Bacillus

J Eukaryot Microbiol, 1993 May-Jun, 40(3), 336 - 40
Lipophosphoglycan antigen shedding by Leishmania donovani; Kaneshiro ES et al.; The biochemical characterizations of lipophosphoglycans from various Leishmania species reported by other workers may or may not contain several types of lipophosphoglycan molecules . This is the first report in which a specific lipophosphoglycan has been defined by both its antigenic and electrophoretic properties . Furthermore, a purification procedure for this specific lipophosphoglycan is described and some biochemical characterizations are presented . Phospholipase C and the so-called phosphatidylinositol-specific phospholipase C of Bacillus cereus convert the amphipathic form of the lipophosphoglycan antigen to the hydrophilic form . Under equivalent incubation conditions, other phospholipases tested were not effective in conversion of the amphipathic to the hydrophilic form . Since the amphipathic form is present in conditioned media, antigen shedding cannot be explained by phospholipase C digestion of the amphipathic form, which would result in the release of only the hydrophilic form into the medium . Both the pellet and the supernatant fractions of conditioned media contained both forms of the antigen and did not differ in the relative amounts of the two . This observation rules out membrane blebbing as the major mechanism for the release of the amphipathic form.

J Eukaryot Microbiol, 1993 May-Jun, 40(3), 323 - 8
Antagonistic action of the bacterium Bacillus licheniformis M-4 toward the amoeba Naegleria fowleri; Cordovilla P et al.; Free-living amoebae belonging to the species Naegleria fowleri are known to be the etiological agents for a form of fulminant meningoencephalitis that is generally fatal (primary amoebic meningoencephalitis) . In a broad bacterial screening from soil and water we have isolated three strains (M-4, D-13 and A-12) belonging to the species Bacillus licheniformis that have remarkable amoebicidal activity against Naegleria sp . and also against different Gram-positive and Gram-negative bacteria . Physical-chemical characteristics, partial purification and biological activities of a substance produced by the M-4 strain have been investigated . This substance (m-4) is stable at high temperature (up to 100 degrees C) and extremes of pH (2.5-9.5) and also at -20 degrees C for months . Its production is greatly influenced by oxygenation of the cultures and is probably related to the sporulation process of the bacterium . Scanning electron microscope observations reveal that amoebae are lysed after a few minutes contact with m-4.

Plant Mol Biol, 1993 May, 22(2), 313 - 21
Genetically improved potatoes: protection from damage by Colorado potato beetles; Perlak FJ et al.; Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var . tenebrionis . A modified gene that dramatically improved plant expression of this protein was utilized . Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations . Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.

J Clin Microbiol, 1993 May, 31(5), 1136 - 42
Intracellular bacteria of porcine proliferative enteropathy: cultivation and maintenance in vitro; Lawson GH et al.; An obligate intracellular bacterium was isolated from the intestines of all 10 cases of porcine proliferative enteropathy from four different pig farms . The organism grew in a rat enterocyte cell line (IEC-18) and was maintained over 20 passages . The growth of the bacteria was assessed by immunostaining of cells exposed to infection . Infection was not associated with morphological cell change, and growth was confined to cells infected at the time of each transfer of infection and the progeny of these cells . The bacterium is a microaerophilic, cell dependent, curved or rod-shaped, gram-negative bacillus that multiplies freely in the enterocyte cytoplasm . Cell cultures containing the intracellular bacteria appear to be free of other microorganisms, including chlamydiae and viruses.

J Pediatr, 1993 May, 122(5 Pt 1), 697 - 702
Safety and immunogenicity of bacille Calmette-Guérin, diphtheria-tetanus-pertussis, and oral polio vaccines in newborn children in Zaire infected with human immunodeficiency virus type 1; Ryder RW et al.; OBJECTIVE: To determine the safety and immunogenicity of childhood vaccines in children with perinatally acquired human immunodeficiency virus type 1 (HIV-1) infection . DESIGN: Nonrandomized, prospective cohort study; 12-month follow-up period . SETTING: Obstetric wards and outpatient pediatric clinics at two large hospitals in Kinshasa, Zaire . PATIENTS: A total of 8108 pregnant women were screened for HIV-1 antibodies . The 474 children born to 466 seropositive women identified during screening and the 616 children born to 606 seronegative, age- and parity-matched women were vaccinated . INTERVENTION: The following vaccines were administered at the stated ages: bacille Calmette-Guerin (BCG) vaccine (2 days); trivalent oral Sabin poliomyelitis vaccine (2 days and 6, 10, and 14 weeks); and adsorbed diphtheria-tetanus-pertussis (DTP) vaccine (6, 10, and 14 weeks) . MEASUREMENTS AND MAIN RESULTS: Protective antibody titers to tetanus and poliovirus types 1, 2, and 3 were achieved in 95% of all children . Among children with HIV-1 infection, 70.8% had protective antibody titers to diphtheria compared with 98.5% of uninfected children (p < 0.05) . Geometric mean antibody titers to diphtheria and poliovirus types 1, 2, and 3 were significantly lower in children with HIV-1 infection than in uninfected children . Vaccine-associated side effects were similarly low in all children . CONCLUSIONS: The low incidence of side effects and the high proportion of children with HIV-1 infection who achieved protective postimmunization antibody titers support the continuing use of BCG, DTP, and oral polio vaccines in childhood immunization programs in HIV-1 endemic areas.

Curr Opin Oncol, 1993 May, 5(3), 574 - 80
Advances in bladder cancer; Ozen H; Intravesical chemotherapy and immunotherapy are administered to patients with bladder cancer in the hope of decreasing the rate of recurrence and, more importantly, reducing the rate of progression . So far, bacille Calmette-Guerin is the only agent to decrease progression rate and thus increase survival . Bacille Calmette-Guerin has been shown to achieve this outcome by selectively sparing certain sequences during fibronectin degradation; these critical fragments were shown to have motility-stimulating activity, which may then enhance tumor invasion . In vivo and in vitro studies have shown that cathepsin B inhibitors prevent degradation of human basement membrane laminin, which is the first step of tumor invasion . Cathepsin B inhibitors either alone or in combination with bacille Calmette-Guerin may be valuable in preventing the progression of superficial bladder tumors in the future . Clinical studies on bacille Calmette-Guerin have repeatedly shown that a second course of bacille Calmette-Guerin to patients with local failure did not jeopardize patient survival . Adjuvant chemotherapy for patients with locally advanced disease and for those with regionally involved lymph nodes improves progression-free survival . Further controlled studies will establish the verdict for adjuvant chemotherapy . With the advancement of molecular genetic studies relevant to modern technology, our understanding of tumor behavior improves dramatically, and classic knowledge becomes more outdated every day.

J Bacteriol, 1993 May, 175(10), 2952 - 60
Full expression of the cryIIIA toxin gene of Bacillus thuringiensis requires a distant upstream DNA sequence affecting transcription; de Souza MT et al.; The cryIIIA gene encoding a coleopteran-specific toxin is poorly expressed in Bacillus thuringiensis when cloned in a low-copy-number plasmid . This weak expression is observed when the gene is cloned only with its promoter and its putative terminator . cryIIIA gene expression was analyzed by using deletion derivatives of a larger DNA fragment carrying the toxin gene and additional adjacent sequences . The results indicate that a 1-kb DNA fragment located 400 bp upstream of the promoter strongly enhances CryIIIA production in B . thuringiensis sporulating cells . Similar results were obtained when the low-copy-number plasmid pHT304 carrying transcriptional fusions between upstream regions of cryIIIA and the lacZ gene was used . Analysis of the start sites, the sizes, and the amounts of cryIIIA-specific mRNAs shows that the enhancement occurs at the transcriptional level by increasing the number of cryIIIA-specific transcripts from the onset of sporulation to about 6 h after the onset of sporulation . The nucleotide sequence of the 1-kb activating fragment and of the 700 bp containing the promoter region and the 5' end of cryIIIA were determined . No potential protein-coding sequences were found upstream of the promoter . The major characteristic of the 1-kb activating fragment is the presence of a 220-bp A + T-rich region.

J Infect Dis, 1993 May, 167(5), 1160 - 7
The major secreted antigen complex (Ag 85) from Mycobacterium bovis bacille Calmette-Guérin is associated with protective T cells in leprosy: a follow-up study of 45 household contacts; Launois P et al.; T cell proliferation and interferon (IFN)-gamma secretion were analyzed in 45 leprosy contacts stimulated with antigen 85 (Ag85), the major culture filtrate antigen from Mycobacterium bovis bacille Calmette-Guerin . All 14 Mitsuda reaction-positive contacts reacted to Mycobacterium leprae and Ag85 . Three Mitsuda reaction-negative contacts reacted weakly to M . leprae and Ag85 . The other 28 Mitsuda reaction-negative contacts did not react to M . leprae, but 9 reacted to Ag85 . Thirty-four contacts were retested 16 months later . Eleven contacts initially positive by the Mitsuda test remained lepromin positive and reactive to M . leprae and Ag85 . Fourteen contacts initially negative by the Mitsuda test converted, and all reacted in vitro to M . leprae and Ag85 . Finally, 9 contacts remained Mitsuda test-negative, and 7 were unreactive to Ag85 . In vitro reactivity to Ag85 at baseline in Mitsuda test-negative contacts was associated with subsequent conversion to lepromin reactivity in 7 of 9 subjects . These data suggest that reactive T cells against Ag85 develop very early during M . leprae infection and that Ag85 is a potentially protective T cell immunogen.

J Appl Bacteriol, 1993 May, 74(5), 578 - 82
The application of laser diffractometry to study the water content of spores of Bacillus sphaericus with different heat resistances; De Pieri LA et al.; The distribution of water in the protoplast and integument of three populations of Bacillus sphaericus spores was determined by laser diffractometry together with the sizes of their integuments and protoplasts . The spores were produced at 15, 20 and 30 degrees C . Spores grown at 15 degrees C had protoplast and integument water contents similar to those produced at 20 degrees C, while those grown at 30 degrees C had significantly lower water contents than the other two populations . The inner (or protoplast) radii of the spores produced at 15, 20 and 30 degrees C were 0.41 +/- 0.02 microns, 0.42 +/- 0.01 microns and 0.38 +/- 0.02 microns whilst the outer (or whole spore) radii were 0.56 +/- 0.01 microns, 0.58 +/- 0.01 microns and 0.52 +/- 0.02 microns respectively . The ratios of integument to protoplast radius were 0.40 +/- 0.02, 0.43 +/- 0.07 and 0.41 +/- 0.03 respectively.

Cancer Res, 1993 May 1, 53(9), 2123 - 7
Genetic construction, expression, and characterization of a single chain anti-carcinoma antibody fused to beta-lactamase; Goshorn SC et al.; We report the genetic construction and expression of a fusion protein between an antibody single chain-linked variable domain fragment specific for human carcinomas and beta-lactamase II from Bacillus cereus . Sequences encoding the variable regions of the L6 monoclonal antibody were assembled so as to be separated from each other by an 18-amino acid linker and from the mature form of beta-lactamase by a 6-amino acid linker . The construct was placed under the transcriptional regulation of the lac promoter, and the PelB signal sequence was used to direct export of the fusion protein to the periplasmic space of Escherichia coli . After induction, biologically active material was recovered from both culture supernatants and cell lysates . Affinity chromatography yielded about 2.5 micrograms of protein/ml of initial culture volume . The fusion protein was shown to bind to tumor cells at least as well as chemically prepared F(ab') and to maintain beta-lactamase activity at a level similar to that of the native enzyme . Tumor cells coated with the fusion protein were sensitive to a cephalosporin mustard prodrug in a dose-dependent fashion comparable to that of enzyme chemically conjugated to F(ab') . This article demonstrates the feasibility of using single chain-linked variable domain-enzyme fusion proteins for the activation of anticancer prodrugs.

J Bacteriol, 1993 May, 175(9), 2762 - 6
Large-scale recrystallization of the S-layer of Bacillus coagulans E38-66 at the air/water interface and on lipid films; Pum D et al.; S-layer protein isolated from Bacillus coagulans E38-66 could be recrystallized into large-scale coherent monolayers at an air/water interface and on phospholipid films spread on a Langmuir-Blodgett trough . Because of the asymmetry in the physiochemical surface properties of the S-layer protein, the subunits were associated with their more hydrophobic outer face with the air/water interface and oriented with their negatively charged inner face to the zwitterionic head groups of the dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine (DPPE) monolayer films . The dynamic crystal growth at both types of interfaces was first initiated at several distant nucleation points . The individual monocrystalline areas grew isotropically in all directions until the front edge of neighboring crystals was met . The recrystallized S-layer protein and the S-layer-DPPE layer could be chemically cross-linked from the subphase with glutaraldehyde.

Int J Biochem, 1993 May, 25(5), 689 - 96
Proof of alkaline phosphodiesterase I as a phosphatidylinositol-anchor enzyme; Nakabayashi T et al.; 1 . Ectoenzyme release from kidney brush border membranes of Rattus norvegicus and Sus scrofa domesticus by phosphatidylinositol-specific phospholipase C (PIPLC) of Bacillus thuringiensis was studied . 2 . The levels of specific activities of ectoenzymes in R . norvegicus kidney brush border membranes were higher than those in S . scrofa domesticus . About 10-fold higher values were found for specific activities of alkaline phosphatase and gamma-glutamyl transpeptidase in R . norvegicus . 3 . Alkaline phosphodiesterase I, alkaline phosphatase and 5'-nucleotidase were released from both R . norvegicus and S . scrofa domesticus brush border membranes, while gamma-glutamyl transpeptidase and dipeptidyl peptidase IV were not solubilized . The enzyme release by the action of PIPLC was suppressed when purified anti-PIPLC antibody was added to the reaction mixture . This suggests that enzyme release must be due to the direct action of PIPLC on kidney brush border membranes . 4 . The released alkaline phosphodiesterase I from kidney of S . scrofa domesticus had a molecular weight of 240,000 and was activated by Mg2+ and Ca2+, but strongly inhibited by EDTA.

Res Immunol, 1993 May, 144(4), 233 - 43
Leishmania major infection in BALB/c mice: protection or exacerbation by treatment with different doses of BCG; Stefani MM et al.; The effect of live bacillus Calmette-Guerin (BCG), administered intraperitoneally to BALB/c mice, upon the development of lesions induced by subcutaneous infection with Leishmania major was examined . Lesions in mice given 10(7) BCG colony-forming units (CFU) 9 days before challenge with L . major were less severe and contained significantly fewer parasites than those of similarly infected control mice not given BCG . This effect of treatment with high doses of BCG upon the development of leishmanial lesions was observed using L . major promastigotes and amastigotes, whether or not 10(6) live BCG was included in the parasite inoculum . Lesions in mice given 5 x 10(4) BCG CFU 14 days before infection with L . major contained significantly fewer parasites than those of control mice not given BCG . Mice treated with low doses of BCG and infected with an L . major inoculum also comprising BCG exhibited larger lesions that contained more parasites . Interestingly, compared to naive mice infected with L . major, infection of naive mice with L . major mixed with live BCG consistently led to the development of more severe lesions that contained higher numbers of parasites . No correlation was found between the effect of BCG on the development of lesions induced by L . major and the amounts of IFN gamma, IL5 and TNF produced after in vitro antigenic challenge of either draining lymph node or spleen cells, the antigenic challenge being either live BCG or live L . major.

J Invertebr Pathol, 1993 May, 61(3), 260 - 6
Use of synthetic peptides to probe functional domains of a Bacillus thuringiensis toxin; Pang AS; Two 10-residue peptides exhibiting sequence homology to CryIA(a) toxin were chemically synthesized . One corresponds to a segment from residues 48-57 (peptide A) and is common to all CryIA toxins and the second corresponds to a segment from residues 348-357 (peptide B) and is specific to the CryIA(a) toxin . Antibodies were raised in rabbits against both peptides . Antipeptide A did not protect either Bombyx mori or Choristoneura fumiferana larvae against CryIA(a) toxin . However, antipeptide B offered significant protection to B . mori and somewhat less protection to C . fumiferana larvae . Neither antiserum interfered with the binding of CryIA(a) toxin to brush border membrane vesicles of either insect . The results suggest that the segment from residues 348-357 in CryIA(a) toxin is important for expression of toxicity in both insects but not for binding.

Mikrobiol Zh, 1993 May-Jun, 55(3), 104 - 10
{The role of Bacillus thuringiensis in natural biocenoses}; Golovko AE et al.; The results of the author's data and those from literature on ecology and genetic exchange of Bacillus thuringiensis strains have been analyzed in this brief review . It has been concluded that there is no strict confinement of B . thuringiensis strains of the particular serotypes to certain habitat and that the bacteriocinogenicity is of great importance in the intraspecies competition and stability of strains in nature . It has been emphasized that the plasmid conjugation process in B . thuringiensis can serve as a main factor of genetic variability to establish ecological placement of these bacteria.

Rinsho Byori, 1993 May, 41(5), 543 - 7
{The role of the clinical microbiology laboratory in the past}; Kudo H; Over the past 150 years, since Koch first isolated the tubercle bacillus, clinical microbiologists have used culturing techniques to demonstrate of the presence of pathogenic microorganisms associated with infectious diseases in clinical specimens . The development of medicine and chemotherapy and the improvement of sanitary conditions resulted in a marked decrease in infectious diseases . In stead of infections due to virulent pathogens, opportunistic infections in the compromised hosts by non-virulent or weakly virulent bacteria have been on the rise . As a result, determining the pathogenicity of an isolated bacteria becomes important . The past two decades have seen extensive efforts to exploit the potential of automation in clinical microbiology and to develop increasingly rapid procedures . New technology, such as molecular genetics, has also been introduced into clinical microbiology . For more correct and precise diagnoses of infectious diseases, the clinical microbiology laboratory should be enlarged as an "Infectious Disease Laboratory" to include an extended work on not only the detection of pathogens from the specimens of infectious disease, but also serodiagnostic tests and immunological tests of hosts . The "Infectious Disease Laboratory" would also play a key role in the control of nosocomial infection working with the infection control committee and infectious disease clinicians.

