product of which is highly active against the larvae of the major pests Ostrinia nubilalis (European corn borer) and Heliothis virescens (tobacco budworm) . This new strain had greater potency and a broader activity spectrum than the parent strain . The crystals produced by the asporogenic strain remained encapsulated within the cells, which protected them from UV degradation . The cry1C/Ab gene was introduced into the B . thuringiensis host via a site-specific recombination vector so that unwanted DNA was eliminated . Therefore, the final construct contained no sequences of non-B . thuringiensis origin . As the recombinant strain is a mutant blocked at late sporulation, it does not produce viable spores and therefore cannot compete with wild-type B . thuringiensis strains in the environment . It is thus a very safe biopesticide . In field trials, this new recombinant strain protected cabbage and broccoli against a pest complex under natural infestation conditions. Appl Environ Microbiol, 1999 Sep, 65(9), 3942 - 9 Long-chain polyphosphate causes cell lysis and inhibits Bacillus cereus septum formation, which is dependent on divalent cations; Maier SK et al.; We investigated the cellular mechanisms that led to growth inhibition, morphological changes, and lysis of Bacillus cereus WSBC 10030 when it was challenged with a long-chain polyphosphate (polyP) . At a concentration of 0.1% or higher, polyP had a bacteriocidal effect on log-phase cells, in which it induced rapid lysis and reductions in viable cell counts of up to 3 log units . The cellular debris consisted of empty cell wall cylinders and polar caps, suggesting that polyP-induced lysis was spatially specific . This activity was strictly dependent on active growth and cell division, since polyP failed to induce lysis in cells treated with chloramphenicol and in stationary-phase cells, which were, however, bacteriostatically inhibited by polyP . Similar observations were made with B . cereus spores; 0.1% polyP inhibited spore germination and outgrowth, and a higher concentration (1.0%) was even sporocidal . Supplemental divalent metal ions (Mg(2+) and Ca(2+)) could almost completely block and reverse the antimicrobial activity of polyP; i . e., they could immediately stop lysis and reinitiate rapid cell division and multiplication . Interestingly, a sublethal polyP concentration (0.05%) led to the formation of elongated cells (average length, 70 microm) after 4 h of incubation . While DNA replication and chromosome segregation were undisturbed, electron microscopy revealed a complete lack of septum formation within the filaments . Exposure to divalent cations resulted in instantaneous formation and growth of ring-shaped edges of invaginating septal walls . After approximately 30 min, septation was complete, and cell division resumed . We frequently observed a minicell-like phenotype and other septation defects, which were probably due to hyperdivision activity after cation supplementation . We propose that polyP may have an effect on the ubiquitous bacterial cell division protein FtsZ, whose GTPase activity is known to be strictly dependent on divalent metal ions . It is tempting to speculate that polyP, because of its metal ion-chelating nature, indirectly blocks the dynamic formation (polymerization) of the Z ring, which would explain the aseptate phenotype. Antimicrob Agents Chemother, 1999 Sep, 43(9), 2126 - 30 Analysis of rifapentine for preventive therapy in the Cornell mouse model of latent tuberculosis; Miyazaki E et al.; Rifapentine is a long-acting rifamycin which may be useful for intermittent drug therapy against tuberculosis . In this study we measured the efficacies of rifapentine-containing intermittent drug regimens for preventive therapy using the Cornell mouse model of latent tuberculosis . We infected groups of mice intravenously with Mycobacterium tuberculosis and then treated them with isoniazid and pyrazinamide for 12 weeks according to the Cornell latency development protocol . After a 4-week interval of no treatment, experimental preventive therapy was administered by esophageal gavage for 12 or 18 weeks . After equilibration and dexamethasone amplification treatment, mouse organs were analyzed by quantitative colony counts to measure the effectiveness of therapy . Our results showed that once-weekly isoniazid plus rifapentine combination therapy for 18 weeks was an effective preventive regimen with sterilizing potency and bacillary load reduction comparable to those of daily isoniazid therapy for 18 weeks . Monotherapy with rifapentine weekly or fortnightly or with rifampin twice weekly for up to 18 weeks did not offer advantages in reducing bacillary load or in sterilizing organs compared to the effects of a placebo . These results with the Cornell mouse model indicate that once-weekly, short-course preventive therapy with isoniazid plus rifapentine is effective and may warrant investigation in humans with latent tuberculosis infection. J Antibiot (Tokyo), 1999 Jun, 52(6), 559 - 71 Molecular cloning of the gene for the key carbocycle-forming enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics and its comparison with dehydroquinate synthase; Kudo F et al.; The 2-deoxystreptamine aglycon is a common structural feature found in aminocyclitol antibiotics including neomycin, kanamycin, tobramycin, gentamicin, sisomicin, butirosin and ribostamycin . A key enzyme involved in the biosynthesis of the 2-deoxystreptamine moiety is 2-deoxy-scyllo-inosose (DOI) synthase which catalyses the carbocycle formation from D-glucose-6-phosphate to 2-deoxy-scyllo-inosose . The recent success of isolating the 2-deoxy-scyllo-inosose synthase from Bacillus circulans prompted us to clone the gene responsible for this important enzyme by the use of reverse genetics approach . With the aid of DNA probes constructed on the basis of the amino-terminal sequence of the purified 42 kDa subunit of the enzyme, the responsible gene btrC was successfully cloned . Subsequently the btrC gene was heterologously expressed in Escherichia coli, and the 2-deoxy-scyllo-inosose synthase activity of the recombinant polypeptide was confirmed by chemical analysis . The btrC gene encodes a protein composed of 368 amino acids with a molecular mass of 40.7 kDa . Our previous proposal for the similarity of 2-deoxy-scyllo-inosose synthase to dehydroquinate synthase has been confirmed on the basis of their amino acid sequences . Significant differences in the sequences can also be observed however, particularly in the crucial substrate recognition regions . Comparison of the BtrC sequence with those of biosynthetic enzymes for other related microbial products is also discussed. J Control Release, 1999 Aug 27, 61(1-2), 203 - 17 Gamma irradiation for terminal sterilization of 17beta-estradiol loaded poly-(D,L-lactide-co-glycolide) microparticles; Mohr D et al.; 17beta-Estradiol-loaded microparticles using poly-(D, L-lactide-co-glycolide) polymer (PLG) were prepared by a modified spray-drying method and the effects of gamma-irradiation on drug substance, polymer and microparticles were investigated . Irradiation doses ranging from 5.1 to 26.6 kGy were applied using a 60Co-radiation source . 