J Biochem (Tokyo), 1993 May, 113(5), 607 - 13
Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells; Suzuki K et al.; A glycosylphosphatidylinositol (GPI)-anchored protein, 5'-nucleotidase {EC 3.1.3.5}, was released from the membrane of bovine liver by use of phosphatidylinositol-specific phospholipase C (PI-PLC) of Bacillus thuringiensis and purified by several column chromatographies to a homogeneous state . The purified protein has an apparent molecular mass of 61 kDa, as estimated by SDS-polyacrylamide gel electrophoresis . From the partial amino acid sequence of a tryptic peptide, mixed oligonucleotides were synthesized and used to screen a lambda gt11 liver cDNA library, and one positive clone, pE1, was isolated . Since the insert of the clone lacked the NH2-terminal coding region, another lambda gt11 liver cDNA library was screened by using a synthetic probe corresponding to the 5' region of the insert of pE1 . Three additional cDNA clones were obtained . Sequencing of these cDNAs revealed an open reading frame that encodes a 574-residue polypeptide with a calculated mass of 63,084 Da . The predicted structure showed two highly hydrophobic stretches at both ends of the protein, like those of rat and human 5'-nucleotidases . The NH2-terminal 26 residues comprise a signal peptide and the COOH-terminal hydrophobic stretch may serve as a signal for the posttranslational GPI modification . An expression vector of the cDNA, pSVNT, was constructed in a mammalian expression vector pSVL and the 5'-nucleotidase activity was transiently expressed in COS-1 cells . The expressed activity was about 8 times higher than the pSVL-transfected control activity . PI-PLC released 45% of the transiently expressed 5'-nucleotidase activity, indicating that the cDNA isolated here encodes this enzyme expressed as a GPI-anchored protein.

Hinyokika Kiyo, 1993 May, 39(5), 463 - 5
{Kidney-conservative surgery followed by bacillus Calmette-Guerin instillation therapy for transitional cell carcinoma of solitary renal pelvis: a case report}; Ao T et al.; We report a case (62-year-old male) of transitional cell carcinoma (grade 2) of solitary right renal pelvis which was successfully treated with tumor excision followed by bacillus Calmette-Guerin instillation therapy . He had a past history of left radical nephroureterectomy for renal pelvic tumor 11 years earlier . Kidney-preserved surgery with BCG instillation therapy is expected to be an alternative for radical forms of therapy, especially in the patients with a solitary kidney.

Anticancer Res, 1993 May-Jun, 13(3), 705 - 8
The anti-metastatic effect of intravenously-inoculated BCG on prostate tumor cells; Pollard M et al.; The complication of metastasis from the primary tumor site to the distant organ is difficult to control . Tumor cells usually spread through the vascular system: lymphatics and/or blood; or by direct extension into adjacent sites . Transplantable prostate adenocarcinoma (PA-III) cells in L-W rats produces a tumor in the implant site and then spreads via the ipsilateral lymphatic route to the lungs in which new visible tumors develop . Viable bacillus Calmette-Guerin (BCG) was inoculated SC or IV into rats which were then inoculated either SC or IV with PA-III cells . They were examined thereafter for primary and for lung tumors . IV-inoculated BCG generated a strong intravascular intervention mechanism on metastatic PA-III cells in L-W rats . This immobilization mechanism was not demonstrable in rats that had been inoculated SC with BCG.

Res Microbiol, 1993 May, 144(4), 271 - 8
Immunological localization of Bacillus thuringiensis serovar israelensis toxins in midgut cells of intoxicated Anopheles gambiae larvae (Diptera: Culicidae); Ravoahangimalala O et al.; Fourth instar larvae of Anopheles gambiae were intoxicated with doses of purified crystals from Bacillus thuringiensis serovar israelensis (Bti) corresponding to 50-fold the LC50 after 24 h . Midguts were dissected after various contact times, then processed for immuno-light and -electron microscopy . Immunodetection on thin sections was performed using affinity-purified rabbit IgG against Bti crystal CryIVD or CytA polypeptides, in combination with anti-rabbit IgG/peroxidase . Both polypeptides were detected by optical and electron microscopy after 15 min of contact with Bti crystals on the apical brush border of midgut cells, but only in the gastric caeca and posterior stomach . No specific signal was detected in the other parts of the midgut, i.e . the cardia cells and the anterior stomach . These results confirm that mosquito midgut cells are the primary target for the toxins and that binding to specific receptors on the apical microvilli membrane is the initial step of delta-endotoxin action.

Nippon Saikingaku Zasshi, 1993 May, 48(3), 541 - 50
{Fragmentation and solubilization of filamentous appendages of Bacillus cereus spores}; Kozuka S; Filamentous appendages were removed from the surfaces of Bacillus cereus spores by treatment with sodium thioglycolate and purified by filtration through a glass microfiber paper and a membrane filter . The basic structure of the isolated appendages resembled that of the native appendages on the spores . The results of electron microscopical analysis, amino acid analysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) attested the purity of the preparation . The purified filamentous appendages were fragmented by treatment with 2N sodium hydroxide at 30C but neither with SDS nor mercaptoethanol, and the fragmentation progressed with the prolongation of the treatment period . When observed under an electron microscope, each fragmented appendage appeared to be rod shaped with an apparent axial hole and helical or disk-like arrangement of subunits . In the agar gel immunodiffusion test, a soluble fraction of the appendages obtained by treatment with sodium hydroxide formed only a single precipitation line against the antibody to the intact appendages . The molecular mass of the subunit separated by SDS-PAGE was approximately 10kDa . The 10-kDa protein was distributed over the surface of the intact appendages in immunoelectron microscopy . The antigenicity of the appendage subunits disappeared upon treatment with 2N sodium hydroxide for longer than 24h . Neither intact appendages nor intact spores agglutinated sheep or guinea-pig erythrocytes.

Appl Microbiol Biotechnol, 1993 May, 39(2), 197 - 203
Cloning and sequence analysis of the cyclomaltodextrinase gene from Bacillus sphaericus and expression in Escherichia coli cells; Oguma T et al.; The gene for cyclomaltodextrinase (CDase; EC 3.2.1.54) from Bacillus sphaericus E-244 was cloned in the recombinant plasmid pCD629 . Sequencing a portion of pCD629 revealed a unique open reading frame of 1,773 nucleotides coding for a 591-amino-acid polypeptide . The deduced polypeptide sequence showed about 50% homology with that of a neopullulanase, and was slightly homologous to those of the cyclodextrin glucanotransferases and the alpha-amylases . The optimum pH, specific activity and Km value for beta-cyclodextrin of the CDase that has been produced in Escherichia coli cells were 8.0, 16.4 units/mg protein, and 0.41 mM, respectively . These values were almost identical to those from B . sphaericus E-244.

J Biochem (Tokyo), 1993 May, 113(5), 637 - 41
Induction of antibiotic production by protease in Bacillus brevis (ATCC 8185); Oyama M et al.; Bacillus brevis (ATCC 8185) produces an antibiotic peptide, linear gramicidin, in the early stationary growth phase . Since we observed that preculture in milk medium is essential for production of the antibiotic in broth medium, we studied the role of the preculture in the antibiotic production . We found that addition of the supernatant of the precultured milk medium was sufficient to induce gramicidin production in broth medium . Fresh milk medium had no effect . The effector substance in the overnight cultured milk medium was labile at both acidic and alkaline pH and was destroyed by heat . We also found that addition of a protease (for example, bovine pancreas chymotrypsin or Streptomyces griseus protease) instead of the supernatant could induce the gramicidin production . Addition of Mn2+ was not required for the protease-induced production of gramicidin . It is known that B . brevis cells secrete protease into milk medium . But neither use of the protease-pretreated broth medium nor addition of casamino acids to broth medium induced gramicidin production . These results suggest that B . brevis cells secrete a factor for linear gramicidin production, that the inducing factor is protease and that the target of the protease is a substance(s) produced by the bacteria.

J Am Vet Med Assoc, 1993 Apr 15, 202(8), 1249 - 54
Field trial of four cowside antibiotic-residue screening tests; Sischo WM et al.; Four commercially available screening tests for antibiotic residues in milk were evaluated for their ability to correctly identify the antibiotic status of cows . A field trial, which included 199 cows from 2 herds, was conducted . Sensitivity, specificity, and likelihood ratios for a positive test result were calculated by using the Bacillus stearothermophilus var calidolactis disk assay as the reference test . The relationship of risk factors to the probability of a false-positive result for each screening test was modeled by use of unconditional logistic regression . The risk factors evaluated in these models were loge somatic cell count (scc), intramammary infection, herd, milk appearance, time milk sample frozen before tested, days in lactation, parity, and manufacturer's lot number . The risk factors log(e) scc and intramammary infection were forced into all models . The overall specificities for the 4 tests ranged from 0.78 to 0.95, whereas likelihood ratios for a positive test result ranged from 4.54 to 20.0 . When the confounding of cofactors was controlled in the logistic model, there was a positive effect of log(e) scc on the probability of a false-positive result for 3 of the screening tests, that is, for incremental increases in log(e) scc, there was an increasing likelihood for a false-positive result . In some of the tests, parity and intramammary infection also influenced the likelihood of a false-positive result . The goal of cowside testing is to assist in the production of high-quality, antibiotic residue-free milk from dairies.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1993 Apr 15, 268(11), 8240 - 5
Evidence that the CryIA crystal protein from Bacillus thuringiensis is associated with DNA; Bietlot HP et al.; Toxin generated by activation of the Bacillus thuringiensis CryIA(c) crystal protein (protoxin) with bovine trypsin was separated into two components by anion-exchange chromatography . One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (T1) . Only one major protoxin component was observed, and it was found to be associated with DNA . The DNA from the T2 toxin varied in size from 100 to 300 base pairs, whereas the crystal and the solubilized protoxin contained 20-kilobase DNA as the major DNA component . DNase treatment converted the T2 toxin to the DNA-free T1 toxin . In contrast, the DNA in the crystal and the solubilized protoxin was resistant to DNase digestion and was not dissociated from the protein by 1.5 M NaCl . The protoxin and DNA appeared to elute as a complex with a molecular mass of > 2 x 10(6) Da on gel-filtration chromatography . No toxin was generated from the protoxin with trypsin after extensive digestion of the protoxin with DNase or dissociation of the DNA by succinylation of the lysine residues . It is proposed that DNA binds to the COOH-terminal half of the crystal protein and is essential for maintaining the conformational integrity required for crystal formation and generation of toxin.

Eur J Biochem, 1993 Apr 15, 213(2), 683 - 7
Identification of charge-transfer transitions in the optical spectrum of low-spin ferric cytochrome P-450 Bacillus megaterium; McKnight J et al.; The optical, low temperature magnetic circular dichroism (MCD) and EPR spectra of low-spin Fe(III) cytochrome P-450 BM-3 from Bacillus megaterium, and its imidazole adduct have been measured . The MCD spectra locate new transitions over 600-700 nm and 800-1300 nm . The latter are assigned to porphyrin (a1u)-Fe(III) (dyz) charge-transfer (CT) transitions . In the case of the imidazole adduct the energy of this transition fits well to the theoretical model of Gadsby and Thomson {Gadsby, P . M . A . & Thomson, A . J . (1990) J . Amer . Chem . Soc 112, 5003-5011} . For the native enzyme, the energy of the CT band suggests co-ordination by a strongly H-bonded water ligand and the axial thiolate form of cysteine . Two transitions between 600-700 nm are detected in both derivatives . A theoretical analysis and fit of the MCD magnetisation properties shows that these transitions are polarised XY and XZ, respectively . They are assigned as thiolate sulphur py-d-shell and pz-d-shell CT transitions, analogous to the well known 695 nm band of methionine-histidine co-ordinated haem as in cytochrome c . They should prove usefully diagnostic of cysteine/Fe(III) conformational changes or protonation.

Carbohydr Res, 1993 Apr 7, 242, 173 - 80
Identification of 2-amino-2,6-dideoxy-D-glucose (D-quinovosamine), isolated from the cell walls of the alkaliphilic Bacillus sp . Y-25, by 500-MHz 1H NMR spectroscopy; Ito M et al.; Cell walls of alkaliphilic Bacillus strain Y-25 are composed of gamma-peptidoglycan and two acidic polymers . An amino sugar, which was a min component of one acidic polymer, did not correspond to any of the commercially available hexosamines . The amino sugar was isolated from the hydrolysate of the acidic polymer, purified, and identified as D-quinovosamine (2-amino-2,6- dideoxy-D-glucose) by 500-MHz NMR spectroscopic analysis and polarimetry.

Biochemistry, 1993 Apr 6, 32(13), 3429 - 36
Structural and functional characterization of the alpha 5 segment of Bacillus thuringiensis delta-endotoxin; Gazit E et al.; One of the most conserved sequences in various delta-endotoxins is the 30 amino acid long block I . Block I of cryIIIA delta-endotoxin contains a 23 amino acid amphiphilic alpha-helix termed alpha 5 . The potential involvement of this alpha 5 helix in the toxic mechanism of delta-endotoxin was examined . For this purpose, a peptide corresponding to the alpha 5 segment and its proline incorporated analogue (P-alpha 5) were synthesized and characterized . The alpha-helical content of the peptides, assessed in methanol by circular dichroism (CD), was 58% and 24% for alpha 5 and P-alpha 5, respectively . To monitor the interaction of alpha 5 peptides with phospholipid membranes, they were selectively labeled at their N-terminal amino acids with the fluorescent probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or carboxyfluorescein . Fluorometric studies allowed the calculation of membrane surface partition constants, which were about 10(4) M-1 for both alpha 5 and P-alpha 5, and revealed that their N-terminals are located within the lipid bilayers . The shape of the binding isotherms indicated that alpha 5 aggregated in both zwitterionic and acidic vesicles . Functional characterization of the alpha 5 peptides was determined by assessing their ability to dissipate a diffusion potential from sonicated small unilamellar vesicles (SUV) composed of zwitterionic or acidic phospholipids and to lyse human erythrocytes . alpha 5 was much more active than P-alpha 5 in both assays . Moreover, membrane-bound alpha 5 was more protected from enzymatic proteolysis than P-alpha 5.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1993 Apr 5, 268(10), 7553 - 61
Critical residues involved in FMN binding and catalytic activity in cytochrome P450BM-3; Klein ML et al.; Cytochrome P450BM-3 from Bacillus megaterium is a soluble, catalytically self-sufficient fatty acid mono-oxygenase that, in structural organization and amino acid sequence, resembles the Class II (microsomal) P450 systems . Its single polypeptide chain contains both a P450 heme domain and an NADPH:P450 reductase domain, each of which bears significant homology with its microsomal counterparts . We report here the critical nature of three amino acids in the reductase domain of this enzyme with respect to FMN binding and catalytic activity . We used site-directed mutagenesis to change glycine 570 to bulkier amino acids; none of these mutant enzymes contained FMN after purification . We also made substitutions for tryptophan 574 and tyrosine 536, which by sequence analogy (Porter, T . D . (1991) Trends Biochem . Sci . 16, 154-158) were proposed to bind FMN through stacking of the aromatic rings with the isoalloxazine ring of the flavin . Mutants of tryptophan 574 which retained the aromatic side chain contained no less than 0.85 mol of FMN per mol of enzyme, while aspartate and glycine substitutions yielded enzymes which did not incorporate FMN . Substitution of tyrosine 536 with aspartate gave an enzyme which contained 0.44 mol of FMN per mol of enzyme but was inactive as a fatty acid hydroxylase and had only 2% of wild-type cytochrome c reductase activity, while the glycine mutant at this position bound no FMN . Furthermore, although all of the mutant enzymes contained 1 mol of FAD per mol of enzyme, the Y536D mutant and those entirely lacking FMN retained no more than 40% of wild-type ferricyanide reductase activity . By assaying these enzymes in the presence of added FMN, we were able to assess the relative importance of the residues in the wild-type sequence with respect to their contribution to FMN binding . In addition, the aromatic mutants of tryptophan 574, which were nearly as active in cytochrome c reduction as wild-type P450BM-3, were only 20% as active in myristate hydroxylation as the wild-type enzyme, suggesting that this amino acid plays an important role in the flow of electrons between the P450 heme and reductase domains.

J Biol Chem, 1993 Apr 5, 268(10), 6932 - 8
Kinetic and stereochemical comparison of wild-type and active-site K145Q mutant enzyme of bacterial D-amino acid transaminase; Bhatia MB et al.; D-Amino acid transaminase (EC 2.6.1.21), from Bacillus sp . YM-1, a thermostable enzyme with pyridoxal 5'-phosphate as coenzyme and a target for the design of novel antimicrobial agents, catalyzes the reversible transfer of an amino group between D-alanine and alpha-ketoglutarate to form pyruvate and D-glutamate, respectively . To explore the catalytic role of Lys-145, which binds the coenzyme, a site-specific mutant enzyme, K145Q (in which Lys-145 had been mutated to glutamine) constructed earlier (Futaki, S., Ueno, H., Martinez del Pozo, A., Pospischil, M . A., Manning, J . M., Ringe, D., Stoddard, B., Tanizawa, K., Yoshimura, T., and Soda, K . (1990) J . Biol . Chem . 265, 22306-22312) was compared to the wild-type enzyme for its kinetic parameters . Initial velocity studies and partial reaction isotope exchange experiments showed that the low activity of the mutant enzyme (about 1.5% the activity of the wild-type enzyme with saturating substrates) is an intrinsic property, confirming that contaminating enzymes do not account for the low activity of the K145Q mutant enzyme . The rates of the forward reaction for both wild-type and mutant enzymes were 30-40 times higher than the rates of the reverse reaction . KM values for the four substrates were 10 to 100 higher for the mutant compared to the wild-type enzyme . Whereas D-alanine is preferred over L-alanine by the wild-type enzyme (10(3) higher kcat/KM for D- over L-alanine), the K145Q enzyme does not efficiently discriminate between L- and D-alanine . Both wild-type and mutant enzymes also catalyze the slow racemization of L- and D-alanine . Proton NMR studies showed that wild-type enzyme catalyzed a time-dependent exchange of the C alpha proton of D-alanine with solvent D2O and a slow exchange of the alpha proton of L-alanine; the latter slow exchange rate is the same for the C alpha proton of both L- and D-alanine with the K145Q mutant enzyme . Thus, in addition to binding pyridoxal 5'-phosphate, the active-site Lys-145 of D-amino acid transaminase is involved in several other important functions, i.e . it optimizes catalytic efficiency and it maintains stereochemical fidelity . The steady-state kinetic results on the K145Q mutant enzyme together with the findings on the relative racemization rates and the NMR protein exchange data suggest that an alternate base catalyzes abstraction of the alpha proton of substrate in this mutant D-amino acid transaminase.

J Gen Microbiol, 1993 Apr, 139 ( Pt 4), 775 - 80
Properties of the cold-labile NAD(+)-specific glutamate dehydrogenase from Bacillus cereus DSM 31; Jahns T et al.; Nicotinamide-adenine-dinucleotide-specific glutamate dehydrogenase (NAD-GDH; EC 1.4.1.3) from Bacillus cereus DSM 31 was enriched 260-fold . The molecular mass was determined by gel filtration to be 270 kDa (+/- 25 kDa) . The enzyme was highly specific for the coenzyme NAD(H) and catalysed both the formation and the oxidation of glutamate . Apparent Km values of 7.7 mM for glutamate and 0.56 mM for NAD+ during oxidative deamination were measured . Both in crude cell-free extracts and in enriched preparations the enzyme was extremely unstable, especially at low temperatures . The loss of activity in the cold was found to be due to the dissociation of the holoenzyme into catalytically inactive subunits of molecular mass 48 kDa (+/- 5 kDa), indicating that the native enzyme has a hexameric structure . The activity was restored under certain conditions, and no instability of the enzyme in the cold was observed in undisrupted cells.

An Med Interna, 1993 Apr, 10(4), 188 - 94
{Prevention of tuberculosis infection}; Prados C et al.; Tuberculosis (TB) is a disease caused by a mycobacterium, whose incidence has increased in the past years . This increase is related to the adquired immunodeficiency syndrome (AIDS) . Due to its high prevalence, Spain is considered a developing country . The tuberculous infection depends on the degree of functionality of the alveolar macrophages that stimulate the lymphocytes and isolate the bacillus . The infection by mycobacterias can be quantified by means of the cutaneous reaction against tuberculin and mantoux, allowing us to select the subjects that must receive prophylaxis . For its correct interpretation, it is currently recommended to avoid BCG vaccination of children, except in countries with high prevalence of TB.

J R Coll Physicians Lond, 1993 Apr, 27(2), 169 - 74
Microbes, molecules and man . The Mitchell Lecture 1992; Moxon ER; Robert Koch, the discoverer of the tubercle bacillus, has had a seminal influence on the extraordinary progress in the field of infectious diseases in the past 100 years . Koch's postulates defined the germ theory of disease causation . They have now been confirmed and brought up to date by the application of molecular techniques . Developments in molecular genetics have helped in the elucidation of the pathogenesis of meningitis caused by H influenzae and the role of type b capsule as a determinant of bacterial virulence--and satisfied the requirements of Koch's molecular postulates . This new knowledge has contributed to the development of a successful immunoprophylactic strategy for eliminating Hib disease . Studies in Oxford over the past eight years have confirmed the effectiveness and safety of a routine immunisation programme for the UK.

Int J Syst Bacteriol, 1993 Apr, 43(2), 221 - 31
Characterization of Bacillus brevis with descriptions of Bacillus migulanus sp . nov., Bacillus choshinensis sp . nov., Bacillus parabrevis sp . nov., and Bacillus galactophilus sp . nov; Takagi H et al.; Thirty-five Bacillus brevis strains obtained from culture collections, including protein-producing isolates, were taxonomically studied by using numerical analysis, DNA base composition, and DNA-DNA hybridization . Six DNA relatedness groups were represented, and these groups correlated well with clusters based on the numerical analysis . The B . brevis strains were separated into B . brevis sensu stricto, four new species, and an unidentified species of the genus Bacillus . Bacillus migulanus sp . nov., Bacillus choshinensis sp . nov., Bacillus parabrevis sp . nov., and Bacillus galactophilus sp . nov . are proposed.

Nippon Hinyokika Gakkai Zasshi, 1993 Apr, 84(4), 656 - 61
{Long-term results and risk factors of tumor recurrence in patients with superficial bladder cancer who were treated by intravesical bacillus Calmette-Guerin (BCG) instillation}; Tachibana M et al.; An evaluation was made of the long-term results and risk factors of tumor recurrence in patients with superficial bladder cancer who were treated with intravesical bacillus Calmette-Guerin (BCG) and who had a mean follow-up period of 57 months . Eligible for the study were a total of 102 patients who were treated by transurethral resection of tumors and were considered to be free from tumor from 1984 to 1989 . A suspension containing 80 mg Tokyo 172 strain BCG in 50 ml normal saline was given intravesically to 50 patients once a week for six weeks without further maintenance instillation . To the other 52 patients was additionally given monthly intravesical BCG instillation for 12 months . The actuarial nonrecurrent rates according to sex, different BCG treatment protocol, tumor grade, tumor status including primary or recurrent tumors, and solitary or multiple tumors were estimated by Kaplan-Meier method . The estimated three-, five- and seven-year actuarial nonrecurrent rates in all 102 cases were 77.3%, 68.5% and 60.6%, respectively . When nonrecurrent rates were compared to tumor characteristics, no statistically significant differences were observed between primary and recurrent tumors or between solitary and multiple tumors estimated by generalized Wilcoxon method . On the other hand, when nonrecurrent rates were compared with tumor grades, grade 3 tumor showed a 40.0% three-year nonrecurrent rate . The differences between grade 3 and grade 1 and between grade 3 and grade 2 were statistically significant (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1993 Apr 1, 108(2), 139 - 43
Expression of bacteriophage PhiX174 lysis gene E in Staphylococcus carnosus TM300; Halfmann G et al.; Expression of the cloned PhiX174 gene E causes lysis of the Gram-negative bacterium Escherichia coli, which led to the proposal that a two-membrane system is necessary for the protein E lysis function . Gene E was cloned in an E . coli/Bacillus subtilis shuttle vector and expressed in the Gram-positive bacterium Staphylococcus carnosus TM300 . Regulated gene E expression had a lethal effect on S . carnosus; however, no lysis was detected, lending support to the hypothesis.

Biotechnol Appl Biochem, 1993 Apr, 17 ( Pt 2), 205 - 16
Cyclomaltodextrin glucanotransferase from Bacillus circulans E 192: nitration with tetranitromethane; Villette JR et al.; Nitration of tyrosine residues was performed on Bacillus circulans E 192 cyclomaltodextrin glucanotransferase (CGTase) using tetranitromethane (TNM) . A maximum of 15 out of 28 tyrosine residues is modified with 8 mM TNM, entailing a concomitant loss of enzymic activity and tryptophan fluorescence . Spectroscopic studies suggest that these two phenomena are related to an impairment of the enzyme conformation as a consequence of the tyrosine nitration . The presence of 5 mM acarbose during the CGTase nitration results in the protection of one tyrosine residue and the rate of inactivation is reduced 9.4-fold . These results support a contribution of a tyrosine residue in the CGTase catalytic site . The nitration of CGTase also entails a decrease in the enzyme's affinity for a beta-cyclodextrin (beta-CD) co-polymer . Kinetic and analytical investigations on isolated modified enzymes support the concept that this phenomenon is unrelated to the modification of tyrosine residues, but rather concerns a side reaction of the reagent occurring at the raw-starch-binding site of the CGTase.

Appl Environ Microbiol, 1993 Apr, 59(4), 1253 - 8
Expression and secretion of Bacillus amyloliquefaciens alpha-amylase by using the yeast pheromone alpha-factor promoter and leader sequence in Saccharomyces cerevisiae; Southgate VJ et al.; Replacement of the regulatory and secretory signals of the alpha-amylase gene (AMY) from Bacillus amylolique-faciens with the complete yeast pheromone alpha-factor prepro region (MF alpha 1p) resulted in increased levels of extracellular alpha-amylase production in Saccharomyces cerevisiae . However, the removal of the (Glu-Ala)2 peptide from the MF alpha 1 spacer region (Lys-Arg-Glu-Ala-Glu-Ala) yielded decreased levels of extracellular alpha-amylase.

Appl Environ Microbiol, 1993 Apr, 59(4), 1131 - 7
Cloning of a novel cryIC-type gene from a strain of Bacillus thuringiensis subsp . galleriae; Kalman S et al.; A novel cryIC-type gene was isolated from a strain of Bacillus thuringiensis subsp . galleriae . A new polymerase chain reaction (PCR) technique with a set of several oligonucleotide primer pairs specific to the cryIC gene was used to screen a number of B . thuringiensis strains . PCR amplified several DNA fragments ranging from 100 bp to 1 kb for B . thuringiensis strains containing a cryIC gene . PCR fragments amplified from the Bacillus thuringiensis subsp . galleriae HD29 DNA differed from the fragments amplified from other cryIC-containing strains, indicating strain HD29 contained a novel cryIC-type gene . To isolate crystal genes homologous to cryIC, an HD29 gene library was probed with a 984-bp fragment of the amino-terminal coding region of the cryIC gene cloned from Bacillus thuringiensis subsp . aizawai HD229 . A putative toxin gene was isolated from a phage that hybridized strongly to the cryIC probe . Translation of the putative toxin DNA sequence revealed an open reading frame of 1,176 amino acids whose predicted molecular mass was 132.8 kDa . Comparisons of the toxin gene sequence with sequences of other cry genes indicated that this gene is a subclass of cryIC . We propose to designate this gene cryIC(b) . In Escherichia coli, the cryIC(b) gene produced a protein of approximately 130 kDa toxic to Spodoptera exigua and Trichoplusia ni.

Biochem J, 1993 Apr 1, 291 ( Pt 1), 151 - 5
An overview of the kinetic parameters of class B beta-lactamases; Felici A et al.; The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme . The A . hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific 'carbapenemase' . No relationships were found between sequence similarities and catalytic properties . The problem of the repartition of class B beta-lactamases into sub-classes is discussed . Improved purification methods were devised for the P . maltophilia and A . hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column.

Vet Hum Toxicol, 1993 Apr, 35(2), 141 - 3
Sump additives as a source of bioaerosols in a school building; Thorne PS; An investigation was launched following complaints of poor air quality and building-related illness in a public elementary school . Occlusion of air intakes put the building under negative pressure and caused vents from a below-ground sump to become air intakes . Outside air drawn through the sump pit traveled into the adjacent main air handling unit and was disseminated throughout the building . Sump additives introduced in an attempt to counteract foul odors contained spores of Bacillus species, which appeared as bioaerosols throughout the school . Viable microbial sampling identified B subtilis, B cereus, and B licheniformis in the sump room and classrooms at levels as high as 760 colony forming units/m3 (CFU/m3) . Concentrations of CO2 in classrooms were 1250 ppm, indicating inadequate makeup air . Remediation was accomplished by opening the air intakes, isolating the sump room from the air handling system, venting the sump to the outside, and flushing the sump with fresh water on a regular basis.

J Bacteriol, 1993 Apr, 175(8), 2271 - 7
Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth; Tojo N et al.; The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation . A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E . coli as a probe . The nucleotide sequence of the M . xanthus lon gene was determined . It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues . The deduced amino acid sequence of the M . xanthus lon gene product showed 60 and 56% identity with those of the E . coli and Bacillus brevis lon gene products, respectively . Analysis of an M . xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M . xanthus . In contrast to that of E . coli, the M . xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.

J Bacteriol, 1993 Apr, 175(8), 2248 - 54
Relevance of charged groups for the integrity of the S-layer from Bacillus coagulans E38-66 and for molecular interactions; Sara M et al.; In this paper, the importance of charged amino and carboxyl groups for the integrity of the cell surface layer (S-layer) lattice from Bacillus coagulans E38-66 and for the self-assembly of the isolated subunits was investigated . Amidination of the free amino groups which preserved their positive net charge had no influence on both . On the other hand, acetylation and succinylation, which converted the amino groups into either neutral or negatively charged groups, and amidation of carboxyl groups were accompanied by the disintegration or at least by the loss of the regular structure of the S-layer lattice . Treatment of S-layer monolayers with the zero-length cross-linker carbodiimide led to the introduction of peptide bonds between activated carboxyl groups and amino groups from adjacent subunits . This clearly indicated that in the native S-layer lattice the charged groups are located closely enough for direct electrostatic interactions . Under disrupting conditions in which the S-layer polypeptide chains were unfolded, 58% of the Asx and Glx residues could be amidated, indicating that they occur in the free carboxylic acid form . As derived from chemical modification of monolayer self-assembly products, about 90% of the lysine and 70% of the aspartic and glutamic acid residues are aligned on the surface of the S-layer protein domains . This corresponded to 45 amino groups and to 63 carboxyl groups per S-layer subunit . Labelling experiments with macromolecules with different sizes and charges and adsorption studies with ion-exchange particles revealed a surplus of free carboxyl groups on the inner and on the outer faces of the S-layer lattice . Since the carboxyl groups on the outer S-layer face were accessible only for protein molecules significantly smaller then the S-layer protomers or for positively charged, thin polymer chains extending from the surface of ion-exchange beads, the negatively charged sites must be located within indentations of the corrugated S-layer protein network . This was in contrast to the carboxyl groups on the inner S-layer face, which were found to be exposed on elevations of the S-layer protein domains (D . Pum, M . Sara, and U.B . Sleytr, J . Bacteriol . 171:5296-5303, 1989).

Am Rev Respir Dis, 1993 Apr, 147(4), 958 - 61
Quantitative bacillary response to treatment in HIV-associated pulmonary tuberculosis; Brindle RJ et al.; A group of 122 patients with culture-proven pulmonary tuberculosis were recruited to examine the concentrations of Mycobacterium tuberculosis in sputum and the relationship to HIV-1 antibody status . They were followed for up to 28 days from the start of antituberculous chemotherapy to assess the early bacillary response to two chemotherapeutic regimens . Of 67 treated with streptomycin, thiacetazone, and isoniazid 17 were HIV positive, and subsequently 55, of whom 20 were HIV positive, were treated with streptomycin, rifampin, isoniazid, and pyrazinamide . The mean initial concentration of M . tuberculosis in the sputum of the HIV-negative patients was significantly higher than in HIV-positive patients (6.95 and 6.34 log colony-forming units respectively; p = 0.019) . The HIV-positive patients had less radiologic evidence of disease and significantly fewer zones of lung affected with cavities . The response to treatment was similar, but with HIV-positive patients more likely to become culture negative by 28 days . The differences that exist between HIV-positive and HIV-negative patients are minor, and standard regimens are at least as effective in HIV-positive patients in the first month of treatment.

J Clin Microbiol, 1993 Apr, 31(4), 972 - 4
Rapid screening test for enterotoxin-producing Bacillus cereus; Jackson SG; Culture supernatants of 30 enterotoxin-producing Bacillus cereus isolates produced a characteristic progressive destruction of McCoy cell monolayers . Enterotoxin-negative B . cereus and other group 1 Bacillus spp . caused no monolayer disruption . The McCoy cell tissue culture system appears to provide a rapid screening assay for detection of enterotoxin-producing B . cereus.

Am J Public Health, 1993 Apr, 83(4), 583 - 5
Complications of BCG vaccinations in rural Haiti; Bonnlander H et al.; This study investigated an outbreak of axillary lymphadenitis and abscesses after Bacillus Calmette-Guerin vaccination among rural Haitian children treated at the Hospital Albert Schweitzer from January 1986 through March 1991 . Seventy-seven cases of vaccine-related complications were identified, all among children immunized before the age of 1 year . The proportions of children with complications were 0.017% for 1986 through 1989, 0.91% for 1990, and 2.2% for January through March 1991.

Virology, 1993 Apr, 193(2), 621 - 30
Nucleotide sequence and genomic organization of rice tungro spherical virus; Shen P et al.; Rice tungro disease is caused by a combination of two viruses: rice tungro spherical virus (RTSV) and rice tungro bacilliform virus (Jones et al . (1991) J . Gen . Virol . 72, 757-761.) . The genome of RTSV is a single-stranded polyadenylated RNA . We present here the 12,433-nucleotide complete sequence of RTSV genomic RNA and its deduced coding regions . This sequence contains a large open reading frame (ORF) which initiates following a 514-nucleotide 5' leader sequence and is capable of encoding a viral polyprotein of 390.3 kDa . Two viral subgenomic RNAs of ca . 1.2 and 1.4 kb, respectively, were detected in RTSV-infected leaf tissues and mapped by S1 nuclease protection assay . These RNAs were determined to be congruent with the genomic RNA sequence proximal to the 3' terminus and could contain up to two small ORFs in their 5' to 3' orientation . There are at least three capsid protein subunit cistrons near the N-terminus of the large ORF . A computer-aided search of the C-terminal half of the large ORF revealed conserved protein sequence motifs for a viral RNA polymerase, proteinase, and a putative NTP-binding protein . These sequence motifs are arranged in a manner that resembles those of picorna-like viruses . Taken together, these data indicate that RTSV is a distinct type of positive-strand RNA virus . The evolutionary relationships between RTSV and other picorna-like plant viruses are discussed.

J Urol, 1993 Apr, 149(4), 744 - 8
Intravesical bacillus Calmette-Guerin immunoprophylaxis of superficial bladder cancer: results of a controlled prospective trial with modified treatment schedule; Melekos MD et al.; A controlled prospective trial on 94 patients evaluated the efficacy of intravesical Pasteur strain bacillus Calmette-Guerin (BCG) administration as prophylaxis against tumor recurrences after complete endoscopic resection of superficial bladder cancer . The treatment schedule, consisting of an initial 6-week course of instillations and a single quarterly maintenance dose to the responders, was modified to those of the latter who were at high risk for recurrence and who received an additional separate 4-week course of therapy . The percentage of the patients treated prophylactically with BCG and who remained free of recurrences (68%, mean followup 33.8 months) was significantly higher than that of the controls who underwent transurethral resection only (41%, mean followup 30.2 months) . In terms of relative risk of recurrences, recurrence rate per 100 patient-months and disease-free interval, comparisons between the 2 groups of patients revealed a significant benefit for the BCG group overall as for those subjects having stages Ta and T1 tumors, multifocal tumors, a history of disease, and grades 2 and 3 carcinoma . Drug-induced toxicity was acceptable . Our study suggests that our modified treatment protocol is notably safe and effective against recurrent superficial bladder cancer.

J Immunol, 1993 Apr 1, 150(7), 3101 - 7
Immunization with recombinant BCG-SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys; Yasutomi Y et al.; Because the transmission of HIV is likely to occur through cell-associated virus, an effective HIV vaccine should be capable of eliciting HIV-specific CTL . We have employed the simian immunodeficiency virus (SIV)/rhesus monkey model to explore the use of the attenuated tuberculosis bacillus, Calmette Guerin bacillus (BCG), as a vaccine vehicle to elicit AIDS virus-specific CTL . BCG was engineered to express SIVmac gag under the control of hsp70 regulatory sequences . Immunization with this rBCG-SIVmac gag elicited MHC class I-restricted, CD8+ SIVmac gag-specific CTL in rhesus monkeys . In fact, SIVmac gag-specific CTL could be cloned readily from peripheral blood lymphocytes of these immunized monkeys . These findings provide further evidence for the power of BCG as a vaccine vector and its continued exploration as a vehicle for eliciting HIV-specific immunity.