17beta-Estradiol drug substance showed excellent stability against gamma-irradiation in the investigated dose range, whereas microencapsulated estradiol seems to be converted to conjugation products with PLG, and to a lesser extent to the degradation product 9,11-dehydroestradiol . The weight-average molecular weight of the PLG polymers decreased with increasing irradiation dose while polydispersity indices (M(w)/M(n)) remained nearly unchanged, compatible with a random chain scission mechanism in lactide/glycolide-copolymer degradation . In vitro drug release studies showed accelerated kinetics with increasing irradiation doses due to dose dependent polymer degradation . Microbiological process monitoring showed decreasing bioburden with increasing spraying time, which was successfully further reduced by applying irradiation sterilization . Microencapsulated test spore suspensions of Bacillus pumilus ATCC 27142, the official test specimen for the gamma-sterilization process, revealed effective reduction of bioburden, confirming its published D(10) value . In conclusion, our studies demonstrated efficacy of gamma-irradiation as terminal sterilization method for poly-(D,L-lactide-co-glycolide) polymer-based drug delivery systems . The sterilization conditions need to be carefully adjusted for the final dosage form. J Biochem (Tokyo), 1999 Sep, 126(3), 566 - 71 Protein design of geranyl diphosphate synthase . Structural features that define the product specificities of prenyltransferases; Narita K et al.; Geranyl diphosphate synthase catalyzes the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to give a C(10) compound, geranyl diphosphate, which is a precursor of all monoterpenoids . However, the gene has not been isolated from any organisms . To examine the possibility that geranyl diphosphate synthase has evolved from a common ancestor of the prenyltransferase family and to predict the active site structure, we tried to convert Bacillus stearothermophilus farnesyl diphosphate synthase to geranyl diphosphate synthase, according to our previous findings . Several mutated farnesyl diphosphate synthases that have single amino acid substitutions before the first aspartate-rich motif were constructed . A mutated enzyme that has the replacement of serine by phenylalanine at the fourth position before the motif exclusively produced geranyl diphosphate when dimethylallyl diphosphate was used as the primer, and hardly accepted geranyl diphosphate as a primer, indicating that this mutation causes the conversion to geranyl diphosphate synthase . This result supports the idea that the product specificities of all members of the E-prenyltransferase family are mainly defined by a few structural features: the amino acids at the fourth position and the fifth position before the first aspartate-rich motif, and the insertion of two amino acids in the motif . This suggests that natural geranyl diphosphate synthases might have an active site structure similar to that of the mutated enzyme. Biotechnol Appl Biochem, 1999 Aug, 30 ( Pt 1), 35 - 40 Characterization of a novel stable biocatalyst obtained by protein engineering; Van den Burg B et al.; Protein engineering is a powerful tool for the improvement of the properties of biocatalysts . Previously we have applied protein engineering technologies to obtain an extremely stable variant of the thermolysin-like protease from Bacillus stearothermophilus {Van den Burg, Vriend, Veltman, Venema and Eijsink (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 2056-2060} . This variant is much more resistant to denaturing conditions (temperature and denaturing agents) than the wild-type enzyme . An extensive enzymic characterization was undertaken to explore the suitability of the variant as a biocatalyst at high temperatures . By comparing a range of variants with increasing thermal stabilities we show that the additivity of the mutations is accompanied by an increase in activity at elevated temperatures in accordance with the Arrhenius law . The results suggest that the constructed protease variants could be suitable alternatives to proteases that are currently used industrially . Our studies demonstrate how protein engineering can be exploited to obtain high-performance biocatalysts. Arch Insect Biochem Physiol, 1999 Sep, 42(1), 51 - 63 Resistance to bacillus thuringiensis Cry1Ac toxin in three strains of heliothis virescens: proteolytic and SEM study of the larval midgut Forcada C, Alcacer E, Garcera MD, Tato A, Martinez R. In a previous study, we demonstrated that resistance to Bacillus thuringiensis toxins in Heliothis virescens might be related to differences in the composition of the proteolytic extracts from insect midgut . There, we found specific proteolytic bands present in the gut extracts of the resistant strain and absent from the susceptible one . Here we report related facts using a new resistant strain (KCB) and a cross between the two strains used in our previous study . As would be expected, no quantitative differences in total proteolytic activity were found between the strains, although qualitative differences related to the presence or absence of specific proteolytic activity bands using SDS-PAGE could be responsible for the observed resistance phenomenon . Moreover, an SEM study made at different time intervals after intoxication shows that in the initial hours following intoxication, both the susceptible and the resistant strains show significant damage to the midgut epithelium . In the interval between 3 and 48 h, however, the resistant strain recovered such that by 48 h it had fully recovered whereas the susceptible strain did not . Arch . Arch Insect Biochem Physiol, 1999 Sep, 42(1), 1 - 12 Protease interactions with bacillus thuringiensis insecticidal toxins Oppert B. The microbe Bacillus thuringiensis (Bt) produces crystals that contain insecticidal crystal proteins (ICPs) used to control many major pests . ICPs are degraded by proteases from a variety of sources, including those endogenous to the bacterium, those purified from animals and plants, or those found in insects . Proteases in the bacterium function in protein metabolism during sporulation; in some cases they hydrolyze ICPs . Insect proteases are implicated in Bt toxin specificity, mode of action and insect adaptation to Bt . This review describes the current knowledge of protease interactions with ICPs with special emphasis on the role of proteases in insect resistance to Bt toxins . Arch . Am J Trop Med Hyg, 1999 Mar, 60(3), 449 - 52 A continuing focus of Hansen's disease in Texas; Taylor JP et al.; To describe epidemiologic and clinical characteristics of Hansen's disease cases in Texas, information was abstracted from records of 810 patients reported from 1973 through 1997 . Annually, from 18 to 54 patients were reported . Average annual incidence rates ranged from 1.9 to 2.4 cases per million population . A majority of the patients were male (63%) and white (77%) . More than half (53%) of the patients were born in the United States; a majority (83%) of the patients born in the United States were born in Texas . Most (76%) patients were diagnosed with multi-bacillary leprosy . Foreign-born patients were more likely to be younger at onset and have multi-bacillary disease compared with patients born in the United States . Within Texas, an endemic focus of Hansen's disease exists along the Gulf of Mexico coast. J Gen Virol, 1999 Aug, 80 ( Pt 8), 2229 - 37 Sequence changes in six variants of rice tungro bacilliform virus and their phylogenetic relationships; Cabauatan PQ et al.; The DNA of three biological variants, G1, Ic and G2, which originated from the same greenhouse isolate of rice tungro bacilliform virus (RTBV) at the International Rice Research Institute (IRRI), was cloned and sequenced . Comparison of the sequences revealed small differences in genome sizes . The variants were between 95 and 99% identical at the nucleotide and amino acid levels . Alignment of the three genome sequences with those of three published RTBV sequences (Phi-1, Phi-2 and Phi-3) revealed numerous nucleotide substitutions and some insertions and deletions . The published RTBV sequences originated from the same greenhouse isolate at IRRI 20, 11 and 9 years ago . All open reading frames (ORFs) and known functional domains were conserved across the six variants . The cysteine-rich region of ORF3 showed the greatest variation . When the six DNA sequences from IRRI were compared with that of an isolate from Malaysia (Serdang), similar changes were observed in the cysteine-rich region in addition to other nucleotide substitutions and deletions across the genome . The aligned nucleotide sequences of the IRRI variants and Serdang were used to analyse phylogenetic relationships by the bootstrapped parsimony, distance and maximum-likelihood methods . The isolates clustered in three groups: Serdang alone; Ic and G1; and Phi-1, Phi-2, Phi-3 and G2 . The distribution of phylogenetically informative residues in the IRRI sequences shared with the Serdang sequence and the differing tree topologies for segments of the genome suggested that recombination, as well as substitutions and insertions or deletions, has played a role in the evolution of RTBV variants . The significance and implications of these evolutionary forces are discussed in comparison with