Zhonghua Bing Li Xue Za Zhi, 1993 Apr, 22(2), 80 - 2
{Localization and significance of lysozyme in tuberculosis}; Zhang SF; The relationship between expression of lysozyme and tuberculosis lesions was studied in 132 patients . The positive rate of lysozyme expression in 132 tuberculosis cases was 83.3% . Lysozyme was mainly distributed intracellularly, and the positive rates in different types, i.e . in cases with lesions of caseous necrosis, cellular nodes, and fibroid nodes were 90.1%, 79.3% and 63.3% respectively (P < 0.05 between lesions of caseous necrosis and fibroid nodes) . The positive rate of lysozyme in cases of infection associated with L-form tubercule bacillus was 64.4%, which was higher in cases with positive L-form antigen than that in antigen negative cases (P < 0.025) . The results indicated that expression of lysozyme was rather active, and might be considered as a marker for the prognosis of tuberculosis treatment.

J Korean Med Sci, 1993 Apr, 8(2), 135 - 44
Analysis of the immunologic mechanism of intravesical bacillus Calmette-Guerin therapy for superficial bladder tumors: distribution and function of immune cells; Chung JY et al.; Intravesical bacillus Calmette-Guerin (BCG) administration has been used as an adjuvant therapy after transurethral resection for superficial bladder cancer, but the exact mechanisms of its antitumor activity are not yet known . The aim of this study was to characterize the immunologic aspects of antitumor activity of BCG using an animal model . C3H/He inbred mice and murine bladder tumor cell line, MBT-2 were used . The changes in immune cells such as helper T cells, suppressor T cells, macrophages and natural killer cells in the bladder and spleen were analysed by immunohistochemical method in intravesical BCG instilled in normal bladder, MBT-2 implanted after electrocauterization of the bladder mucosa and MBT-2 implanted and intravesical BCG treated group . The changes in natural killer cell activity of the splenocytes and peritoneal lymphocytes were evaluated using 51chromium release assay at regular time intervals following intraperitoneal BCG instillation . The prophylactic anticancer effect was evaluated by observing the tumor growth in the intravesically BCG treated group after intravesical MBT-2 implantation . In immunohistochemical examination, a remarkable infiltration of macrophage and helper T cell was observed in the lamina propria of the bladder, and the helper and suppressor T cells ratio (Th/Ts ratio) was increased after intravesical BCG therapy . In 51chromium release assay, enhanced natural killer cell activity of the splenocytes and peritoneal lymphocytes was observed after intraperitoneal BCG inoculation . The growth of implanted tumor was suppressed following intravesical instillation of BCG . These results suggest that the antitumor activity of BCG is not related to the simple inflammatory reaction but to the local and systemic immune response in which helper T lymphocytes and mononuclear cells play an important role.

J Gen Microbiol, 1993 Apr, 139 ( Pt 4), 661 - 7
A partial physical map for the chromosome of alkalophilic Bacillus sp . strain C-125; Sutherland KJ et al.; Bacillus sp . strain C-125 has been chosen as a model alkalophilic bacterium to understand how adaptation to growth at high pH is achieved . To aid genetic analysis, we have started characterization of its genome . By using the two infrequently-cutting restriction endonucleases, AscI and Sse8387I, in conjunction with pulsed-field electrophoretic techniques, the size of the genome was found to be 3.7 Mb . Southern blot analysis of single, double and partial digests of Bacillus sp . strain C-125 DNA, using AscI-linking clones, gene probes and purified Bacillus sp . strain C-125 restriction fragments, allowed a putative chromosome map to be constructed.

Int J Immunopharmacol, 1993 Apr, 15(3), 371 - 82
delta-9-Tetrahydrocannabinol inhibits cell contact-dependent cytotoxicity of Bacillus Calmétte-Guérin-activated macrophages; Burnette-Curley D et al.; The effect of delta-9-tetrahydrocannabinol (delta-9-THC), the major psychoactive component of marijuana, on the capacity of Bacillus Calmette-Guerin (BCG)-activated macrophages to lyse L929 tumor cells, Naegleria fowleri amoebae, and herpes simplex virus-infected cells was examined . Delta-9-THC inhibited tumoricidal and amoebicidal activity in a dose-related manner . Antiviral activity was decreased when mice received 25 and 50 mg/kg delta-9-THC . The cannabinoid did not directly suppress the activation of macrophages as determined by levels of 5'-nucleotidase activity and did not inhibit splenic T-lymphocytes of BCG-recipient mice from producing interferon gamma . Nomarski optics microscopy, scanning electron microscopy, and radiolabeling binding studies demonstrated that macrophages from delta-9-THC-treated mice retained their capacity to attach to their targets . These results suggest that delta-9-THC suppresses cell contact-dependent amoebicidal, tumoricidal, and antiviral activities of activated macrophages at a stage following effector cell-target cell conjugation.

J Bacteriol, 1993 Apr, 175(7), 2137 - 42
Two glucose transport systems in Bacillus licheniformis; Tangney M et al.; Bacillus licheniformis NCIB 6346 showed active accumulation of glucose which was inhibited by agents which affect the transmembrane proton gradient . Phosphotransferase (PTS) activity, identified as phosphoenolpyruvate-dependent phosphorylation of glucose, was found in cell extracts but could not be demonstrated in cells permeabilized with toluene when assays were conducted at pH 6.6 . The same was true for mannitol and fructose phosphotransferase activities . Cells grown on fructose accumulated glucose at a slower rate than glucose-grown cells, and extracts prepared from them did not contain glucose PTS activity . Examination of the effects of analogs on glucose uptake and phosphorylation showed that 2-deoxyglucose was not a PTS substrate, but did markedly inhibit glucose uptake, with stronger inhibition in cells grown on fructose . Glucose accumulation by whole cells grown on glucose became less sensitive to the uncoupler tetrachlorosalicylanilide (TCS) as the pH was raised from 6.6 to 8.0, while in fructose-grown cells TCS was equally effective across this pH range . PTS activity was exhibited by toluene-treated cells at pH 7.5 and above, although the system itself in extracts was not affected by pH in the range of 5.0 to 8.0 . The results are consistent with the presence of two glucose transport systems, one a PTS and the other operating by an alternative mechanisms, and suggest that the PTS in B . licheniformis may be regulated in a pH-dependent manner.

Wei Sheng Wu Xue Bao, 1993 Apr, 33(2), 115 - 21
{Purification and properties of beta-mannanases from alkalophilic Bacillus N16-5}; Tian X et al.; Three extracellular alkaline beta-mannanases from an alkalophilic Bacillus N16-5 were purified to electrophoretic homogeneity by (NH4)2SO4 precipitation, DEAE-Sephadex A-25 chromatography, hydroxyapatite chromatography and preparative electrophoresis . Molecular weights and pI values of the beta-mannanases (M-1, M-2 and M-3) were 51000, 38000 and 34700 by SDS-PAGE and 4.3, 2.5 and 2.5 by PAGEIEF, respectively . The optimum pH for enzyme catalysis were 9.0 for M-1 and 10.0 for M-2 and M-3, respectively . All of three enzymes were the most active at 70 degrees C . The activities of three enzymes were strongly inhibited by Ag1+, Hg2+ and Mn2+ . Michaelis constants (Km) of the enzyme M-1, M-2 and M-3 for mannase from konjak were 2.9, 1.7 and 12.5 mg/ml, maximum velocities (Vmax) for the saccharide were 27500, 47500 and 15700 mumol.min-1.mg-1, respectively . Konjak was hydrolyzed by the enzyme M-1, and major components in digests were mono-, di-, tri- and tetra-saccharides.

Mol Ecol, 1993 Apr, 2(2), 65 - 78
Variable stability of antibiotic-resistance markers in Bacillus cereus UW85 in the soybean rhizosphere in the field; Halverson LJ et al.; We compared the stability of antibiotic-resistance markers in strains derived from Bacillus cereus UW85 in culture media and in the soybean rhizosphere in a growth chamber and in the field . We studied two independent, spontaneous mutants resistant to neomycin, three independent, spontaneous mutants resistant to streptomycin, and strains carrying plasmid pBC16, which encodes tetracycline resistance . Antibiotic-resistance markers were maintained in populations of all UW85 derivatives in culture and in the rhizosphere of soybeans grown in soil in a growth chamber . In two field experiments, antibiotic resistance was substantially lost in rhizosphere populations of B . cereus as early as 14 or as late as 116 days after planting . To distinguish between death of the inoculated strain and loss of its marker, we tested populations of B . cereus for other phenotypes (orange pigmentation, plasmid-borne resistance to tetracycline, and biocontrol activity) that are typical of UW85-derivatives used as inoculum, but atypical of the indigenous populations of B . cereus, and these phenotypes were maintained in populations from which the marker was lost . In general, neomycin-resistance markers were maintained at a higher frequency than streptomycin-resistance markers, and maintenance of antibiotic-resistance markers varied with position on the root and with the year of the experiment . In a semi-defined medium, the UW85 derivatives grew at the same rate as the wild type at 28 degrees C, but most grew more slowly than the wild type at 16 degrees C, demonstrating that antibiotic resistance can affect fitness under some conditions . The results suggest that the stability of antibiotic-resistance markers should be assessed in the ecosystems in which they will be studied.

Chest, 1993 Apr, 103(4), 1087 - 90
Use of BCG vaccine in shelters for the homeless . A decision analysis; Nettleman MD; As a result of many interacting variables, including crowded shelters and limited access to health care, homeless persons are at high risk for tuberculosis . Using traditional approaches, control of tuberculosis in this population has been difficult . Decision analysis was used to investigate the cost-effectiveness of BCG (bacillus Calmette-Guerin) vaccination in persons attending homeless shelters . This vaccination was cost-effective over a wide range of assumptions . Using conservative assumptions, a vaccine that was at least 40 percent effective would result in a net cost savings . If the efficacy of the vaccine were 50 percent, $4,000 would be saved, 12 life-years gained, and 23 cases of active tuberculosis prevented for every 1,000 persons vaccinated . Further study of the BCG vaccine in homeless persons and other populations at risk is warranted.

J Formos Med Assoc, 1993 Apr, 92(4), 317 - 23
Gram-negative bacillary meningitis in adults; Hsu GJ et al.; To evaluate the clinical aspects of gram-negative bacillary meningitis (GNBM), we reviewed 41 adult patients with bacteriologically proven gram-negative bacillary meningitis, seen from 1985 to 1990 . Thirty-two patients had post-neurosurgical GNBM and nine patients had spontaneous GNBM . Spontaneous GNBM appeared to have a sudden onset, a relatively fulminant course, and was caused most often by Escherichia coli . Post-neurosurgical GNBM, however, had a more insidious onset, a more protracted course, and was more often caused by nosocomial organisms which were resistant to multiple antibiotics . The overall mortality was 39% . Patients treated with combined aminoglycoside therapy had a lower mortality rate than those treated with intravenous aminoglycoside (17% vs 48%) . The use of third-generation cephalosporins has made a significant therapeutic advance in the treatment of GNBM, with a lower mortality of 21% . We recommend treatment of GNBM with third-generation cephalosporins and aminoglycosides . If aminoglycosides are to be employed, it is suggested that they be administered both intravenously and directly into the central nervous system.

Tuber Lung Dis, 1993 Apr, 74(2), 96 - 9
DNA analysis demonstrates that mycococcus forms are not mycobacteria; de Wit D et al.; Previous experimental evidence has suggested that the mycobacteria may exist in morphological forms other than the well characterised acid-fast bacillus . However none of these studies have been able to show conclusively that these variant forms are derived from the mycobacteria and therefore much debate has centered around the exact nature of these organisms . In this study we have examined stored cultures of the mycococcus form of Mycobacterium bovis BCG and M . phlei which were prepared by Csillag in 1972 and 1969 . Restriction fragment patterns of the DNA of the variant forms and the parent mycobacteria were not similar and chromosomal DNA from the parent mycobacteria did not hybridise with the DNA of the variant forms . Furthermore biochemical studies indicate that the variant forms are environmental contaminants . Although this study shows conclusively that the mycococcus is not derived from the mycobacteria, we believe that the search for variant morphological or metabolic forms of the mycobacteria should remain an active area of investigation, but that no study should be considered complete without the full use of newer molecular technology.

J Bacteriol, 1993 Apr, 175(8), 2314 - 20
Cytotoxicity and ADP-ribosylating activity of the mosquitocidal toxin from Bacillus sphaericus SSII-1: possible roles of the 27- and 70-kilodalton peptides; Thanabalu T et al.; Clones expressing regions of the 100-kDa Bacillus sphaericus SSII-1 mosquitocidal toxin (Mtx) as fusion proteins with glutathione S-transferase were constructed, and the toxin-derived peptides were purified . The in vitro ADP-ribosylation activities of these peptides and their effects on larvae and cells in culture were studied . Mtx25 (amino acids 30 to 493) was found to ADP-ribosylate two proteins with molecular masses of 38 and 42 kDa, respectively, in Culex quinquefasciatus (G7) cell extracts, in addition to ADP-ribosylating itself . Mtx21 (amino acids 30 to 870; or a combination of Mtx25 and Mtx26 (amino acids 259 to 870) caused mortality in C . quinquefasciatus larvae . Mtx25, Mtx26, or Mtx24 (amino acids 30 to 276) alone and Mtx24 in combination with Mtx26 were not toxic to larvae . Mtx21 and Mtx26 produced marked morphological changes in G7 cells and to a lesser extent in Aedes aegypti cells but had no effect on Anopheles gambiae or HeLa cells . Thus, a domain in the N-terminal region of the Mtx protein is sufficient for ADP-ribosylation of C . quinquefasciatus cell protein, and a domain in the C-terminal region is sufficient for toxicity to cultured C . quinquefasciatus cells; however, both regions are necessary for toxicity to mosquito larvae.

Biosci Biotechnol Biochem, 1993 Apr, 57(4), 584 - 90
Importance of the central region of 130-kDa insecticidal proteins of Bacillus thuringiensis var . israelensis for their activity in vivo and in vitro; Yoshida K et al.; To delineate the mosquitocidal regions of the ISRH3 (CryIVB) and ISRH4 (CryIVA) proteins, which are two of the mosquitocidal 130-kDa proteins contained in the crystalline protein bodies (CPBs) of Bacillus thuringiensis var . israelensis (BTI), a deletion analysis of these protein genes has been done . Based on the evidence that each 130-kDa protein had two mosquitocidal regions, N-terminal and C-terminal ones, and these two regions shared a common part in the center of the 130-kDa proteins, deleted genes on this region were constructed . As the protein products which lacked the central region had reduced activities, the central region could be important for the mosquitocidal activity . The mosquitocidal and non-mosquitocidal truncated gene products of 130-kDa protein genes were also applied to a cultured lepidopteran cell line, TN-368 . The mosquitocidal proteins caused the swelling and disruption of the cells in spite of the insecticidal specificity of CPBs of BTI, but the non-mosquitocidal proteins did not . Therefore, TN-368 cells were sensitive to the mosquitocidal fragments of 130-kDa proteins of BTI under the assay conditions used.

Appl Microbiol Biotechnol, 1993 Apr, 39(1), 63 - 8
Production and immobilization of a proteinase-reduced cyclodextrin glycosyltransferase preparation; Steighardt J et al.; Cyclodextrin glycosyltransferase (CGTase) was produced by a 3-day cultivation of Bacillus macerans growing in a natural medium containing grated-potatoes . Besides CGTase the culture supernatant contained a mixture of serine proteinases with a predominant subtilisin-like activity . By fractionated precipitation with ammonium sulphate the CGTase (molecular mass approximately 70 kDa) was concentrated and largely separated from the proteinases (molecular mass approximately 28 kDa) . Among the various immobilization methods and carrier materials tested the enriched CGTase was covalently bound preferably onto porous glass beads using glutardialdehyde as cross-linker . The discontinuous conversion of soluble starch into beta-cyclodextrin was carried out with native as well as immobilized CGTase over 24 h . The batch re-usability of the fixed enzyme proved to be at least 20 times with a residual CGTase activity of 65%.

Immunology, 1993 Apr, 78(4), 635 - 42
Induction of an auto-anti-IgE response in rats . IV . Effects on mast cell degranulation; Jaffery G et al.; Induction of an auto-anti-IgE (auto-aIgE) response in the rat inhibits both total and specific IgE production and alters the distribution of mast cell (MC) subpopulations identified by differential Alcian blue/safranin staining . We have extended these observations by characterizing the auto-aIgE antibodies and determining their effects on MC degranulation in vitro and in vivo . An auto-aIgE response was induced in bacillus Calmette-Guerin (BCG)-primed rats by injecting a conjugate of highly purified rat IgE myeloma (IR2) coupled to tuberculin-derived purified protein derivative (PPD) . Anti-IgE autoantibodies were almost exclusively IgG2a . The intradermal injection of auto-aIgE into untreated rats induced local MC degranulation as shown by a strong immediate skin response . Histologically there was evidence of significant degranulation of safranin staining connective tissue MC (SMC) in the skin but not of the Alcian blue staining MC (ABMC) in the sub-epidermal region . The induced degranulation was epsilon-chain specific; immunopurified anti-idiotypic antibodies raised to the IgE IR2 myeloma had no MC degranulating activity . When administered locally, auto-aIgE inhibited a subsequent passive cutaneous anaphylaxis (PCA) response elicited by anti-ovalbumin IgE . In addition, the PCA response was significantly decreased in animals with an ongoing auto-aIgE response . Immunopurified auto-aIgE also induced histamine release in vitro from rat peritoneal MC . These results are discussed in the context of naturally occurring autoantibodies to IgE present in patients with allergic disease.

Biochem Biophys Res Commun, 1993 Mar 31, 191(3), 922 - 7
Specific interaction of guanidine hydrochloride with essential carboxyl group of xylanase from alkalothermophilic Bacillus sp; Chauthaiwale J et al.; Experimental evidence for the specific interaction of guanidine hydrochloride with the carboxyl group of xylanase has been presented for the first time . Guanidine hydrochloride (0.1 M) inactivated the xylanase from alkalothermophilic Bacillus sp . to 50% without affecting the conformation of the protein as determined by the fluorometric profile . The kinetic analysis indicated a competitive type of inhibition and a requirement of 1.4 molecules of guanidine hydrochloride per molecule of the enzyme for inhibition . Maximum inhibition occurred at the pH which is optimum for the enzyme activity . The reaction of guanidine hydrochloride with the enzyme prior to modification by Woodward's Reagent K, a specific inhibitor of the carboxyl group, made it inaccessible for modification as indicated by absorbance data at 340 nm . Urea, sodium dodecyl sulphate, LiCl, KCl and NaCl at 0.1 M concentration each had negligible effect on the enzyme activity.

Biochemistry, 1993 Mar 23, 32(11), 2845 - 52
Significance of hydrophobic S4-P4 interactions in subtilisin 309 from Bacillus lentus; Bech LM et al.; The subtilisins have an extended substrate binding cleft comprising at least 8 subsites . Two pockets at the S1 and S4 sites are particularly conspicuous, and the interactions between substrate and these two pockets are very important for the substrate specificity . Phe residues have mutationally been introduced at one of positions 102, 128, 130, and 132 of the subtilisin Savinase from Bacillus lentus to investigate the effects of introducing bulky groups along the rim of the S4 binding pocket . It is shown that the marked P4 preference of wild-type Savinase for aromatic groups is eliminated by the Gly102-->Phe and Ser128-->Phe mutations, indicating that bulky groups at positions 102 and 128 block the S4 binding site . In contrast, the activity toward hydrophilic P4 residues is not nearly as affected by these mutations, suggesting that the binding mode of the P4 side chain is dependent on its properties . Introduction of a bulky -CH2-S-CH2-CH2-pyridyl group at position 128, by mutational incorporation of Cys followed by chemical modification with 2-vinylpyridine, has essentially the same effect . The Ser130-->Phe mutation hardly affects the activity of the enzyme while the Ser-->Phe mutation at position 132 renders the preference for hydrophobic groups in P4 even more pronounced . This mutation furthermore affects the size of the S4 pocket . An analysis of double mutants at positions 132 and 104 suggests that the S4 region is flexible and is adjusted upon binding of substrates.

Eur J Biochem, 1993 Mar 15, 212(3), 801 - 9
Structural studies of the O-antigenic polysaccharide of Fusobacterium necrophorum; Hermansson K et al.; The O-specific polysaccharide component of the lipopolysaccharide produced by Fusobacterium necrophorum is of the teichoic acid type, with repeating units connected by phosphoric diester linkages . Dephosphorylation of the polysaccharide by treatment with aqueous hydrogen fluoride yielded a carbohydrate composed of a trisaccharide linked to an acidic component . This product, and the polysaccharide, were investigated by chemical methods and 1H-, 13C-, 31P- and 15N-NMR spectroscopy and the former also by fast-atom-bombardment mass spectrometry . It is proposed that the polysaccharide is composed of repeating units having the following structure, in which Fuc represents fucose (6-deoxy-galactose), Am represents an acetamidino group and Sug 2,4-diamino-2,4,6-trideoxy-D-glucose ('bacillosamine') acetylated at the 2-position and acylated with a (S)-3-hydroxybutanoic acid at the 4-position . The acid was identified as a 2-amino-2-deoxy-2-C-methyl-pentonic acid (2-amino-2-methyl-3,4,5-trihydroxypentanoic acid) . The configuration of this acid remains to be determined . {formula: see text}

Proc Natl Acad Sci U S A, 1993 Mar 15, 90(6), 2355 - 9
Structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei determined from Laue data; Vellieux FM et al.; The three-dimensional structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase {D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.12.1.12} from the sleeping-sickness parasite Trypanosoma brucei was solved by molecular replacement at 3.2-A resolution with an x-ray data set collected by the Laue method . For data collection, three crystals were exposed to the polychromatic synchrotron x-ray beam for a total of 20.5 sec . The structure was solved by using the Bacillus stearothermophilus enzyme model {Skarzynski, T., Moody, P . C . E . & Wonacott, A . J . (1987) J . Mol . Biol . 193, 171-187} with a partial data set which was 37% complete . The crystals contain six subunits per asymmetric unit, which allowed us to overcome the absence of > 60% of the reflections by 6-fold density averaging . After molecular dynamics refinement, the current molecular model has an R factor of 17.6% . Comparing the structure of the trypanosome enzyme with that of the homologous human muscle enzyme, which was determined at 2.4-A resolution, reveals important structural differences in the NAD binding region . These are of great interest for the design of specific inhibitors of the parasite enzyme.

Brain Res, 1993 Mar 5, 605(1), 155 - 63
Membrane-bound choline-O-acetyltransferase in rat hippocampal tissue is anchored by glycosyl-phosphatidylinositol; Smith LK et al.; In an earlier study, we presented evidence to suggest that some of the particulate choline-O-acetyltransferase (ChAT) in rat hippocampal tissue might be linked to membranes by a glycosyl-phosphatidylinositol (GPI) anchor . In the present report, we attempted to determine if any of this GPI-anchored ChAT might be intracellular . Internalization of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis into rat hippocampal synaptosomes by the DMSO (dimethyl sulfoxide) freeze/thawing procedure caused an increase in cytosolic and a decrease in membrane-bound ChAT . Incubation of a plasma membrane enriched subcellular fraction at 16 degrees C relative to 4 degrees C led to a conversion of the membrane-bound, amphiphilic ChAT into hydrophilic ChAT . This conversion was blocked by zinc, an inhibitor of GPI-PLC . The cytosolic fraction of ChAT immunoreacted on western blots with an antibody directed against the cross-reacting determinant (CRD) of the GPI anchor . We suggest that some of the membrane-bound ChAT in rat hippocampal tissue is GPI-anchored intracellularly; also, that an endogenous GPI-PLC-like enzyme acts to release it into the cytosol.

J Mol Biol, 1993 Mar 5, 230(1), 323 - 41
The high-resolution structure of the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus; Kalia YN et al.; The three-dimensional structure of a 43-residue active, synthetic peptide encompassing the peripheral subunit-binding domain of dihydrolipoamide acetyltransferase from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of a multi-cooling dynamical simulated annealing protocol using restraints derived from 1H nuclear magnetic resonance spectroscopy . A total of 442 experimentally derived restraints including 13 dihedral angle (phi, chi 1) restraints were used . A final set of 35 structures was calculated with a root-mean-square deviation from the mean co-ordinates of 0.36 A for the backbone atoms and 0.96 A when side-chain heavy atoms were included for the well-defined region comprising residues Val7 to Leu39 . Although assignments were made and sequential connectivities observed for the N-terminal six and C-terminal four residues, the absence of long-range NOEs suggests that the terminal regions are largely unstructured . The binding domain contains two short parallel alpha-helices (residues Val7 to Lys14 and Lys32 to Leu39), a3(10)-helix (residues Asp17 to Val21) and a structured loop made up of overlapping beta-turns (residues Gln22 to Leu31), which enclose a close-packed hydrophobic core . The loop is stabilized to a large extent by Asp34 . This residue is conserved in all peripheral subunit-binding domains and its carboxylate side-chain forms a set of side-chain-main-chain hydrogen bonds with the main-chain amide protons of Gly23, Thr24, Gly25 and Leu31 and a side-chain-side-chain hydrogen bond with the hydroxyl group of Thr24 . We propose that a peripheral subunit-binding site may be located in the loop region, which contains a series of highly conserved residues and provides a number of potential recognition sites . The structured region of the binding domain, comprising 33 residues, represents an exceptionally short amino acid sequence with defined tertiary structure that has no disulphide bond, ligand or cofactor to stabilize the fold . It may be approaching the lower size limit for a three-dimensional structure possessing features characteristic of larger structures, including a close-packed, non-polar interior . The organization of the side-chains in the hydrophobic core may have implications for de novo protein design.

Yakugaku Zasshi, 1993 Mar, 113(3), 264 - 71
{Inducible resistance to macrolide-lincosamide-streptogramin type B antibiotics in Bacillus licheniformis: common structures of macrolide antibiotics capable of inducing the resistance}; Kobayashi H et al.; Whether or not resistance to macrolide-lincosamide-streptogramin type B antibiotics (MLS) can be induced by many macrolide antibiotics (Mac), was inquired in Bacillus licheniformis EMR . Resistance to MLS in the strain was induced by erythromycin, oleandomycin, clarithromycin, roxithromycin, narbomycin, picromycin, kujimycin A or B, mycinamicin I, or rosamicin . On the contrary, josamycin, spiramycin, tylosin, rokitamycin, midecamycin, and miokamycin as well as lincosamide and streptogramin type B antibiotics could not induce MLS-resistance . The results suggest that two common chemical residues of the inducer Mac, that is, 1) a single monosaccharide at C5 in the 14- and 16-membered lactone rings, and 2) one polar group such as dimethylamino or methoxyl at C3' in the sugar, are likely to be responsible for showing the activity of MLS-resistance inducer in Bacillus licheniformis EMR.

Plant Mol Biol, 1993 Mar, 21(6), 1131 - 45
The reconstruction and expression of a Bacillus thuringiensis cryIIIA gene in protoplasts and potato plants; Adang MJ et al.; A Bacillus thuringiensis (B.t.) cryIIIA delta-endotoxin gene was designed for optimal expression in plants . The modified cry gene has the codon usage pattern of an average dicot gene and does not contain AT-rich nucleotide sequences typical of native B.t . cry genes . We assembled the 1.8 kb cryIIIA gene in nine blocks of three oligonucleotide pairs . For two DNA blocks, the polymerase chain reaction was used to enrich for correctly ligated pairs . We compared modified cryIIIA gene with native gene expression by electroporation of dicot (carrot) and monocot (corn) protoplasts . CryIIIA-specific RNA and protein was detected in carrot and corn protoplasts only after electroporation with the rebuilt gene . Transgenic potato lines were generated containing the redesigned cryIIIA gene under the transcriptional control of a chimeric CaMV 35S/mannopine synthetase (Mac) promoter . Out of 63 transgenic potato lines, 58 controlled first-instar Colorado potato beetle (CPB) larvae in bioassays . Egg masses which produced ca . 250,000 CPB larvae were placed on replicate clones of 56 transgenic potatoes . No CPB larvae developed past the second instar on any of these plants . Plants expressing high levels of delta-endotoxin were identified by their toxicity to more resistant third-instar larvae . We show there was good correlation between insect control and the levels of delta-endotoxin RNA and protein.

Mol Biol (Mosk), 1993 Mar-Apr, 27(2), 416 - 28
{Comparison of the heat stability and structure close homologs--Bacillus amyloliquefaciens ribonuclease and Bacillus intermedius 7P ribonuclease}; Makarov AA et al.; Parameters of heat denaturation and intrinsic fluorescence of barnase and its close homologue, binase, in the pH region 2-6 have been determined . Barnase heat denaturation (pH 2.8-5.5) proceeds according to the "all-or-none" principle . Barnase denaturation temperature is lower than that of binase and this difference increases from 2.5 degrees C at pH 5 to 7 degrees C at pH 3 . Enthalpy values of barnase and binase denaturation coincide only at pH 4.5-5.5, but as the pH decreases the barnase denaturation enthalpy decreases significantly and in this respect it differs from binase . The fluorescence and CD techniques do not reveal any distinctions in the local environment of aromatic residues in the two proteins, and the obtained difference in the parameters of intrinsic fluorescence is due to fluorescence quenching of the barnase Trp-94 by the His-18 residue, which is absent in binase . Secondary structures of both native and denaturated proteins also do not differ . Some differences have been found in the barnase and binase electrostatic characteristics, revealed in the character of the dipole moment distribution.

J Biochem (Tokyo), 1993 Mar, 113(3), 355 - 63
Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: molecular cloning, sequence determination, overproduction, and purification; Koyama T et al.; The structural gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli cells . A 1,260-nucleotide sequence of the cloned fragment was determined . This sequence specifies an open reading frame of 891 nucleotides for farnesyl diphosphate synthase . The deduced amino acid sequence shows a 42% similarity with that of E . coli FPP synthase {Fujisaki et al . (1990) J . Biochem . 108, 995-1000} . Comparison with prenyltransferases from a wide range of organisms, from bacteria to human, revealed the presence of seven highly conserved regions . In contrast to thermolabile prenyltransferases, which have four to six cysteine residues, the thermostable farnesyl diphosphate synthase carries only two cysteine residues . This enzyme is also unique in that some of the amino acids that are fully conserved in equivalents from other sources are replaced by functionally different amino acids . Construction of an overproducing strain provided a sufficient supply of this enzyme and it was purified to homogeneity . The purified recombinant enzyme is immunochemically identical with the native B . stearothermophilus enzyme, and it is not inactivated even after treatment at 65 degrees C for 70 min.

Insect Biochem Mol Biol, 1993 Mar, 23(2), 273 - 83
In vitro and in vivo proteolysis of the Bacillus thuringiensis subsp . israelensis CryIVD protein by Culex quinquefasciatus larval midgut proteases; Dai SM et al.; Proteases with trypsin-, chymotrypsin- and thermolysin-like specificity were detected in Culex quinquefasciatus larval midguts . Their activities were monitored by N-terminal amino acid sequence analysis of the Bacillus thuringiensis subsp . israelensis CryIVD toxin proteolytic fragments . These proteases are located in the larval midgut and in different fractions obtained during the preparation of brush border membrane vesicles . The activity of the midgut proteases increased with an increase in pH . Both the chymotrypsin- and thermolysin-like activities are involved in the processing of solubilized CryIVD toxin, whereas an additional trypsin-like protease is necessary for the CryIVD parasporal inclusion processing . The solubilized CryIVD toxin was first cleaved between Thr347 and Phe348 and between Phe348 and Tyr349, generating a 40-kDa N-terminal fragment and a 32.5-kDa C-terminal fragment . The C-terminal domain was resistant to further processing, with only a small amount of a 31-kDa product appearing due to the action of a thermolysin-like protease . However, the N-terminal domain was very unstable, and was further degraded to about 30 kDa . Unlike the solubilized CryIVD toxin, the processing of the CryIVD parasporal inclusion was very slow at neutral pH . Three protease-resistant products were detected at pHs higher than 9.5 with an overnight incubation at 37 degrees C . The 30- and 28.5-kDa C-terminal peptides are proteolytic products of trypsin- and chymotrypsin-like proteases, respectively; while the 28-kDa N-terminal peptide has 27 amino acids deleted from the N-terminal end by a thermolysin-like protease.

Biokhimiia, 1993 Mar, 58(3), 340 - 7
{Characteristics of phospholipid hydrolysis kinetics by phospholipase C from Bacillus cereus . Hydrolysis of phosphatidylinositol in various aggregated states}; Selishcheva AA et al.; The process of phospholipase C hydrolysis of phosphatidylethanolamine (PE) in the form of mixed micelles phospholipid-detergent or in the form of vesicules in the mixture with phosphatidylcholine (PC) was studied . The size of the micelles was measured by dynamic light-scattering and their structure was determined by 31P-NMR spectroscopy . It was found that the kinetics of PE hydrolysis in the micelles by phospholipase C from Bacillus cereus do not follow Michaelis-Menten equation, but at all concentrations studied PE was hydrolyzed significantly slower than PC . The rate of PC hydrolysis was measured in the previous work . The hydrolysis of PE in PC-vesicules was followed with use of Victoria blue R dye method . It was shown that the rate of PE hydrolysis in the vesicules is similar to that of PC.

Clin Exp Dermatol, 1993 Mar, 18(2), 133 - 7
Bacillary epithelioid angiomatosis in acquired immunodeficiency syndrome (AIDS)--clinicopathological and ultrastructural study of a case with a review of the literature; Innocenzi D et al.; Bacillary epithelioid angiomatosis (BEA) is a rare cutaneous disorder usually affecting patients with human immunodeficiency virus (HIV) infection often misdiagnosed as a vascular tumour . We describe a 51-year-old, HIV-positive, Caucasian, homosexual male who developed scattered papulo-nodular lesions with clinicopathological and ultrastructural features of BEA . He had a dramatic therapeutic response to systemic antibiotics . There has been a lack of such reports in the European literature . The differential diagnosis is discussed and a brief review of the English literature to date is included.

Appl Environ Microbiol, 1993 Mar, 59(3), 927 - 32
Purification, characterization, gene cloning, and sequencing of a new beta-glucosidase from Bacillus circulans subsp . alkalophilus; Paavilainen S et al.; An intracellular beta-glucosidase was purified from cell extracts of Bacillus circulans subsp . alkalophilus by NAD affinity and high-performance anion-exchange chromatographies . The enzyme was active against a wide range of aryl-beta-glucosides and beta-linked disaccharides . The structural gene for beta-glucosidase was cloned in Escherichia coli . The beta-glucosidase gene consisted of an open reading frame of 1,350 bp encoding a protein of 450 amino acids with a calculated M(r) of 51,303 . The enzyme exhibited from 45 to 66% identity with five bacterial beta-glucosidases.

Appl Environ Microbiol, 1993 Mar, 59(3), 815 - 21
High-level cryIVD and cytA gene expression in Bacillus thuringiensis does not require the 20-kilodalton protein, and the coexpressed gene products are synergistic in their toxicity to mosquitoes; Chang C et al.; Interactions among the 20-kDa protein gene and the cytA and cryIVD genes located in a 9.4-kb HindIII fragment were studied . A series of plasmids containing a combination of these different genes was constructed by using the Escherichia coli/Bacillus thuringiensis shuttle vector pHT3101 . The plasmids were then used to transform an acrystalliferous strain, cryB, derived from B . thuringiensis subsp . kurstaki . The results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses suggest that although the 20-kDa protein is required for the efficient CytA protein production in E . coli, it is not required in B . thuringiensis . With or without the truncated 20-kDa protein gene, the CtyA and/or CryIVD proteins are produced and form parasporal inclusions in B . thuringiensis cells . However, more-efficient expression is obtained when a second protein, probably acting as a chaperonin, is present . In addition, the time course studies show that the CytA and CryIVD proteins are coordinately produced . Both the crude B . thuringiensis culture and purified inclusions from each recombinant B . thuringiensis strain are toxic to Culex quinquefasciatus larvae . The parasporal inclusions formed in B . thuringiensis cells are mosquitocidal, with CytA synergizing CryIVD toxicity.

Gut, 1993 Mar, 34(3), 371 - 4
Antibodies to Mycobacterium paratuberculosis and nine species of environmental mycobacteria in Crohn's disease and control subjects; Stainsby KJ et al.; Cultural and serological studies have provided limited, often conflicting, evidence of a role for mycobacteria in the pathogenesis of Crohn's disease . Interest has focussed on Mycobacterium paratuberculosis, previously considered to be common in the environment with no major role as a human pathogen . Whether a specific serum antibody response to mycobacteria occurs in Crohn's disease or ulcerative colitis was investigated . Sera from patients with Crohn's disease (n = 38), ulcerative colitis (n = 15), and a healthy control population (n = 30) were assayed in an enzyme linked immunosorbent assay (ELISA) using eight filtered sonicate mycobacterial preparations and a purified protein derivative made from the bovine tubercle bacillus . In addition, IgG, IgM, and IgA levels to M paratuberculosis were determined in sera from patients with active (n = 24) or inactive (n = 29) Crohn's disease and the control populations . There was strong evidence of contact with environmental mycobacteria in all patients and control populations, with the greatest responses to preparations of M avium, M tuberculosis, and M kansasii . A large proportion of patients with Crohn's disease had antibodies that bound most antigens tested but there were no statistical differences between these values and those of the control population . Similarly, there were no differences in antibody levels to M paratuberculosis in patient and control groups . Although a subset of patients with active Crohn's disease (25%) had IgG concentrations that exceeded the control mean by more than 2 SD, this phenomenon may not be specific to Crohn's disease: 20% of a small group of patients with coeliac disease had similarly raised IgG levels to M paratuberculosis . These findings do not provide serological evidence of a role for this organism in the pathogenesis of Crohn's disease.