badnaviruses and caulimoviruses. J Gen Virol, 1999 Aug, 80 ( Pt 8), 2217 - 28 A short open reading frame terminating in front of a stable hairpin is the conserved feature in pregenomic RNA leaders of plant pararetroviruses; Pooggin MM et al.; In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA . By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5'-leader . The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem-loop structure; both features are known to be inhibitory for downstream translation . Formation of the structure brings the first long ORF into the close spatial vicinity of a 5'-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element . The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses . Both the short ORF and the structure have been implicated in the shunt process for CaMV pgRNA translation . The conservation of these elements among all plant pararetroviruses suggests conservation of the ribosome shunt mechanism . For some of the less well-studied viruses, the localization of the conserved elements also allowed predictions of the pgRNA promoter region and the translation start site of the first long ORF. Tsitol Genet, 1999 Mar-Apr, 33(2), 74 - 8 {The construction and use of inosine-containing primers for searching for and identifying the genes of insecticidal crystal proteins in Bacillus thuringiensis}; Rymar' SE et al.; The specific to 3 types of Cry genes primers containing inosine were constructed to find crystal insecticidal protein Bacillus thuringiensis genes using PCR . A number of new B . thuringiensis strains isolated in Ukraine were investigated using these PCR primers . As a results, Cry genes were found, some of them were identified and demonstrated high homology to Cry1Ba2 and Cry1Bc genes. J Bacteriol, 1999 Sep, 181(17), 5455 - 60 ZmaR, a novel and widespread antibiotic resistance determinant that acetylates zwittermicin A; Stohl EA et al.; ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of Bacillus . The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro . Cell extracts of E . coli containing zmaR inactivated zwittermicin A by covalent modification . Chemical analysis of inactivated zwittermicin A by 1H NMR, 13C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated . Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with {14C}acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases . Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function. Biochem Biophys Res Commun, 1999 Aug 27, 262(2), 359 - 64 Inorganic phosphate regulates CryIVA protoxin expression in Bacillus thuringiensis israelensis; Banerjee-Bhatnagar N; The role of nutritional factors during CryIVA protoxin expression in Bacillus thuringiensis israelensis (Bti) has been investigated . Inorganic phosphate (Pi) was found to stimulate 135 kD protoxin synthesis by Bti cells . There was a corresponding increase in the cryIVA specific mRNA in the presence of Pi . Inorganic phosphate inhibited HPr kinase but activated HPr phosphatase, the two enzymes responsible for regulating the concentration of phosphorylated HPr in the cell . Addition of protein phosphatase inhibitors NaF and calyculin A during resuspension resulted in the inhibition of toxin synthesis by Bti cells . Calyculin A inhibited HPr phosphatase activity in the in vitro assay also . The concentration of phosphorylated HPr was upregulated when the cells were resuspended in the presence of calyculin A, while the levels of the same were lowered in the presence of Pi, as determined by Western blotting the respective cells . The efficiency of sporulation of Bti was not affected when Pi was added alone or along with the phosphatase inhibitor calyculin A . Am J Trop Med Hyg, 1999 Aug, 61(2), 344 - 9 An outbreak of acute bartonellosis (Oroya fever) in the Urubamba region of Peru, 1998; Ellis BA et al.; During May 1998, we conducted a case-control study of 357 participants from 60 households during an outbreak of acute bartonellosis in the Urubamba Valley, Peru, a region not previously considered endemic for this disease . Blood and insect specimens were collected and environmental assessments were done . Case-patients (n = 22) were defined by fever, anemia, and intra-erythrocytic coccobacilli seen in thin smears . Most case-patients were children (median age = 6.5 years) . Case-patients more frequently reported sand fly bites than individuals of neighboring households (odds ratio {OR} = 5.8, 95% confidence interval {CI} = 1.2-39.2), or members from randomly selected households > or = 5 km away (OR = 8.5, 95% CI = 1.7-57.9) . Bartonella bacilliformis isolated from blood was confirmed by nucleotide sequencing (citrate synthase {g/tA}, 338 basepairs) . Using bacterial isolation (n = 141) as the standard, sensitivity, specificity, and positive predictive value of thin smears were 36%, 96%, and 44%, respectively . Patients with clinical syndromes compatible with bartonellosis should be treated with appropriate antibiotics regardless of thin-smear results. J Formos Med Assoc, 1999 Jul, 98(7), 496 - 9 Annual risk of tuberculous infection in Taiwan, 1996-1998; Yu MC et al.; Tuberculosis is still an important public health issue in Taiwan, and monitoring the trend of annual risk of infection (ARI) with Mycobacterium tuberculosis is essential . In this study, we conducted tuberculin skin tests to estimate the prevalence and annual risk of M . tuberculosis infection in first-grade schoolchildren in Taiwan Province . Because mass bacille Calmette-Guerin (BCG) vaccination programs have been carried out here, only non-BCG-vaccinated students were tested . From September 1996 through June 1998, there were 520,866 registered first-grade elementary school students in Taiwan Province . Of them, 15,147 (2.9%) were non-BCG-vaccinated, as determined by the absence of a BCG scar . All of them were tested for M . tuberculosis infection with 1 tuberculin unit (0.1 mL injection) of purified protein derivative RT23, by means of the Mantoux technique . Among the tested schoolchildren, 430 (2.8%) had a positive tuberculin reaction . Thus, the calculated ARI was 0.44% . The ARI varied in different areas of Taiwan, being highest (1.04%) in Nantou County and lowest (0.14%) in Miaoli and Tainan Counties . The ARI in aboriginal areas (1.16%) was 2.7 times that in nonaboriginal areas (0.42%) . Our results indicate that the M . tuberculosis ARI is still high in Taiwan . To achieve the World Health Organization target of less than 0.1% for industrialized countries, we must intensify tuberculosis control programs in Taiwan. Int J Tuberc Lung Dis, 1999 Aug, 3(8), 695 - 702 Treatment of bacillary pulmonary tuberculosis at the chest clinics in the private sector in Korea, 1993; Hong YP et al.; SETTING: Cohort study of bacillary pulmonary tuberculosis patients treated at private sector chest clinics in Korea . OBJECTIVE: To assess the treatment behaviour of physicians in private chest clinics and the treatment outcomes of their patients . DESIGN: 1) A retrospective analysis of a cohort of patients admitted from July through October in 1993, and 2) comparison with results from health centres under the National Tuberculosis Programme (NTP) . RESULTS: Nine hundred and sixty bacillary patients (507 newly diagnosed--'new', and 453 retreatment--'old') were admitted to the study . Initial smears and cultures were not performed in 7% and 21%, and follow-up smears and cultures not done in 19% and 28%, respectively . The regimens prescribed were variable: 23 in 'new' and 72 in 'old' patients, 86 in total . Six-month short-course treatment using HRZE was prescribed for 26.2% of 'new' patients . In many instances, the planned treatment duration was excessive . The success rates (cured plus completed) for 'new' and 'old' patients were 74% and 51%, respectively . The failure rates were less than 1% in 'new' and 9% in 'old' patients . CONCLUSION: Prescribed regimens were variable in terms of drug combinations and treatment duration . Overall treatment outcome was inferior to that of the health centres under the NTP. Med Hypotheses, 1999 Jun, 52(6), 583 - 93 Tuberculosis I: a conceptual frame for the immunopathology of the disease; Maes HH et al.; An analysis of the cellular and humoral immune responses after bacille Calmette-Guerin (BCG) vaccination and during