FEMS Microbiol Lett, 1993 Mar 1, 107(2-3), 179 - 83
Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis; Murakami T et al.; Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA . The antibody against the flagellar antigen of B . cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B . cereus . This common flagellar antigen of B . cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay . Monoclonal antibody H15A5 against common antigenic epitope of B . cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA . This monoclonal antibody reacted with the 61-kDa protein of the flagella of B . cereus H.1 and H.2 and B . thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis . These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B . cereus and B . thuringiensis.

Trop Geogr Med, 1993 Mar, 45(1), 2 - 5
The approach to the leprosy problem in the past in Indonesia . A historical review of leprosy control activities in Indonesia during the last centuries; Zuiderhoek B; Due to the severe disabilities leprosy has always been a disease which appealed to the imagination . In bygone centuries cause and treatment were unknown and the fear to be infected was enormous . In Indonesia medical officers struggled with the problem . As early as the 17th century the disease was described in detail by Ten Rhijne . In the 19th century leprosy was considered hereditary . After the discovery of the leprosy bacillus by Hansen in 1873, confusion continued as the bacillus could not be cultivated . Many therapies were tried, but with no result . At first patients were isolated in leprosaria, later on a more humane system of house-isolation was introduced . Since 1932 Indonesian medical officers have played a prominent part in research and the determination of the future approach to the problem . Seen against the background of our present knowledge about the disease, it is interesting to follow the struggle against leprosy in the past.

J Bacteriol, 1993 Mar, 175(6), 1814 - 22
Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19; Bystrykh LV et al.; The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing . The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa . Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7 . These cofactors are redox active toward alcohol and aldehyde substrates . Both enzymes contain significant amounts of Zn2+ and Mg2+ ions . The primary amino acid sequences of the A . methanolica and M . gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition . The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1.

Chest, 1993 Mar, 103(3), 839 - 43
Empyema of the thorax in adults . Etiology, microbiologic findings, and management; Alfageme I et al.; The etiology, microbiologic findings, and management of 82 episodes of empyema treated by our unit over a period of 6 years were analyzed . Average patient age was 54 years . Eighty-two percent had underlying disease such as alcoholism (29 percent), malignancy (23 percent), and diabetes mellitus (20 percent) . Sixty (73 percent) had an empyema develop secondary to a bronchopulmonary infection . Other etiologies were as follows: infradiaphragmatic sepsis, five cases; iatrogenic, ten cases; and idiopathic, seven cases . Cultures were positive in 76 cases and negative in the remaining 6 (2 positive Gram stains, 1 positive under bacilloscopy, and 3 were sterile) . Anaerobes were isolated from 25 and aerobes from 47 of the positive cultures . A single bacteria was isolated from 43 and multiple organisms (average: 2.63/case) grew on the remaining 33 positive cultures . Length of hospitalization averaged 37 days . Seven patients received antibiotics only, thoracentesis was performed on three, intercostal chest tube drainage was required in 72, and more aggressive surgery was performed on 12 patients (7 with fibrothorax and 5 with pneumonectomy) . Streptokinase was instilled into the pleural space of eight patients with good results . Pleural drainage superinfection occurred at a rate of 8.5 percent . Nine patients died; the remaining recovered . Only three deaths came about as a direct result of the empyema.

Cancer, 1993 Mar 1, 71(5), 1846 - 7
Pulmonary granulomata . A complication of intravesical administration of bacillus Calmette-Guerin for superficial bladder carcinoma; Smith RL et al.; Intravesical administration of bacillus Calmette-Guerin (BCG) is an effective treatment for superficial carcinoma of the bladder . The authors report a pulmonary complication characterized by miliary infiltration on chest roentgenogram and caseating granulomata on lung biopsy specimens . This case and three prior reports suggest that this complication may be a form of hypersensitivity rather than true BCG infection.

Am J Clin Pathol, 1993 Mar, 99(3), 244 - 8
Granulomatous inflammation in bladder wash specimens after intravesical bacillus Calmette-Guerin therapy for transitional cell carcinoma of the bladder; Betz SA et al.; The authors retrospectively reviewed a series of bladder wash cytologic specimens and concurrently available bladder biopsy samples to evaluate granulomatous inflammation in patients receiving intravesical bacillus Calmette-Guerin (BCG) immunotherapy for transitional cell carcinoma of the bladder . Comparisons were made during the 2-month period following six weekly BCG instillations in 25 patients and during the 1-year period following the last dose of BCG in 23 patients . At some point during the 2-month follow-up period, cytologic specimens contained free histiocytes in 64%, histiocyte aggregates (resembling fragments of granulomas) in 76%, and multinucleated histiocytic giant cells in 56% of patients . Similarly, concurrent biopsy samples contained granulomas in 78% and multinucleated giant cells in 56% of patients . During the 1-year period following the last dose of BCG, inflammatory changes peaked within the first 3 months and gradually resolved at 9-12 months . In none of these instances were the cytologic features of granulomatous inflammation misinterpreted as malignant . It was concluded that the cytologic findings after intravesical BCG are more specific than previously realized and closely parallel the histologic changes seen in corresponding bladder biopsy specimens.

J Clin Oncol, 1993 Mar, 11(3), 390 - 9
Adjuvant active specific immunotherapy for human colorectal cancer: 6.5-year median follow-up of a phase III prospectively randomized trial; Hoover HC Jr et al.; PURPOSE: Patients with colon or rectal cancer were entered onto a prospectively randomized, controlled clinical trial of active specific immunotherapy (ASI) with an autologous tumor cell-bacillus Calmette-Guerin (BCG) vaccine . We investigated whether ASI could improve disease-free status and survival . PATIENTS AND METHODS: Ninety-eight patients with Dukes' stage B2-C3 colon or rectal cancer were randomized into groups treated by resection alone or resection plus ASI . Eighty patients met all eligibility criteria . All patients with rectal cancer were to receive 50 Gy of pelvic irradiation . Analysis of distribution of survival and disease-free survival was made on all eligible patients until December 31, 1990 . RESULTS: As a single study, no statistically significant differences were detected in survival or disease-free survival for all 80 eligible patients . However, since it was recognized at the outset that there were treatment differences, in that rectal cancer patients were to receive postimmunotherapy radiation, it was considered that a cohort analysis of the colon and rectal cancer patients might be informative . With a median follow-up of 93 months, there is a significant improvement in survival (two-sided P = .02; hazards ratio, 3.97) and disease-free survival (two-sided P = .039; hazards ratio, 2.67) in all eligible colon cancer patients who received ASI . With a median follow-up of 58 months, no benefits were seen in patients with rectal cancer who received ASI . CONCLUSION: This study suggests that ASI may be beneficial to patients with colon cancer.

J Bacteriol, 1993 Mar, 175(5), 1367 - 74
Prevention of DNA damage in spores and in vitro by small, acid-soluble proteins from Bacillus species; Fairhead H et al.; The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed alpha/beta-type small, acid-soluble spore proteins (SASP) . These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light . In addition, heat treatments (e.g., 85 degrees C for 30 min) which give little killing of wild-type spores of B . subtilis kill > 99% of spores which lack most alpha/beta-type SASP (termed alpha - beta - spores) . Similar large differences in survival of wild-type and alpha - beta - spores were found at 90, 80, 65, 22, and 10 degrees C . After heat treatment (85 degrees C for 30 min) or prolonged storage (22 degrees C for 6 months) that gave > 99% killing of alpha - beta - spores, 10 to 20% of the survivors contained auxotrophic or asporogenous mutations . However, alpha - beta - spores heated for 30 min at 85 degrees C released no more dipicolinic acid than similarly heated wild-type spores (< 20% of the total dipicolinic acid) and triggered germination normally . In contrast, after a heat treatment (93 degrees C for 30 min) that gave > or = 99% killing of wild-type spores, < 1% of the survivors had acquired new obvious mutations, > 85% of the spore's dipicolinic acid had been released, and < 1% of the surviving spores could initiate spore germination . Analysis of DNA extracted from heated (85 degrees C, 30 min) and unheated wild-type spores and unheated alpha - beta - spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA . In contrast, the DNA from heated alpha- beta- spores had more than 10 single-strand breaks per 20 kb . These data suggest that binding of alpha/beta-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival . While the precise nature of the initial DNA damage after heating of alpha- beta- spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination . A role for alpha/beta-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold.

Eur J Biochem, 1993 Mar 1, 212(2), 447 - 55
Dissecting the contributions of a specific side-chain interaction to folding and catalysis of Bacillus stearothermophilus lactate dehydrogenase; Nicholls DJ et al.; X-ray crystallography predicts hydrogen-bonding interactions between the side chains of Thr198 and two other amino acid residues, Glu194 (adjacent to the catalytic His195) and Ser318 (on the alpha-H helix which rearranges on substrate binding) . In order to investigate the contribution of this conserved amino acid residue, Thr198, two mutants of Bacillus stearothermophilus lactate dehydrogenase were created (Val198 and Ile198) . The steady-state kinetic parameters for both mutant enzymes were very similar with increased substrate Km and reduced kcat when compared with the wild-type enzyme . The mutation Val198 allowed non-productive binding of pyruvate to the unprotonated form of His195 . Steady-state kinetic parameters determined for the Val198 mutant enzyme in high solvent viscosity suggested both an altered rate-limiting step in catalysis and implicated Thr198 in allosteric activation by the effector fructose 1,6-bisphosphate (Fru1,6P2) . A shift in the Fru1,6P2 activation constant for the Val198 mutant enzyme suggested that Thr198 stabilises the catalytically competent (Fru1,6P2-activated) form of the enzyme by 6.6 kJ/mol . However, Thr198 was not important for maintaining the thermal stability of the Fru1,6P2-activated form . Equilibrium unfolding in guanidinium chloride indicated that Thr198 contributes 17.2 kJ/mol subunits towards the tertiary structural stability . The results emphasise the importance of the side chain-hydroxyl group of Thr198 which is required for (a) productive substrate binding, (b) allosteric activation and (c) protein conformational stability . The characteristics of the B . stearothermophilus lactate dehydrogenase mutations reported here were significantly different from those of the same mutations made in the corresponding position of the analogous enzyme Thermus flavus malate dehydrogenase {Nishiyama, M., Shimada, K., Horinouchi, S., & Beppu, T . (1991) J . Biol . Chem . 266, 14294-14299}.

Mol Cell Biol, 1993 Mar, 13(3), 1522 - 33
Phosphatidylcholine hydrolysis and c-myc expression are in collaborating mitogenic pathways activated by colony-stimulating factor 1; Xu XX et al.; Stimulation of diglyceride production via phospholipase C (PLC) hydrolysis of phosphatidylcholine was an early event in the mitogenic action of colony-stimulating factor 1 (CSF-1) in the murine macrophage cell line BAC1.2F5 and was followed by a second phase of diglyceride production that persisted throughout the G1 phase of the cell cycle . Addition of phosphatidylcholine-specific PLC (PC-PLC) from Bacillus cereus to the medium of quiescent cells raised the intracellular diglyceride concentration and stimulated {3H}thymidine incorporation, although PC-PLC did not support continuous proliferation . PC-PLC treatment did not induce tyrosine phosphorylation or turnover of the CSF-1 receptor . The major protein kinase C (PKC) isotype in BAC1.2F5 cells was PKC-delta . Diglyceride production from PC-PLC did not target PKC-delta, since unlike phorbol esters, PC-PLC treatment neither decreased the electrophoretic mobility of PKC-delta nor increased the amount of GTP bound to Ras, and PC-PLC was mitogenically active in BAC1.2F5 cells in which PKC-delta was downregulated by prolonged treatment with phorbol ester . PC-PLC mimicked CSF-1 action by elevating c-fos and junB mRNAs to 40% of the level induced by CSF-1; however, PC-PLC induced c-myc mRNA to only 5% of the level in CSF-1-stimulated cells . PC-PLC addition to CSF-1-dependent BAC1.2F5 clones that constitutively express c-myc increased {3H}thymidine incorporation to 86% of the level evoked by CSF-1 and supported slow growth in the absence of CSF-1 . Therefore, PC-PLC is a component of a signal transduction pathway leading to transcription of c-fos and junB that collaborates with c-myc and is independent of PKC-delta and Ras activation.

FEBS Lett, 1993 Mar 1, 318(2), 167 - 71
Modification of the amino acid specificity of tyrosyl-tRNA synthetase by protein engineering; de Prat Gay G et al.; The amino acid specificity of Bacillus stearothermophilus tyrosyl-tRNA synthetase was studied by site-directed mutagenesis of residues close to the active site . X-ray crystallographic studies of the enzyme have suggested that Asp-176 is a major determinant of amino acid specificity, as its carboxylate is observed to make a hydrogen bond with the hydroxyl group of the substrate tyrosine . Previous efforts to test the importance of Asp-176 by site-directed mutagenesis led to inactive enzymes . We have now investigated the catalytic properties of enzymes altered, not at Asp-176 itself, but instead at two amino acids, Asn-123 and Trp-126, that appear in the crystallographic structure to form hydrogen bonds with Asp-176 . Mutation of Trp-126 does not affect the kinetics of activation with respect to ATP but leads to modest increases in the Km for tyrosine . Conversely, position Asn-123 mutants are strongly affected: 160-fold lower kcat and 5-fold higher Km for the Ala-123; and 17-fold decrease and 270-fold increase, respectively, of the same parameters for the Asp-123 mutation . The specificity against phenylalanine was determined from the ratios of kcat/Km for the amino acids in the pyrophosphate exchange reaction . The ratio of 1.2 x 10(5) for the wild-type enzyme decreases 4-fold on mutation of Asn-123 but increases 7-fold on the mutation of Trp-126-->Phe and 2-fold on Trp-126-->Leu . The wild-type enzyme has not reached the maximum limit of discrimination between tyrosine and phenylalanine.

Cancer Res, 1993 Mar 1, 53(5), 1043 - 50
Clinical observations on adoptive immunotherapy with vaccine-primed T-lymphocytes secondarily sensitized to tumor in vitro; Chang AE et al.; The adoptive immunotherapy of human malignancy requires reliable methods to sensitize and expand patients' T-cells reactive to autologous tumors . In animal studies, we have generated therapeutic effector cells against a poorly immunogenic tumor by a two-step procedure: vaccination of the host followed by the secondary stimulation of vaccine-primed lymph node (LN) cells by in vitro sensitization (IVS) with tumor in the presence of interleukin 2 (IL-2) . Based on these observations, we performed a clinical trial in patients with advanced cancer to evaluate the antitumor efficacy of vaccine-primed LN cells which were similarly activated in vitro . Patients were vaccinated with irradiated autologous tumor admixed with Bacillus Calmette-Guerin and had draining LN excised 10 days later for IVS culture . During IVS culture, LN cells expanded up to 14-fold (average of 8.4-fold) . A mean of 6.7 x 10(9) cells was infused in ten patients (seven melanoma, three renal cell cancer) along with the concomitant i.v . administration of IL-2 (180,000 IU/kg every 8 h for 5 days) . Phenotype analysis of IVS-LN cells revealed 78 +/- 4% CD3+ T-cells which were predominantly CD4+ (67 +/- 5%) with expression of HLA-DR and IL-2 receptor . IVS-LN cells displayed relative specificity of autologous tumor lysis in four of ten cases compared to zero of seven IVS-peripheral blood lymphocytes derived from the same patients as measured by the 51Cr release assay . One mo after therapy, seven of nine patients treated with IVS-LN cells and IL-2 developed delayed-type hypersensitivity reactivity to autologous tumor compared to zero of nine patients treated with tumor vaccination and IL-2 only (P < 0.002) . These observations suggest that antitumor reactivity was passively transferred with the IVS-LN cells . Major toxic side effects including fever, hepatic dysfunction, and weight gain associated with the capillary leak syndrome were associated with exogenous IL-2 administration . Tumor vaccination and cell transfer were well tolerated without significant complications . Of the ten patients treated with IVS-LN cells and IL-2, there were one partial and one minor response, and one patient has had stable disease for 27+ mo . There was no evidence of tumor response in ten patients treated with tumor vaccination and IL-2 only . Further clinical studies evaluating the antitumor reactivity of vaccine-primed LN cells are warranted.

J Urol, 1993 Mar, 149(3), 584 - 7
Tuberculous spondylitis as a complication of intravesical bacillus Calmette-Guerin therapy; Fishman JR et al.; We report a case of tuberculous spondylitis following intravesical bacillus Calmette-Guerin (BCG) instillation . A 90-year-old male physician living in South Africa received an uncomplicated 6-week course of intravesical BCG (Japanese 172 strain) for high grade superficial bladder carcinoma . He experienced a sudden onset of debilitating lower back pain 16 months following this treatment . A lytic lesion involving the anterior T11 and T12 vertebral bodies was diagnosed and subsequently biopsied . An acid-fast organism was isolated after 3 weeks of incubation and was confirmed through deoxyribonucleic acid probe hybridization as a mycobacterium . High performance liquid chromatography analysis speciated the organism as Mycobacterium bovis BCG, proving that it was acquired through the intravesical therapy.

Infect Immun, 1993 Mar, 61(3), 876 - 83
In vitro model of adhesion and invasion by Bacillus piliformis; Franklin CL et al.; An in vitro model of Bacillus piliformis infection was developed to investigate the mechanisms of adhesion and internalization of this obligate intracellular bacterium . Adhesion and internalization events were examined by electron microscopic evaluation of infected Caco-2 cell monolayers . A few bacteria were identified in apical surface invaginations and in vacuoles subjacent to the apical surface, whereas the majority of bacteria were observed free within the cytoplasm, suggesting that B . piliformis entered epithelial cells via a phagocytic process and rapidly escaped the phagosome . To confirm that host cell phagocytosis was involved in entry of B . piliformis into mammalian cells, Intestine 407 cells were treated with the phagocytic inhibitor cytochalasin D, infected with B . piliformis, and evaluated for bacterial internalization by double-fluorescence labeling . The results showed decreased intracellular bacteria, suggesting that internalization was dependent on host cell microfilament function . To examine the role of B . piliformis in internalization, growth of live and Formalin-killed bacteria was compared . Dead bacteria were not internalized, suggesting that B . piliformis actively participates in internalization . B . piliformis appears to enter host cells by a bacterially directed phagocytic process . The in vitro system described should prove invaluable in further investigations of B . piliformis pathogenic mechanisms.

Infect Immun, 1993 Mar, 61(3), 868 - 75
Murine peritoneal macrophages activated by the mycobacterial 65-kilodalton heat shock protein express enhanced microbicidal activity in vitro; Peetermans WE et al.; After an intraperitoneal (i.p.) injection of purified protein derivative, peritoneal macrophages from mice infected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) show an enhanced respiratory burst, inhibit the intracellular proliferation of Toxoplasma gondii, and kill Listeria monocytogenes more efficiently than peritoneal macrophages from normal mice . One of the immunodominant antigens of Mycobacterium spp . is the 65-kDa heat shock protein (Hsp 65), and in the present study, we determined whether injection of this protein into mice leads to activation of their peritoneal macrophages . After an i.p . injection of Hsp 65, peritoneal macrophages from BCG-infected CBA/J mice also released more H2O2, inhibited the proliferation of T . gondii, and killed L . monocytogenes faster than peritoneal macrophages from normal mice, although Hsp 65 was less effective than purified protein derivative . When normal mice were injected with Hsp 65 suspended in saline after a booster injection with Hsp 65, their macrophages did not display enhanced antimicrobial activity, indicating that an adjuvant was required for a cellular immune response against Hsp 65 . In the present study, the adjuvant dimethyl dioctadecylammonium bromide (DDA) was preferred because it contains no endotoxin or mycobacterial antigens and because it has been reported that DDA does not induce the production of gamma interferon . Peritoneal macrophages from C57BL/6 and CBA/J mice that had received a subcutaneous injection of Hsp 65 suspended in DDA followed by an i.p . booster injection of Hsp 65 suspended in saline were activated, as indicated by the enhanced production of H2O2, inhibition of the intracellular proliferation of T . gondii, and increased rate of intracellular killing of L . monocytogenes in vitro relative to that by resident peritoneal macrophages and peritoneal macrophages obtained from mice that had received ovalbumin instead of Hsp 65 . The rate of phagocytosis of L . monocytogenes was not affected by Hsp 65 treatment . Despite the in vitro expression of enhanced microbicidal activity of peritoneal macrophages, no difference in the growth of L . monocytogenes in the liver and spleen between Hsp 65-treated and control mice was found.

Ann Intern Med, 1993 Mar 1, 118(5), 331 - 6
Syndrome of Rochalimaea henselae adenitis suggesting cat scratch disease; Dolan MJ et al.; OBJECTIVE: To describe a clinical syndrome of cat scratch disease caused by Rochalimaea henselae, including methods for isolation of the organism from tissue and for identification . DESIGN: Case series . SETTING: U.S . Air Force referral hospital infectious diseases clinic . PATIENTS: Two previously healthy patients . MAIN MEASUREMENTS AND RESULTS: Two immunocompetent patients who had handled cats developed unilateral upper-extremity adenitis associated with a distal papular lesion and fever . The adenitis and distal lesions persisted and progressively worsened . Cultures of the involved lymph nodes from both patients grew R . henselae, a recently described organism associated with bacillary angiomatosis and peliosis hepatis in human immunodeficiency virus-infected patients and with bacteremia in immunocompromised and immunocompetent hosts . The organism was characterized as oxidase negative and X-factor dependent and had a characteristic pattern in analysis of whole-cell fatty acids differing from Afipia felis, a bacterium that has been associated with cat scratch disease . The identity of the isolate was confirmed by analysis of whole-cell fatty acids using gas chromatography and by amplification of the citrate synthetase gene sequence and analysis of the polymerase chain reaction-amplified product . The organisms were broadly susceptible to a variety of antimicrobials by broth microdilution; however in-vitro resistance to first-generation cephalosporins correlated with clinical failure of therapy . CONCLUSION: Rochalimaea henselae can be a cause of cat scratch disease in immunocompetent patients.

Radiology, 1993 Mar, 186(3), 833 - 8
Human immunodeficiency virus infection: musculoskeletal manifestations; Steinbach LS et al.; The authors retrospectively reviewed the charts, radiographs, and other accompanying imaging studies of 45 patients with musculoskeletal abnormalities associated with human immunodeficiency virus (HIV) infection . These included 19 patients with osseous infection, including eight with osteomyelitis, seven with bacillary angiomatosis (six of whom were described in a previous report), and four with septic arthritis; 10 with bacterial myositis (six of whom were described in a previous report); seven with non-Hodgkin lymphoma; five with hypointense marrow signal intensity at magnetic resonance imaging; two with Kaposi sarcoma; one with polymyositis; and one with psoriasis . The musculoskeletal system can be affected by a variety of abnormalities in association with HIV infection . Knowledge of their existence and characteristic appearance is valuable to radiologists for diagnosis and to clinicians for detection and appropriate treatment.

Microb Pathog, 1993 Mar, 14(3), 169 - 76
Resistance of genetically selected mice to MHV3 infection is not dependent on the H2O2 release by macrophages; Vassao R et al.; The genetically selected high antibody responder line of mice (HIII) for a natural immunogen were fully susceptible to mouse hepatitis virus 3 (MHV3) and the corresponding low antibody responder mice (LIII) were fully resistant, regardless of whether they were previously treated or not with the bacillus Calmette-Guerin (BCG) . The resistance or susceptibility correlated with the virus growth in the peritoneum of mice . Peritoneal cells isolated from resistant mice released higher amounts of H2O2 after phorbol myristate acetate (PMA) stimulation than susceptible mice . No spontaneous in vitro H2O2 release by peritoneal exudate cells from HIII mice, shown to be mostly macrophages, were observed during the MHV3 infection . In contrast, the spontaneous in vitro H2O2 release by these cells from MHV3-infected LIII mice increased gradually, reaching a maximal value 3 days after infection, and decreased in parallel to the virus clearance from the peritoneum . The BCG treatment primed the macrophages of HIII mice for the production of H2O2 during the MHV3 infection, but did not confer resistance against the virus infection . The data obtained suggest that the acquired capability of macrophages to release H2O2 does not participate in the anti-MHV3 activity or resistance against the MHV3 infection.

Biophys J, 1993 Mar, 64(3), 784 - 91
Crystallization of phosphatidylinositol-specific phospholipase C from Bacillus cereus; Bullock TL et al.; Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves phosphoinositides into two parts, lipid-soluble diacylglycerol and the water-soluble phosphorylated inositol . Two crystal forms of Bacillus cereus PI-PLC have been obtained by the vapor diffusion technique . Hexagonal crystals were grown from solutions containing polyethylene glycol (PEG; 4,000 to 8,000 D) . The space group of these hexagonal crystals is P6(1)22 (or the enantiomorphic space group P6(5)22), with cell constants a = b = 133 A, and c = 231 A . The crystals diffract to 2.8 A . The second crystalline form was grown from a two-phase PEG (600 D)-sodium citrate solution . The phase diagram and PI-PLC distribution between phases has been determined . The enzyme crystallizes from the PEG-rich phase . The crystals are orthorhombic with space group P2(1)2(1)2(1) (a = 45 A, b = 46 A, c = 160 A), and contain one PI-PLC monomer per asymmetric unit . The orthorhombic crystals diffract to 2.5 A . Both the hexagonal and orthorhombic forms are suitable for crystallographic studies.

Support Care Cancer, 1993 Mar, 1(2), 92 - 7
Hickman catheters in association with intensive cancer chemotherapy; Newman KA et al.; Hickman catheters were the major venous access devices utilized at the University of Maryland Cancer Center from November 1978 to 1987 . This study provided an opportunity to standardize insertion technique, to manage catheter-related activities and daily maintenance procedures in order to examine the progression of Hickman-catheter-related problems, to identify those factors that may minimize them, and to develop guidelines for the management and prevention of complications and malfunctions . In all, 690 Hickman catheters (368 double lumens) were placed in patients with acute leukemia and other cancers: 401 catheters were placed in patients with leukemia; 269 were placed during neutropenia; and 230 at platelet counts of < 50,000/microliters . Two surgeons inserted 490 catheters, and the remaining 200 were placed by a group of rotating surgeons . All catheters were placed with the intention that they would remain in place as long as clinically necessary . Total Hickman catheter days were 134273 . Infectious complications included exit site infections (160), tunnel infections (46) and bacteremias (397) . There were 438 instances of noninfectious complications including thrombosis, lack of function, catheter migration, fracture and hemorrhage . Recommendations for prevention and treatment of Hickman-catheter-related complications include the development of a select group committed to placement, daily maintenance and management of problems; prompt removal of catheters with Candida sp . fungemia and bacteremia due to Bacillus sp . or a bacteremia that persists for > 48 h after initiation of appropriate antibiotics, tunnel infections or Hickman-catheter-associated thrombosis . The majority of bacteremias and exit site infections can be effectively treated with antibiotics and local care.

Indian J Malariol, 1993 Mar, 30(1), 37 - 41
Effect of temperature on toxicity of two bioinsecticides spherix (Bacillus sphaericus) and bactoculicide (Bacillus thuringiensis) against larvae of four vector mosquitoes; Mittal PK et al.; Two bioinsecticide preparations, viz . Spherix (Bacillus sphaericus) and Bactoculicide (Bacillus thuringiensis H-14), were tested in the laboratory against larvae of Anopheles culicifacies, Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti at different temperatures . The LCs50 of Spherix against III instar larvae of these species at 27 +/- 2 degrees C were 2.0, 0.19, 0.05 and > 40 mg/litre, respectively and those of Bactoculicide were 0.32, 0.16, 0.06 and 0.03 mg/litre, respectively . The toxicity of two bioinsecticides, especially Spherix, varied to a great extent when the tests were repeated at 21 +/- 2 degrees C and 31 +/- 2 degrees C . At 21 +/- 2 degrees C, Spherix was almost non-toxic against larvae of An . culicifacies and An . stephensi (LC50 > 10 mg/l) but at 31 +/- 2 degrees C the bioinsecticide was highly toxic against An . culicifacies (LC50 = 0.48 mg/litre) and An . stephensi (LC50 = 0.04 mg/litre) . A similar effect of the temperature was also observed with Bactoculicide.

Indian J Malariol, 1993 Mar, 30(1), 17 - 21
Application of bactoculicide (Bacillus thuringiensis H-14) for controlling mosquito breeding in industrial scrap at BHEL, Hardwar (U.P.); Dua VK et al.; Bactoculicide (Bacillus thuringiensis) was evaluated in field trials for controlling mosquito breeding of Aedes, Culex and Anopheles in industrial scraps such as broken heavy machine parts, iron moulds and discarded drums . A dose of 0.5 g/m2 was controlled 96-100% mosquito breeding up to five weeks.

Aust Vet J, 1993 Mar, 70(3), 92 - 7
Tuberculosis in wild seals and characterisation of the seal bacillus; Cousins DV et al.; Tuberculosis was diagnosed in 3 otariid seals found dead on beaches at 3 locations on the south coast of Western Australian between May 1990 and March 1991 . This confirms that tuberculosis is present in the 2 native seals (Neophoca cinerea and Arctocephalus forsteri) in Western Australian waters . Mycobacterium sp isolated from the lungs of 2 of the seals were studied to determine the similarity of the strains to each other, to the strains isolated during 1986 from Australian sea lions and New Zealand fur seals kept in captivity at a marine park near Perth, Western Australia, and to a strain isolated in 1988 from a seal trainer who worked with the infected captive seals for 3 years . After restriction endonuclease analysis (REA) with the endonucleases Bst EII, Bcl I and Pvu II, one of the wild seal strains appeared to have identical DNA fragment patterns to the strains from the captive seals and the seal trainer . The other wild seal isolate had identical REA profiles using Bst EII and Bcl I, but a minor difference was detected using Pvu II . Differences in these isolates were more clearly seen in restriction fragment length polymorphisms after hybridisation with two DNA probes . The secretory protein MPB70, present in M bovis, was not detected in wild seal isolates using sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting techniques . Analysis of protein and DNA fragment profiles indicated that seal tuberculosis isolates form a unique cluster within the M tuberculosis complex.

J Am Mosq Control Assoc, 1993 Mar, 9(1), 17 - 22
Effect of rice husbandry on mosquito breeding at Mwea Rice Irrigation Scheme with reference to biocontrol strategies; Asimeng EJ et al.; A study was carried out at Mwea Rice Irrigation Scheme, Kenya, to assess the impact of rice husbandry on mosquito breeding and identify indigenous biocontrol agents with potential for controlling mosquito breeding in the scheme . The study established a close relationship between the schedule of the farming practices (particularly the flooding phase) and mosquito breeding . Two groups of agents, entomopathogenic bacteria (Bacillus thuringiensis var . israelensis) and larvivorous fish, were identified . Laboratory evaluation of the agents produced encouraging results . The bacterial isolates showed broad-spectrum larvicidal potency against Anopheles, Culex and Aedes mosquito larvae and 2 of the fish species, Tilapia zilli and Oreochromis niloticus, demonstrated a strong predation for a mosquito larval diet . To facilitate their use in effective biocontrol strategies, the agents would require further evaluation under field conditions.

J Biotechnol, 1993 Mar, 28(1), 31 - 40
Rational protein engineering and industrial application: structure prediction by homology and rational design of protein-variants with improved 'washing performance'--the alkaline protease from Bacillus alcalophilus; Aehle W et al.; The successful attempt is presented to engineer an enzyme with respect to its technical application by the use of computer-aided protein design techniques . Based on a modeled 3-D structure a number of mutants of a subtilisin-like protease was designed with the aim to increase its washing performance . The model of the highly alkaline subtilisin protease OPTICLEAN from Bacillus alcalophilus was developed by the process of 'modeling by homology' starting with the structure of subtilisin Carlsberg 1CSE.BRK from the Brookhaven protein databank . Amino acid changes and deletions were performed with the graphic protein design program BRAGI . Force field calculations and molecular dynamic simulations were made with AMBER 3.0 . The comparison of the model and the later solved X-ray structure of OPTICLEAN shows a high similarity between the two structures . On the other hand, interesting deviations between the two structures were observed in some external loop regions . The comparison shows that the deviations are due to difficulties in the prediction of correct main chain torsion angles of additional prolines and the selection of correct loops in deletion or insertion regions.

Clin Neuropathol, 1993 Mar-Apr, 12(2), 121 - 4
Whipple's disease with tubular aggregates in asymptomatic muscle; Stamboulis E et al.; A patient with Whipple's disease aged 34 years is presented . At muscle biopsy, the patient presented with tubular aggregates of the quadriceps muscle . The patient did not exhibit any clinical and electromyographical indications of muscular involvement nor direct or indirect histological indications of muscular infiltration with Whipple's bacillus . The possible etiopathogenetic factors creating these aggregates are discussed.

J Neuroimmunol, 1993 Mar, 43(1-2), 151 - 9
Schwann cells are able to present exogenous mycobacterial hsp70 to antigen-specific T lymphocytes; Ford AL et al.; Peripheral nerves are frequently damaged during infection with Mycobacterium leprae . Although Schwann cells are host for this obligate intracellular parasite, the mechanisms of immunopathology are unresolved . This study examines the ability of Lewis rat Schwann cells to present an exogenous Mycobacterium leprae protein, the heat shock protein 70 (hsp70), to antigen-specific T lymphocytes isolated from the lymph nodes of immunised rats . Secondary reactivation of hsp70-specific T lymphocytes occurred producing an antigen-specific lymphoproliferative response . This was inhibited by monoclonal antibodies against rat major histocompatibility complex (MHC) class II molecules, but not antibodies against MHC class I molecules . Coculture of Schwann cells with the M.leprae hsp70-specific T lymphocytes and antigen (MLrp70) induced the expression of MHC class II molecules on the Schwann cell's surface . Although M.leprae hsp70 is immunodominant in the host response to the bacillus, there is a high degree of homology between human and M.leprae hsp70 . The M.leprae hsp70-specific T lymphocytes also recognised human hsp70 presented by Schwann cells confirming that antigenic determinants are conserved between the proteins . The ability of Schwann cells to present protein antigens in an MHC class II-restricted manner, to antigen-specific T lymphocytes involved in surveillance of the peripheral nervous system, may play an important role in the activation of an immunological reaction associated with nerve damage seen in tuberculoid leprosy.

J Med Microbiol, 1993 Mar, 38(3), 177 - 82
Antigenic diversity in flagellar epitopes among Bacillus piliformis isolates; Boivin GP et al.; Monoclonal antibodies (MAbs) were developed to Bacillus piliformis isolate-specific flagellar epitopes and used to group B . piliformis isolates on the basis of epitope expression . BALB/c mice immunised with flagella purified from various B . piliformis isolates served as the source of immune spleen cells for fusion with SP2/0Ag14 myeloma cells . Evaluation of hybridoma culture medium by ELISA against various bacterial species and B . piliformis isolates indicated that 482 of 2127 hybridomas secreted antibodies specific for B . piliformis . Specificity of MAbs for flagellar epitopes was demonstrated by indirect fluorescent antibody assays and Western blot analyses . Probing of 10 B . piliformis isolates with MAbs indicated that four B . piliformis isolates each possessed a distinct and isolate-specific flagellar epitope; five other isolates shared a common flagellar epitope . One isolate did not react with any of the MAbs specific for flagellar epitopes . Thus, B . piliformis isolates could be grouped into six antigenically distinct groups based upon flagellar epitope expression . Additionally, a MAb reactive with a cell-associated component recognised all but one isolate . This serological grouping of B . piliformis isolates agrees with the grouping of isolates based upon genetic and physiological characteristics, and supports the assertion that there are different strains among B . piliformis isolates.

Nucleic Acids Res, 1993 Feb 25, 21(4), 987 - 91
A unique restriction endonuclease, BcgI, from Bacillus coagulans; Kong H et al.; We have purified and characterized a new restriction endonuclease, BcgI, which has properties unlike those of the three recognized classes of restriction enzymes . BcgI was isolated from Bacillus coagulans, and it recognizes the sequence CGAN6TGC . BcgI cleaves double stranded DNA on both strands upstream and downstream of the recognition sequence, so that the recognition sequence is released as a 34-base pair fragment with 2-base 3'-extensions . Mg++ and S-adenosylmethionine are required for cleavage . Sinefungin, a structural analogue of AdoMet which generally inhibits methylase activity, can replace AdoMet in the cleavage reaction . The apparent binding constant (Kappd) for AdoMet is about 100 nM, while the KappD for sinefungin is about 500 nM.