tuberculosis treatment favors the hypothesis of an immune defence developed in four overlapping successive stages . The initial immune response is innate . The following two intermingle innate and specific responses against low molecular weight oligopeptidic and nonpeptidic antigens, as muramyldipeptide and trehalose dimycolate, and large molecular weight nonpeptidic antigens such as lipoarabinomannan . The ultimate specific response is directed against protein antigens as Antigen 60 . BCG and primary tuberculosis (TB) infections induce cellular and humoral immune responses essentially against oligopeptidic and small and large molecular weight nonpeptidic antigens . Immune responses against non-peptidic substances contribute to the immunoprotection of the infected person who develops a primary infection . Some infected people allow the expression of the immunosuppressive activity of the pathogen . This results in the synthesis of interleukin-10 (IL-10), which suppresses the formation of interferon-gamma (INF-gamma) and IL-2, and of IL-6, which suppresses T-cell responses . These patients have a skewed immune response against non-peptidic antigens and present with symptoms . They will not recover unless responses directed against proteinic antigens occur, which restore INF-gamma and IL-2 production . The formation of immumoglobulin-G (IgG)-type antibodies and of a cellular immunity against mycobacterial peptidic antigens is essential for a good protection against a post-primary infection. Immunology, 1999 Aug, 97(4), 626 - 33 Enhanced antigen-presenting activity and tumour necrosis factor-alpha-independent activation of dendritic cells following treatment with Mycobacterium bovis bacillus Calmette-Guérin; Kim KD et al.; Dendritic cells (DCs) are most potent among the antigen-presenting cells and are believed to be crucial for the initiation of a primary T-cell response to foreign antigens . Mycobacterial infection within macrophages is controlled by cell-mediated immunity . To elucidate the stimulation of immune response by Mycobacterium bovis bacillus Calmette-Guerin (BCG), we purified DCs from precursor cells in human peripheral blood mononuclear cells (PBMC) by culturing them with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) and characterized their surface antigen expression . The interaction of cultured DCs with BCG resulted in increased surface expression of several DC-related marker antigens . BCG also induced reduction of endocytosis, enhancement of CD83 expression as well as B7 costimulatory molecules and IL-12 production, suggesting that BCG treatment directly induces DCs to mature . BCG-treated DCs were much more potent antigen-presenting cells in allogeneic immune response than untreated DCs . Moreover, while the neutralization of tumour necrosis factor-alpha (TNF-alpha) significantly blocked the DC maturation induced by lipopolysaccharide (LPS), it could not inhibit the induction of DC maturation by the BCG treatment, indicating that TNF-alpha production plays a minor role in the BCG-induced DC maturation . However, the neutralization of TNF-alpha resulted in decreased IL-12 production by activated DCs . These results suggest that infection with BCG might evoke direct activation and maturation of DC and the general immune stimulant effect of BCG might be related with the activation of DCs. Immunology, 1999 Aug, 97(4), 573 - 81 Characterization of the memory/activated T cells that mediate the long-lived host response against tuberculosis after bacillus Calmette-Guérin or DNA vaccination; Silva CL et al.; The memory/activated T cells, which mediate the long-lived host response against tuberculosis, in mice immunized with either bacillus Calmette-Guerin (BCG) or mycobacterium heat-shock protein 65 (hsp 65) antigen expressed from plasmid DNA (DNA-hsp 65), were characterized . Protection against Mycobacterium tuberculosis challenge by DNA-hsp 65 vaccination was associated with the presence of lymph node T-cell populations in which CD8+/CD44hi interferon-gamma (IFN-gamma)-producing/cytotoxic cells were prominent even after 8 or 15 months of plasmid DNA-mediated immunizations, whereas after BCG vaccination the majority were CD4+/CD44lo IFN-gamma-producing T cells . When the cells were separated into CD4+CD8- and CD8+CD4- and then into CD44hi and CD44lo types, CD44lo cells were essentially unable to transfer protection in adoptive transfer experiments, the most protective CD44hi cells were CD8+CD4- and those from DNA-vaccinated mice were much more protective than those from BCG-immunized mice . The frequency of protective T cells and the level of protection were increased up to 8 months and decreased after 15 months following DNA or BCG immunizations. Virus Genes, 1999, 18(3), 277 - 83 Analysis of the sequence of dioscorea Alata bacilliform virus: comparison to others members of the badnavirus group; Briddon RW et al.; The complete nucleotide sequence of the genome of Dioscorea alata bacilliform virus (DaBV) has been determined from cloned fragments . Features of the genome confirm DaBV to be a pararetrovirus of the genus Badnavirus which is more similar to other mealy-bug transmitted badnaviruses, in particular to cacao swollen shoot virus, than to rice tungro bacilliform virus . Sequence variability between cloned fragments suggests that the genetic variability of the virus may be quite high (up to 11% nucleotide sequence variation for some small regions of the genome) although the overall variability detected was 4.2% at the nucleotide level. J Food Prot, 1999 Aug, 62(8), 958 - 61 Thermal inactivation kinetics of Bacillus stearothermophilus spores using a linear temperature program; Leontidis S et al.; A systematic study of the inactivation kinetics of Bacillus stearothermophilus spores was carried out in nonisothermic heating conditions using a linear temperature increase program and analyzing the experimental data by means of a one-step nonlinear regression . The D and z values estimated are close to those obtained in isothermic conditions and estimated by using a two-step model, first D values are calculated, and then in the second step a z value is deduced (D(121 degrees C) = 3.08 and 4.38 min, respectively, and z = 7 and 7.9 degrees C, respectively) . No convergence problems were observed when using the one-step nonlinear regression proposed . The results indicated that the methodology applied in this study can be used to obtain kinetic data for bacterial spores, which could mean a significant reduction in the amount of experimental work employed to generate these data. J Food Prot, 1999 Aug, 62(8), 877 - 82 The microbiological quality of cooked rice from restaurants and take-away premises in the United Kingdom; Nichols GL et al.; The microbiological quality of 4,162 samples of cooked rice from restaurants and take-away premises in the United Kingdom was examined, including ready-to-eat rice purchased at point-of-sale and rice that was stored precooked for reheating on demand . The majority of point-of-sale cooked rice samples (1,855 of 1,972; 94%) were of acceptable microbiological quality, but 15 (1%) samples were of unacceptable quality (Bacillus spp . and B . cereus, > or = 10(5) CFU/g; Escherichia coli, > or = 10(4) CFU/g), indicating a potential risk to health . The prevalence of Bacillus spp., B . cereus, and E . coli was significantly greater in precooked stored rice than in point-of-sale cooked rice (P < 0.005 to 0.0005) . Bacillus spp . (> or = 10(4) CFU/g), B . cereus (> or = 10(4) CFU/g), and E . coli (> or = 10(2) CFU/g) were present in 7%, 2%, and 9% of precooked stored samples, respectively, compared to 2%, 0.5%, and 1%, respectively in point-of-sale samples . Although final heating at the point of sale reduces the levels of microorganisms present in rice it will not inactivate the B . cereus emetic toxin if present . Rice from Indian premises was of poorer microbiological quality than that from Chinese and other premises . Although most point-of-sale cooked rice samples (94%) were of an acceptable microbiological quality, evidence from this study indicates that the microbiological quality of cooked rice sold from certain outlets in the UK is of concern. J Ind Microbiol Biotechnol, 1999 Jun, 22(6), 565 - 574 Antibiotic MIC/MBC analysis of Bacillus-based commercial insecticides: use of bioreduction and DNA-based assays; Seligy V V et al.; Minimum inhibitory concentration (MIC) assays, monitored by colony counts, growth (turbidity) and bioreduction of non-toxic XTT {2,3-bis (2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxanilide, inner salt}, were used to assess the performance of several types of antibiotics against: (1) commercial BT products made from scale-up sporulation phase cultures of Bacillus thuringiensis subsp israelensis (Bti) and subsp kurstaki (Btk); (2) vegetative cells derived from these BT products; and (3) Gram-positive and Gram-negative bacteria used as controls . The XTT-kinetic assay improved sensitivity and early reading of MIC breakpoints . The conventional colony count method for determining minimal bactericidal concentration (MBC) was used to validate a multi-sample dot-blot assay in which organisms in individual MIC assays are trapped free of residual antibiotic and their viability is estimated by in situ conversion of MTT {3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide} to insoluble formazan . Tolerance (MBC/MIC) for most antibiotics was low (</=4) . Resistance to beta-lactams was attributed to beta-lactamase activity in both BT products and cultures derived from them . MIC and MBC breakpoints in spore-based assays were also approximated by changes in genome copy, using delta-endotoxin and beta-lactamase genes as probes . The DNA assays are effective for monitoring and authenticating organisms in microbe-containing biotechnology products. J Mol Biol, 1999 Aug 27, 291(4), 843 - 55 Mutational analysis of the thermostable arginine repressor from Bacillus stearothermophilus: dissecting residues involved in DNA binding properties; Karaivanova IM et al.; Recently the crystal structure of the DNA-unbound form of the full-length hexameric Bacillus stearothermophilus arginine repressor (ArgR) has been resolved, providing a possible explanation for the mechanism of arginine-mediated repressor-operator DNA recognition . In this study we tested some of these functional predictions by performing site-directed mutagenesis of distinct amino acid residues located in two regions, the N-terminal DNA-binding domain and the C-terminal oligomerization domain of ArgR . A total of 15 mutants were probed for their capacity to repress the expression of the reporter argC - lacZ gene fusion in Escherichia coli cells . Substitutions of highly conserved amino acid residues in the alpha2 and alpha3 helices, located in the winged helix-turn-helix DNA-binding motif, reduced repression . Loss of DNA-binding capacity was confirmed in vitro for the Ser42Pro mutant which showed the most pronounced effect in vivo . In E . coli, the wild-type B . stearothermophilus ArgR molecule behaves as a super-repressor, since recombinant E . coli host cells bearing B . stearothermophilusargR on a multicopy vector did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when l -arginine was supplied . All mutants affected in the DNA-binding domain lost this super-repressor behaviour . Replacements of conserved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either derepression or stronger super-repression . Substitution of Leu87 by phenylalanine was found to increase the DNA-binding affinity and the protein solubility in the context of a double Leu87Phe/Leu94Val mutant . Structural modifications occasioned by the various amino acid substitutions were confirmed by circular dichroism analysis and structure modelling . Insect Biochem Mol Biol, 1999 Aug, 29(8), 711 - 21 Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae); Silva-Filha MH et al.; The binary toxin (Bin) from Bacillus sphaericus crystals specifically binds to soluble midgut brush border membrane proteins from Culex pipiens larvae . A single 60 kDa midgut membrane protein is identified as the binding protein . This protein is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol specific-phospholipase C (PI-PLC) . Fractionation of soluble proteins by anion exchange chromatography indicates that the binding protein does not co-elute with leucine aminopeptidase activity . After partial purification, the sequences of internal amino acid fragments of the 60 kDa protein were determined . The peptide sequences were compared with data in GenBank, and showed a very high degree of similarity with enzymes belonging to the alpha-amylase family . Further enzymatic investigation showed that the receptor of the Bin toxin in C . pipiens larval midgut may be an alpha-glucosidase. Eye, 1999 Apr, 13 ( Pt 2), 226 - 30 Disinfection of contact lenses without tap water rinsing: is it effective? Seal DV, Dalton A, Doris D. PURPOSE: To establish the efficacy of the two most popular contact lens disinfecting systems--one-step hydrogen peroxide and multipurpose disinfecting solution--for 1 month's use in practice in the absence of tap water rinsing . METHODS: This was a descriptive, prospective microbiological study of contact lens contamination with ideal hygiene compliance and new lenses and storage cases . One hundred and fifty contact lens wearers were instructed to avoid risk factors identified for Acanthamoeba infection . They were randomly assigned to use one of three disinfecting systems and taught to follow manufacturers' instructions . In addition, they were taught to avoid all use of tap water for contact lens hygiene, except for hand washing . RESULTS: There was no isolation of Acanthamoeba from any lens storage case, precluding the chance of amoebic infection . The multi-purpose solution gave the lowest rate of bacterial contamination, with 78% sterility and 15% of cases with < 10(4) bacteria/ml . For both one-step peroxide and multi-purpose solutions, Gram-negative bacteria were reduced in frequency compared with values expected historically, while Bacillus sp . were found more frequently . Storage cases of both one-step peroxide systems leaked fluid . CONCLUSIONS: On the basis of contamination in previous studies, when hydrogen peroxide and other chemical disinfectants were used together with tap water washing, it was expected that approximately 40% of lens storage cases would yield bacteria, often with a high count, and that up to 8% would yield Acanthamoeba . Such contamination did not occur, however, in this study . The multipurpose solution, for 1 month's use, gave the lowest rate of bacterial contamination with only 7% of storage cases harbouring bacteria at > 10(4)/ml and with 78% sterility . One of the two one-step hydrogen peroxide systems performed equally well . Importantly, Acanthamoeba was not isolated from any of the 150 storage cases . Whether lens storage cases need to be sterile or contain < 10(3) bacteria/ml solution within them is debatable, but it is essential that Acanthamoeba be absent from them. J Clin Microbiol, 1999 Sep, 37(9), 2760 - 5 Fluorescence In situ hybridization assay using peptide nucleic acid probes for differentiation between tuberculous and nontuberculous mycobacterium species in smears of mycobacterium cultures; Stender H et al.; TB PNA FISH is a new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes for differentiation between species of the Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) in acid-fast bacillus-positive (AFB+) cultures is described . The test is based on fluorescein-labelled PNA probes that target the rRNA of MTC or NTM species applied to smears of AFB+ cultures for microscopic examination . Parallel testing with the two probes serves as an internal control for each sample such that a valid test result is based on one positive and one negative reaction . TB PNA FISH was evaluated with 30 AFB+ cultures from Denmark and 42 AFB+ cultures from Thailand . The MTC-specific PNA probe showed diagnostic sensitivities of 84 and 97%, respectively, and a diagnostic specificity of 100% in both studies, whereas the NTM-specific PNA probe showed diagnostic sensitivities of 91 and 64%, respectively, and a diagnostic specificity of 100% in both studies . The low sensitivity of the NTM-specific PNA probe in the Thai study was due to a relatively high prevalence of Mycobacterium fortuitum, which is not identified by the probe . In total, 63 (87%) of the cultures were correctly identified as MTC (n = 46) or NTM (n = 17), whereas the remaining 9 were negative with both probes and thus the results were inconclusive . None of the samples were incorrectly identified as MTC or NTM; thus, the predictive value of a valid test result obtained with TB PNA FISH was 100%. Curr Opin Biotechnol, 1999 Aug, 10(4), 349 - 52 Protein engineering of alpha-amylase for low pH performance; Shaw A et al.; Industrial-scale starch liquefaction is currently constrained to operating at pH 6.0 and above, as the enzyme used in the process, Bacillus licheniformis alpha-amylase, is unstable at lower pH under the conditions used . There is a need to develop an enzyme that can operate at lower pH . Recent progress has been made in engineering the B . licheniformis enzyme for improved industrial performance . The availability of crystal structures and subsequent analysis of improved variants, in a structural context, is revealing common factors and a rationale to make further improvements. Immunology, 1999 Jul, 97(3), 515 - 21 A recombinant BCG vaccine generates a Th1-like response and inhibits IgE synthesis in BALB/c mice; Kumar M et al.; The tubercle vaccine, bacille Calmette-Guerin (BCG), is a strong inducer of T-helper type 1 (Th1) responsiveness, and it has been suggested that recombinant BCG (rBCG), which produces and secretes antigens, may be used to prevent allergic diseases . The effects of rBCG vaccination on allergic responses in a murine model were examined in this study . A BCG-Escherichia coli shuttle vector was developed with the promoter and signal sequence of the alpha-antigen of Mycobacterium bovis, and the vector was tested using E . coli beta-galactosidase as the model antigen and allergen . This vector enabled the expression of the E . coli beta-galactosidase gene in BCG, which was detected in its protein extract by immunoblotting analysis . Vaccination of mice with a single dose of 106 recombinant BCG generated a beta-galactosidase-specific antibody response . The splenocytes of vaccinated mice compared with controls produced significantly higher amounts of interferon-gamma (IFN-gamma) (P<0 . 01) and interleukin-2 (IL-2) (P<0.05) and lower amounts of IL-5 (P<0 . 01) . Mice vaccinated with rBCG had significantly less (P<0.01) serum IgE compared with controls . These results together demonstrate that rBCG secreting antigens or allergens may be utilized for the induction of a Th1-like response and the down-regulation of IgE antibody response. Biochim Biophys Acta, 1999 Aug 17, 1433(1-2), 294 - 306 Thermal unfolding of phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase studied by differential scanning calorimetry; Levashov P et al.; Thermal unfolding parameters were determined for a two-domain tetrameric enzyme, phosphorylating D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and for its isolated NAD(+)-binding domain . At pH 8.0, the transition temperatures (t(max)) for the apoforms of the native Bacillus stearothermophilus GAPDH and the isolated domain were 78.3 degrees C and 61.9 degrees C, with calorimetric enthalpies (DeltaH(cal)) of 4415 and 437 kJ/mol (or 30.7 and 22.1 J/g), respectively . In the presence of nearly saturating NAD(+) concentrations, the t(max) and the DeltaH(cal) increased by 13.6 degrees C and by 2365 kJ/mol, respectively, for the native apoenzyme, and by 2.8 degrees C and 109 kJ/mol for the isolated domain . These results indicate that interdomain interactions are essential for NAD(+) to produce its stabilizing effect on the structure of the native enzyme . The thermal stability of the isolated NAD(+)-binding domain increased considerably upon transition from pH 6.0 to 8.0 . By contrast, native GAPDH exhibited greater stability at pH 6.0; similar pH-dependencies of thermal stability were displayed by GAPDHs isolated from rabbit muscle and Escherichia coli . The binding of NAD(+) to rabbit muscle apoenzyme increased t(max) and DeltaH(cal) and diminished the widths of the DSC curves; the effect was found to grow progressively with increasing coenzyme concentrations . Alkylation of the essential Cys149 with iodoacetamide destabilized the apoenzyme and altered the effect of NAD(+) . Replacement of Cys149 by Ser or by Ala in the B . stearothermophilus GAPDH produced some stabilization, the effect of added NAD(+) being basically similar to that observed with the wild-type enzyme . These data indicate that neither the ion pairing between Cys149 and His176 nor the charge transfer interaction between Cys149 and NAD(+) make any significant contribution to the stabilization of the enzyme's native tertiary structure and the accomplishment of NAD(+)-induced conformational changes . The H176N mutant exhibited dramatically lower heat stability, as reflected in the values of both DeltaH(cal) and t(max) . Interestingly, NAD(+) binding resulted in much wider heat capacity curves, suggesting diminished cooperativity of the unfolding transition. J Enzyme Inhib, 1999, 14(3), 175 - 92 Molecular modelling of lanosterol 14 alpha-demethylase (CYP51) from Saccharomyces cerevisiae via homology with CYP102, a unique bacterial cytochrome P450 isoform: quantitative structure-activity relationships (QSARs) within two related series of antifungal azole derivatives; Lewis DF et al.; The construction of a three-dimensional molecular model of the fungal form of cytochrome P450 (CYP51) from Saccharomyces cerevisiae, based on homology with the haemoprotein domain of CYP102 from Bacillus megaterium (a unique bacterial P450 of known crystal structure) is described . It is found that the endogenous substrate, lanosterol, can readily occupy the putative active site of the CYP51 model such that the known mono-oxygenation reaction, leading to C14-demethylation of lanosterol, is the preferred route of metabolism for this particular substrate . Key amino acid contacts within the CYP51 active site appear to orientate lanosterol for oxidative attack at the C14-methyl group, and the position of the substrate relative to the haem moiety is consistent with the phenyl-iron complexation studies reported by Tuck et al . {J . Biol . Chem., 267, 13175-13179 (1992)} . Typical azole inhibitors, such as ketoconazole, are able to fit the putative active site of CYP51 by a combination of haem ligation, hydrogen bonding, pi-pi stacking and hydrophobic interactions within the enzyme's haem environment . The mode of action of azole antifungals, as described by the modelling studies, is supported by quantitative structure-activity relationship (QSAR) analyses on two groups of structurally related fungal inhibitors . Moreover, the results of molecular electrostatic isopotential (EIP) energy calculations are compatible with the proposed mode of binding between azole antifungal agents and the putative active site of CYP51, although membrane interactions may also have a role in the antifungal activity of azole derivatives. J Pept Sci, 1999 Jul, 5(7), 289 - 97 Preparation and characterization of novel bioactive peptides responsible for angiotensin I-converting enzyme inhibition from wheat germ; Matsui T et al.; Reported is the preparation of wheat germ (WG) hydrolyzate with potent angiotensin I-converting enzyme (ACE) inhibitory activity, and the characterization of peptides responsible for ACE inhibition . Successful hydrolyzate with the most potent ACE inhibitory activity was obtained by 0.5 wt.%-8 h Bacillus licheniformis alkaline protease hydrolysis after 3.0 wt.%-3 h alpha-amylase treatment of defatted WG (IC50; 0.37 mg protein ml(-1)) . The activity of WG hydrolyzate was markedly increased by ODS and subsequent AG50W purifications (IC50; 0.018 mg protein ml(-1)) . As a result of isolations by high performance liquid chromatographies, 16 peptides with the IC50 value of less than 20 microM, composed of 2-7 amino acid residues were identified from the WG hydrolyzate . Judging from the high content (260 mg in 100 g of AG50W fraction) and powerful ACE inhibitory activity (IC50; 0.48 microM), Ile-Val-Tyr was identified as a main contributor to the ACE inhibition of the hydrolyzate. Curr Microbiol, 1999 Sep, 39(3), 159 - 62 Differential activity and activation of Bacillus thuringiensis insecticidal proteins in diamondback moth, Plutella xylostella; Monnerat R et al.; Whole-crystal preparations from strains HD-1 and HD-133, activated Cry1Ab and Cry1C toxins as well as Cry1Aa, Cry1Ac, Cry1D, and Cry2Aa protoxins were tested for toxicity to 2nd-instar larvae of the diamondback moth, Plutella xylostella . Mortality data recorded after 2 and 5 days provided different results that were related to differential rates of solubilization, activation, and degradation of insecticidal crystal proteins . The two most active proteins are Cry1Ab and Cry1C, which are both present in HD-133 . The Cry1Ab protoxin is activated within 2 days, whereas activation of the Cry1C protoxin occurs between 2 and 5 days . HD-133 is more active than HD-1 immediately after infection and remains toxic over 5 days owing to the sequential activation of its crystal components . Solubility properties of crystals and rates of activation of protoxins influence the overall toxicity of HD-1 and HD-133 to the diamondback moth. Curr Microbiol, 1999 Sep, 39(3), 134 - 40 Purification and characterization of chitinase from Bacillus circulans No.4.1; Wiwat C et al.; Bacillus circulans No.4.1 produced a high level of chitinase when cells were grown in tryptic soy broth supplemented with 0.3% colloidal chitin at 35 degrees C for 5 days . Purification was carried out by protein precipitation with 80% saturation ammonium sulfate, anion-exchange chromatography with DEAE-Sephacel, and gel filtration with Sephadex G-100, sequentially . The purified enzyme could be demonstrated as a single band on SDS-PAGE, estimated to be 45 kDa . This enzyme could hydrolyze colloidal chitin, purified chitin, glycol chitin, carboxymethyl-chitin (CM-chitin), and 4-methylumbelliferyl-beta-D-N,N'-diacetylchitobioside {4-MU-(GlcNAc)(2)} . The optimal conditions for this chitinase were pH 8.0 and 40 degrees C . The isoelectric point of the chitinase was 5.1 . The amino acid composition of the purified chitinase was determined . The initial 20 amino acid residues of the N-terminal were found to be alanine (A), proline (P), tryptophan (W), asparagine (N), serine (S), lysine (K), glycine (G), asparagine (N), tyrosine (Y), alanine (A), leucine (L), proline (P), tyrosine (Y), tyrosine (Y), arginine (R), glycine (G), alanine (A), tryptophan (W), alanine (A), and valine (V) . Knowledge of these properties of chitinase from B . circulans No . 4.1 should be useful in the development of genetically engineered Bacillus sp . as biopesticides. Arch Biochem Biophys, 1999 Aug 15, 368(2), 367 - 74 Unsaturated glucuronyl hydrolase of Bacillus sp . GL1: novel enzyme prerequisite for metabolism of unsaturated oligosaccharides produced by polysaccharide lyases; Hashimoto W et al.; The bacterium Bacillus sp . GL1 assimilates two kinds of heteropolysaccharides, gellan and xanthan, by using extracellular gellan and xanthan lyases, respectively, and produces unsaturated saccharides as the first degradation products . A novel unsaturated glucuronyl hydrolase (glycuronidase), which was induced in the bacterial cells grown on either gellan or xanthan, was found to act on the tetrasaccharide of unsaturated glucuronyl-glucosyl-rhamnosyl-glucose produced from gellan by gellan lyase, and the enzyme and its gene were isolated from gellan-grown cells . The nucleotide sequence showed that the gene contained an ORF consisting of 1131 base pairs coding a polypeptide with a molecular weight of 42,859 . The purified enzyme was a monomer with a molecular mass of 42 kDa and was most active at pH 6.0 and 45 degrees C . Because the enzyme can act not only on the gellan-degrading product by gellan lyase, but also on unsaturated chondroitin and hyaluronate disaccharides produced by chondroitin and hyaluronate lyases, respectively, it is considered that the unsaturated glucuronyl hydrolase plays specific and ubiquitous roles in the degradation of oligosaccharides with unsaturated uronic acid at the nonreducing terminal produced by polysaccharide lyases . Biotechnol Prog, 1999 Jul-Aug, 15(4), 640 - 5 Production of alpha-amylase in fed-batch cultures of vgb+ and vgb- recombinant Escherichia coli: some observations; Enayati N et al.; Synthesis and excretion of Bacillus stearothermophilus alpha-amylase is analyzed in fed-batch cultivations of Escherichia coli JM103{pMK79} and E . coli JM103{pMK57}, the former strain containing the plasmid-encoded Vitreoscilla hemoglobin (VHb) gene (vgb) and the latter strain being devoid of this gene . Fed-batch operation is observed to be substantially superior to batch operation as concerns the alpha-amylase production rate and the extent of excretion of the enzyme . Faster feeding of a nutrient medium (LB or M9) discourages synthesis of alpha-amylase . While synthesis of alpha-amylase in the vgb(-) strain is discouraged when oxygen availability is reduced, the reverse is the case with the vgb(+) strain, the promotion of alpha-amylase synthesis in the latter strain being linked to the synthesis of VHb . Increased availability of the principal carbon source (glucose) in a defined medium leads to overproduction of both alpha-amylase and VHb under oxygen limitation, which may be responsible for the segregational instability observed with the vgb(+) strain . The very high extents of excretion of alpha-amylase attained in fed-batch cultures are encouraging for downstream processing of the recombinant protein. J Bacteriol, 1999 Aug, 181(16), 4780 - 9 Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by allelic exchange; Pavelka MS Jr et al.; Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange . Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guerin (BCG) and M . tuberculosis . In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M . bovis BCG and M . tuberculosis H37Rv . In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient . The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis . We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria . The M . smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium . The M . tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80 . The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism. Nat Toxins, 1998, 6(6), 219 - 33 Fine structural changes in the ileum of mice fed on delta-endotoxin-treated potatoes and transgenic potatoes; Fares NH et al.; The present work has been designed to study the effect of feeding on transgenic potatoes, which carry the CryI gene of Bacillus thuringiensis var . kurstaki strain HD1, on the light and electron microscopic structure of the mice ileum, in comparison with feeding on potatoes treated with the 'delta-endotoxin' isolated from the same bacterial strain . The microscopic architecture of the enterocytes of the ileum of both groups of mice revealed certain common features such as the appearance of mitochondria with signs of degeneration and disrupted short microvilli at the luminal surface . However, in the group of mice fed on the 'delta-endotoxin', several villi appeared with an abnormally large number of enterocytes (151.8 in control group versus 197 and 155.8 in endotoxin and transgenic-treated groups, respectively) . Fifty percent of these cells were hypertrophied and multinucleated . The mean area of enterocyte was significantly increased (105.3 microm(2) in control group versus 165.4 microm(2) and 116.5 microm(2) in endotoxin and transgenic-treated groups, respectively) . Several forms of secondary lysosomes or auotophagic vacuoles were recognized in these cells . These changes were confirmed with the scanning electron microscope which revealed a remarkable increase in the topographic contour of enterocytes (23 microm in control group versus 44 microm and 28 microm in endotoxin and transgenic-treated groups, respectively) at the divulged surface of the villi . The basal lamina along the base of the enterocytes was damaged at several foci . Several disrupted microvilli appeared in association with variable-shaped cytoplasmic fragments . Some of these fragments contained endoplasmic reticulum, as well as ring-shaped annulate lamellae . In addition, the Paneth cells were highly activated and contained a large number of secretory granules . These changes may suggest that delta-endotoxin-treated potatoes resulted in the development of hyperplastic cells in the mice ileum . Although mild changes are reported in the structural configuration of the ileum of mice fed on transgenic potatoes, nevertheless, thorough tests of these new types of genetically engineered crops must be made to avoid the risks before marketing . Microbiology, 1999 Jul, 145 ( Pt 7), 1575 - 83 The diacetamidodideoxyuronic-acid-containing glycan chain of Bacillus stearothermophilus NRS 2004/3a represents the secondary cell-wall polymer of wild-type B . stearothermophilus strains; Schaffer C et al.; The diacetamidodideoxymannuronic-acid-containing glycan of Bacillus stearothermophilus NRS 2004/3a with the repeating unit structure {-->4)-beta-D-ManpA2,3(NAc)2-(1-->6)-alpha-D-Glcp-(1-->4)-beta-D-+ ++ManpA2,3 (NAc)2-(1-->3)-alpha-D-GlcpNAc-(1-->}, was examined to identify its linkage to the bacterial cell wall . In a previous paper it was suggested that this glycan is covalently linked to the surface layer (S-layer) glycoprotein of that organism . By improved chromatographic techniques (gel permeation over Sephacryl S-1000 SF; C4 reversed-phase HPLC) the diacetamidodideoxyuronic-acid-containing material was completely separated from the S-layer glycoprotein . This implicates only low, if any, specific affinity between these cell-wall components . To obtain sufficient amounts for the chemical characterization of its linkage region, the identical diacetamidodideoxyuronic-acid-containing material was isolated from sonicated cells of that organism by a purification procedure different to that for preparation of S-layers . This method allowed collection of the intact molecule including its linkage region . From the combined results of the chemical characterization and 600 MHz NMR spectroscopy it is proposed that the diacetamidodideoxyuronic-acid-containing glycan chain, consisting of approximately six tetrasaccharide repeating units, is directly linked via a pyrophosphate bridge to carbon 6 of muramic acid residues of the peptidoglycan sacculus . About 20-25% of the muramic acid residues are substituted with these polysaccharide chains . Thus, the diacetamidodideoxyuronic-acid-containing glycan represents a secondary cell-wall polymer of B . stearothermophilus NRS 2004/3a. Microbiology, 1999 Jul, 145 ( Pt 7), 1563 - 73 An unusual cytochrome o'-type cytochrome c oxidase in a Bacillus cereus cytochrome a3 mutant has a very high affinity for oxygen; Contreras ML et al.; Bacillus cereus strain PYM1 is a mutant unable to synthesize haem A or spectrally detectable cytochromes aa3 or caa3 . The nature of the remaining oxidase(s) catalysing oxygen uptake has been studied . Respiratory oxidase activities and the levels of cytochromes b and c increased 2.6- to 4.2-fold on transition from exponential growth, in either of two media, to sporulation stage III, as previously observed for the parent wild-type strain . NADH oxidase activity at both stages of culture was several-fold higher than ascorbate plus tetramethyl-p-phenylenediamine (TMPD) oxidase activity, consistent with the TMPD- phenotype of strain PYM1 . Oxidase activity with ascorbate as substrate was significant even in the absence of TMPD as electron mediator, suggesting that the terminal oxidase receives electrons from a cytochrome c . Carbon monoxide (CO) difference spectra of membranes were obtained using various reductants (ascorbate +/- TMPD, NADH, dithionite) and revealed a haemoprotein resembling cytochrome o' . The CO complex of this cytochrome was photodissociable: the photodissociation spectrum (photolysed minus CO-ligated) exhibited a trough at 416 nm and a peak at 436 nm, together with minor features in the alpha/beta region of the spectrum, consistent with the presence of a cytochrome o'-like pigment . CO recombination occurred at -85 to -95 degrees C . No other haemoproteins showing photoreversible CO binding under these conditions were detected . Evidence that this pigment was the oxidase responsible for substrate oxidation was obtained by photodissociating the CO complex at subzero temperatures in the presence of oxygen; this resulted in faster ligand recombination, attributed to oxygen binding, and extensive oxidation of cytochromes c and b . The oxygen affinity of the oxidase was determined by using the deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissociated oxygen concentration . A single oxidase was revealed with a K(m) for oxygen of about 8 nM; this is one of the highest affinities yet reported for a terminal oxidase. J Immunol, 1999 Aug 15, 163(4), 2249 - 55 Newborns develop a Th1-type immune response to Mycobacterium bovis bacillus Calmette-Guérin vaccination; Marchant A et al.; Data obtained in animals indicate that neonatal immune responses are biased toward Th2 . This could reduce the efficacy of vaccines against viral and mycobacterial diseases . The ability of human newborns to develop a Th1 immune response upon immunization has not been studied . Since the vaccine Mycobacterium bovis bacillus Calmette-Guerin (BCG) triggers a Th1-type response in adults, we investigated whether it induces a similar response in newborns and whether age at vaccination influences immunogenicity . We found that BCG vaccination at birth induces a memory Th1-type response of similar magnitude to that when given later in life . This study demonstrates that human newborns can be immunized against pathogens controlled by a Th1 immune response. J Mol Biol, 1999 Jul 30, 290(5), 917 - 28 The switch from early to late transcription in phage GA-1: characterization of the regulatory protein p4G; Horcajadas JA et al.; The transcription program of the Bacillus phage GA-1, a distant relative of phage Phi29, has been studied . Transcription of the GA-1 genome occurred in two stages, early and late . Early genes were expressed from two promoters equivalent to the Phi29 A2b and A2c promoters, whereas late transcription started at a site equivalent to the Phi29 late A3 promoter . The activity of the GA-1 early A2b and A2c promoters diminished 10 minutes after infection, a time at which expression of the late promoter increased significantly . The switch from early to late transcription required protein synthesis, suggesting the need for viral protein(s) . An open reading frame was found in the GA-1 genome coding for a protein showing a 53 % similarity to Phi29 regulatory protein p4, and was named p4G . In Phi29, protein p4 represses the early A2b and A2c promoters and activates the late A3 promoter by recruiting RNA polymerase to it . A binding site for protein p4Gwas localized upstream from the GA-1 late A3 promoter, overlapping with the early A2b promoter . In vitro, protein p4Gprevented the binding of RNA polymerase to the GA-1 early A2b promoter but, unlike in Phi29, had no effect on the expression of the late A3 promoter: RNA polymerase could efficiently bind and initiate transcription from the A3 promoter in the absence of protein p4G . Therefore, activation of late transcription occurs differently in GA-1 and Phi29 . We propose that protein p4Gis an anti-repressor which inhibits the binding to the late promoter of an unknown repressor factor present in the host strain . Zhonghua Nei Ke Za Zhi, 1997 Oct, 36(10), 661 - 4 {A study on the effects of three kinds of treatment in chronic hepatitis B}; Si C et al.; To study the effects of three kinds of treatment: Zhuling polysaccharides combined with hepatitis B vaccine (group I), LAK cells, induced by interleukin-2 (IL-2) in vitro, transfusion therapy, (group II), bacille Calmette-Guerin (BCG) combined with persatin (group III) in chronic hepatitis B and the mechanism of their effects, we observed 286 patients with chronic hepatitis B, diagnosed according to the criteria made by The 6th National Meeting for Viral Hepatitis and Substantiated by the results of liver biopsy . The patients were divided into 3 treatment groups randomly, with interferon 3 MIU, thrice a week and 10% glucose 500 ml (i.v.) dripping everyday as two different control groups . The ALT recovery rates in group I, II, III were 64%, 35% and 57%, respectively . At the end of the treatment, the HBeAg and HBV DNA clearence rates in group I, II, III were 43% and 44%, 34% and 30%, 57% and 61%, respectively . After one year of follow up, the HBeAg and HBV DNA clearence rates in group I, II, III were 59%, 58%, 61% and 55%, 70%, 60%, respectively . The effects of these kinds of treatment were similar to these of interferon . We also injected Zhuling polysaccharides combined with h