J Mol Biol, 1993 Feb 20, 229(4), 1037 - 48
Three-dimensional structure of the lipoyl domain from Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex; Dardel F et al.; The structure of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of nuclear magnetic resonance spectroscopy . A total of 452 nuclear Overhauser effect distance constraints and 76 dihedral angle restraints were employed as the input for the structure calculations, which were performed using a hybrid distance geometry-simulated annealing strategy and the programs DISGEO and X-PLOR . The overall structure of the lipoyl domain (residues 1 to 79 of the dihydrolipoamide acetyltransferase polypeptide chain) is that of a flattened eight-stranded beta-barrel folded around a core of well-defined hydrophobic residues . The lipoylation site, lysine 42, is located in the middle of a beta-turn, and the N and C-terminal residues of the domain are close together in adjacent beta-strands at the opposite end of the molecule . The polypeptide backbone exhibits a 2-fold axis of quasi-symmetry, with the C alpha atoms of residues 15 to 39 and 52 to 76 being almost superimposable on those of residues 52 to 76 and 15 to 39, respectively (root-mean-square deviation = 1.48 A) . The amino acid residues at key positions in the structure are conserved among all the reported primary structures of lipoyl domains, suggesting that the domains all fold in a similar way.

Eur J Biochem, 1993 Feb 15, 212(1), 145 - 50
Immunologically unrelated Heliothis sp . and Spodoptera sp . midgut membrane-proteins bind Bacillus thuringiensis CryIA(b) delta-endotoxin; Oddou P et al.; The Bacillus thuringiensis toxins, CryIA(a), CryIA(b), CryIA(c) and CryIC were used in a ligand-blot assay to detect specific toxin-binding proteins in the brush-border membranes of Heliothis virescens, Helicoverpa zea, Spodoptera littoralis, Spodoptera exigua and Spodoptera litura . While CryIA(a) and CryIA(b) always recognize the same protein(s) in a given species, CryIA(c) and CryIC were found to bind to other proteins . Polyclonal antibodies directed against the CryIA(b) binding protein of H . virescens and polyclonal anti-idiotype antibodies recognizing some determinants of the CryIA(b)-binding protein involved in the interaction with the toxin, were used to analyse immunological relationships among the toxin-binding proteins . The results showed that the 170-kDa toxin-binding proteins from the H . virescens and H . zea are immunologically related . However, the toxin-binding proteins from the Spodoptera species did not cross-react with either type of antibodies . Therefore, we conclude that the CryIA(b) toxin has different binding determinants on the toxin molecules itself which can interact with specific binding sites on the toxin-binding proteins from Heliothis sp . and Spodoptera sp.

JAMA, 1993 Feb 10, 269(6), 770 - 5
The epidemiology of bacillary angiomatosis and bacillary peliosis; Tappero JW et al.; OBJECTIVE--To determine environmental risk factors for bacillary angiomatosis-bacillary peliosis (BAP), and to confirm infection with Rochalimaea species . DESIGN--Case-control study . SETTING--Community and university hospitals and clinics . PATIENTS--Case patients (N = 48) had biopsy-confirmed BAP . Controls (N = 94) were matched to patients by institution and by human immunodeficiency virus (HIV) serological status . MAIN OUTCOME MEASURES--Clinical information was obtained from medical records . Subjects were queried about environmental exposures . Univariate odds ratios (ORs) with 95% confidence intervals (CIs) were determined . Bivariate analyses were performed on variables associated with disease by univariate analysis . DNA from 22 available case-patient tissues and from 22 control tissues was amplified with the polymerase chain reaction (PCR) using primers designed to detect Rochalimaea species . RESULTS--We identified five HIV-negative, immunocompetent case patients; one HIV-negative, immunodeficient case patient; and 42 HIV-positive case patients . There were no significant differences between case patients and controls by race, sex, age, or risk factors for HIV infection . Owning a cat (OR, 2.8; CI, 1.4 to 5.8) and history of a recent cat lick (OR, 1.95; CI, 1.0 to 3.8), cat scratch (OR, 3.7; CI, 1.7 to 8.0), or cat bite (OR, 3.9; CI, 1.8 to 8.9) were associated with disease in the univariate analysis . In bivariate analyses, only the variables representing traumatic contact with a cat (bite or scratch) remained associated with disease . No other environmental exposure was associated with disease . The PCR amplified a DNA fragment of the size expected for Rochalimaea species in all 22 case-patient tissue specimens . CONCLUSIONS--These data suggest that BAP is a new zoonosis associated with both traumatic exposure to cats and infection with Rochalimaea species or a closely related organism.

FEBS Lett, 1993 Feb 8, 317(1-2), 89 - 92
Stabilization of Bacillus stearothermophilus neutral protease by introduction of prolines; Hardy F et al.; The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein . In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by proline . The mutations caused large positive (Ser-65-->Pro, Ala-69-->Pro) or negative (Thr-63-->Pro, Tyr-66-->Pro) changes in thermostability, which were explained on the basis of molecular modelling of the mutant proteins . The data show that the introduction of prolines at carefully selected positions in the protein can be a powerful method for stabilization.

J Biol Chem, 1993 Feb 5, 268(4), 2997 - 3004
Inhibition by barbiturates of the binding of Bm3R1 repressor to its operator site on the barbiturate-inducible cytochrome P450BM-3 gene of Bacillus megaterium; Shaw GC et al.; In our previous publication (Shaw, G.-C., and Fulco, A . J . (1992) J . Biol . Chem . 267, 5515-5526), we reported that Bm3R1, a protein encoded in an open reading frame just upstream from the cytochrome P450BM-3 gene, is a repressor critically involved in the barbiturate-inducible expression of this gene in Bacillus megaterium . We now describe the purification of the Bm3R1 protein from an overproducing Escherichia coli strain harboring a bm3R1 gene-carrying plasmid and report the effect of barbiturate inducers on the interaction of Bm3R1 with a fragment of B . megaterium DNA containing the bicistronic operator and promoter sequences . Gel filtration analysis revealed that, under our experimental conditions, most of the Bm3R1 protein exists in highly aggregated forms . Gel mobility shift assays showed that Bm3R1 protein bound specifically to a segment of DNA containing the promoter-operator region of the bm3R1 gene while DNase I footprinting experiments established that Bm3R1 protected a region of DNA that covers and flanks the palindromic operator sequence . The interaction between Bm3R1 repressor and its operator, in vitro, was strongly inhibited by the addition of 2 mM pentobarbital or 2 mM methohexital (strong in vivo inducers of P450BM-3) but not by the same concentration of phenobarbital (a relatively weak inducer) or by mephobarbital (a non-inducer) . A detailed comparison of pentobarbital and methohexital at concentrations lower than 2 mM indicated that methohexital was 5-10 times more effective as an inhibitor of Bm3R1 binding in vitro, as compared with its 7-fold greater inducer potency in vivo . The observation that the in vitro inhibition effects of barbiturates on the interaction of Bm3R1 repressor and its operator correlate strongly with their in vivo potency as inducers of cytochrome P450BM-3 suggests a mechanism for the induction process . It seems plausible that the barbiturate inducers might bear a conformational resemblance to and mimic the mode of action of an as yet unidentified endogenous inducer(s) in B . megaterium that functions by releasing the binding of Bm3R1 repressor from its operator site.

J Biol Chem, 1993 Feb 5, 268(4), 2431 - 4
NMR studies of phospholipase C hydrolysis of phosphatidylcholine in model membranes; Bhamidipati SP et al.; Hydrolysis of phospholipids in biological membranes by phospholipase C (PLC) produces an important second messenger molecule, 1,2-diacylglycerol (DAG), that is essential for the activation of protein kinase C (PKC) . While the effects of DAG on model membranes have been investigated earlier, studies on physical properties of DAG introduced into phospholipid bilayers by PLC have been lacking . We present an NMR approach for studying structural and kinetic aspects of PLC-mediated hydrolysis of 13 carbonyl-enriched phospholipids in model membranes (small unilamellar vesicles) . The product DAG is readily detected by 13C NMR, and its structural properties as well as those of the model membrane can be monitored continuously . PLC hydrolysis was limited to a low proportion of the model membrane by incorporating a small amount of ester phospholipid into a nonhydrolyzable ether-linked phospholipid matrix . Under these conditions, PLC (Bacillus cereus) hydrolyzed only the monolayer of phosphatidylcholine to which it was exposed (the outer monolayer) . The 1,2-DAG product remained associated with the membrane bilayer and did not alter bilayer structure in any detectable way . From the chemical shift data, it is inferred that the DAG has an interfacial conformation similar to that of phosphatidylcholine . These results show that DAG could activate PKC by direct interaction with the enzyme rather than by perturbation of the membrane bilayer.

J Chromatogr, 1993 Feb 5, 630(1-2), 151 - 7
Sequence-specific DNA affinity chromatography: application of a group-specific adsorbent for the isolation of restriction endonucleases; Pozidis C et al.; The use of sequence-specific DNA affinity adsorbents for the isolation of restriction endonucleases EcoRI and SphI to near homogeneity has been reported . However, the high cost of these adsorbents is a limiting factor for their wider application . This paper reports the application of sequence-specific DNA affinity ligands containing recognition sequences for 34 restriction endonucleases as group-specific ligands in the isolation of restriction endonucleases . Crude samples of six restriction endonucleases, namely BshFI, BamHI, SmaI, SacII, PvuII and SalI, were shown to bind to these adsorbents and could be eluted at different KCl concentrations . High purification factors and recoveries were obtained . Restriction endonuclease BshFI, an isoschizomer of HaeIII, from the microorganism Bacillus sphaericus was purified to near homogeneity employing a two-step procedure which involves DNA-cellulose chromatography and oligonucleotide-ligand affinity chromatography . The enzyme exists as a monomer with an apparent relative molecular mass of 34,000 as determined by both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion chromatography.

Biochemistry, 1993 Feb 2, 32(4), 1040 - 6
Suppression of protein structure destabilizing mutations in Bacillus thuringiensis delta-endotoxins by second site mutations; Almond BD et al.; Reciprocal exchange of a small region (residues 428-450) within the specificity determining region of two Bacillus thuringiensis delta-endotoxins, CryIAa and CryIAc, resulted in two recombinant proteins that possess a decreased insecticidal activity to Bombyx mori and Manduca sexta . Site-directed mutations introduced in this region of one of the recombinant proteins, with the intent of restoring insecticidal activity, resulted in further reduction of toxicity . We determined that the loss of insecticidal activity in the mutants and the original recombinants was associated with altered toxin protein structure, as measured by sensitivity to intracellular and exogenous proteases . The structural instability of the site-directed mutant proteins could be suppressed genetically by subcloning the mutated region into cryIAc or by introducing second site mutations in defined regions of the mutated cryIAa gene . The second site mutations, by themselves, also produced unstable proteins . In the course of this study, we demonstrated that this small region does not suffice as a specificity determining region for M . sexta.

J Endod, 1993 Feb, 19(2), 68 - 70
Effect of sterilization on contaminated sponges; Kuritani RH et al.; Sponges are routinely used as a storage medium for endodontic files during clinical practice; however, very little research has been done to determine the effectiveness of sterilization procedures for these contaminated sponges . The purpose of this in vitro study was to determine the efficacy of chemical vapor sterilizers (chemiclaves), steam pressure sterilizers (autoclaves), and dry heat sterilizers on laboratory contaminated sponges . Four different types of sponges were used in this study: a black, relatively nonporous sponge; a red, semiporous stationary sponge; a blue, endodontic sponge, and a yellow, common household sponge . Natural sponges were eliminated from the study, because their large pore size made them unsuitable as a storage medium for endodontic instruments . The sponges were divided into three groups: chemiclave, autoclave, and dry heat . Five samples of each sponge type were impregnated with biological indicating strips containing spores of Bacillus stearothermophilus . Each sponge was subjected to 25 cycles of sterilization . The spore strip indicator was inserted into the sponges at 1, 5, 10, 15, 20, and 25 cycles . The spore strip was cultivated in trypticase soy broth medium solution at 55 +/- 1 degree C for 7 days . At 7 days the culture vials were read for turbidity; its presence indicating a positive culture . The samples that were subjected to chemiclaving demonstrated positive cultures of 0.00%, 0.00%, and 30.00% and those to autoclaving 3.33%, 0.00%, and 0.00% positive cultures for the black, red, and blue sponge types, respectively . None of the sponges survived dry heat sterilization . The O-Cell-O sponges become unusable when subjected to all of the sterilization methods used in this study.

Wei Sheng Wu Xue Bao, 1993 Feb, 33(1), 62 - 8
{Studies on insecticidal ingredients from culture supernatant of Bacillus thuringiensis}; Cui Y et al.; Studies on the insecticidal activity of culture supernatant of Bacillus thuringiensis var . kurstaki HD-1, by centrifugation, column chromatography, protein sequence determination, bioassay and other methods . Found that insecticidal activity of culture supernatant appeared on synchronism with cells lysis . Main insecticidal ingredients of culture supernatant is toxin protein of 60 kilodaltons (kDa) . Analyse hat purified the toxin protein, we find that its amino acid composition was similar with 135-kDa P1 protein, and its sequence of N-terminal 18 amino acid was same with that of part of the P1 protein . The results presented here provided that the toxin protein was the same source with the P1 protein . Further found that supernatant have synergizing action on the sediment of culture.

Int J Food Microbiol, 1993 Feb, 17(4), 269 - 79
Analysis of enterotoxin production by Bacillus cereus from dairy products, food poisoning incidents and non-gastrointestinal infections; Granum PE et al.; Of 85 strains of Bacillus cereus isolated in Norway from dairy products, 59% were found to be enterotoxigenic, and 15% were psychrotrophic . Six of the isolates (7%) were identified as potential psychrotrophic food-poisoning strains as they were both enterotoxigenic and exhibited good growth at 6 degrees C . Enterotoxin production was detected using the Western immunoblot technique, and a commercially available reversed passive latex agglutination (RPLA) assay (Unipath BCET-RPLA TD950) . Both methods gave essentially the same results . In a separate study, the Western immunoblot and RPLA assays were used in a conjunction with the in vivo vascular permeability reaction (VPR) assay to determine enterotoxin production among 25 isolates of Bacillus cereus referred to the PHLS Food Hygiene Laboratory from incidents of diarrhoeal- and emetic-syndrome food poisoning and non-gastrointestinal infections . Eighty-four percent of these isolates were found to be enterotoxigenic by the Western immunoblot and the RPLA assays, and these results were in good agreement with those obtained by the VPR assay . In both studies, the BCET-RPLA kit proved to be a simple and reliable means for determining enterotoxin production by strains of Bacillus cereus.

Br J Urol, 1993 Feb, 71(2), 179 - 82
Peripheral blood lymphocyte response in patients with superficial transitional cell carcinoma of the bladder treated with intravesical Bacillus Calmette-Guérin--a useful marker of response?
Schmidt AC, Bouic PJ, Heyns CF, De Kock ML.
Intravesical instillation of Bacillus Calmette-Guerin (BCG) offers safe and effective short-term/long-term treatment for superficial transitional cell carcinoma (TCC) and TCC in situ of the bladder . However, 17 to 42% of patients may experience recurrence in spite of this therapy and a marker of effective treatment is of paramount importance . In this study the in vitro response of peripheral blood lymphocytes (PBL) to BCG was analysed in 10 patients with superficial TCC and TCC in situ before and during BCG instillations . The in vitro response of PBL to BCG, expressed as a stimulatory index (SI), revealed that 6 patients had a SI > 5 and 4 patients had a SI < 5 . None of the former patients had recurrence of TCC during a mean follow-up of 17 months, while all of the latter patients experienced recurrence of TCC within 9 months . It was concluded that the in vitro response of PBL to BCG may be used as a marker of response to intravesical BCG treatment.

Am J Infect Control, 1993 Feb, 21(1), 28 - 33
Effects of steam sterilization on the contents of sharps containers; Palenik CJ et al.; BACKGROUND: One form of medical waste known to be capable of transmitting disease is the contaminated sharp . Safe handling and disposal of sharps is an essential element of any infection control program . Many areas allow the on-site treatment of sharps containers . However, little information currently exists as to the most effective sterilization procedures and container designs . METHODS: This study was intended to evaluate the effect treatment with various autoclaves had on bacterial endospores present on strips or needled syringes . Strips contained 1.7 x 10(5) Bacillus stearothermophilus spores; syringes were soiled with equal numbers of spores or with spores plus blood . Syringes were tested capped and uncapped . A gravity-displacement autoclave and a high-vacuum autoclave were used . Strips and syringes were placed within sharps containers three quarters filled with representative materials . Six types of containers were tested . Containers were processed sitting up or on their sides . Processed strips and needles were aerobically cultured at 56 degrees C for 7 days . If sterilization was not accomplished initially, additional exposure time was added . RESULTS: (1) Soiled syringes were more difficult to sterilize than strips . (2) Capping or the presence of blood did not affect sterilization efficiency . (3) Container positioning was important only for the gravity-displacement autoclave . (4) Additional exposure time was required in the gravity displacement autoclave when sterilizing soiled syringes but not strips . (5) High-vacuum autoclaving killed all spore challenges within the normal processing interval . CONCLUSIONS: The data indicate that processing of sharps containers within a gravity-displacement autoclave appears to require extended exposure intervals to achieve sterilization.

Eur J Immunol, 1993 Feb, 23(2), 454 - 60
Protective immunity induced in rat schistosomiasis by a single dose of the Sm28GST recombinant antigen: effector mechanisms involving IgE and IgA antibodies; Grezel D et al.; Rats immunized by a single dose of the recombinant Sm28GST antigen, using either aluminium hydroxide or Bacillus Calmette-Guerin adjuvant, were significantly protected (up to 59% reduction in worm burden) against a challenge infection with Schistosoma mansoni cercariae . A follow-up study of the humoral response revealed the presence of high levels of IgE and IgA antibodies together with specific IgG . Sera from once Sm28GST-immunized rats induced a cytotoxic response for schistosomula targets in the presence of normal rat eosinophils, similar to the one induced by sera from twice immunized rats . Depletion or competition studies indicated the participation of both IgE and IgA antibodies in eosinophil-dependent cytotoxicity mechanisms . These results suggest the existence, in immunized rats exhibiting protection against schistosomiasis, of an original effector mechanism implying eosinophils and IgA antibodies, together with documented effector mechanisms involving IgE and eosinophils . In addition, they raise questions concerning the role of IgA antibodies in schistosomiasis.

Eur J Biochem, 1993 Feb 1, 211(3), 899 - 902
Effect of mutation of an amino acid residue near the catalytic site on the activity of Bacillus stearothermophilus alpha-amylase; Takase K; Site-directed mutagenesis of a thermostable alpha-amylase from Bacillus stearothermophilus was performed to assess the role of amino acid residues near the catalytic site in catalysis . Asn329 is presumed to be adjacent to the proposed catalytic residue Asp331 . Its mutation to Lys, which is found at the corresponding position in pullulanase, resulted in the loss of 99.7% of the activity, while the mutation to Asp or Val did not drastically reduce the activity . The mutation to Val altered the temperature/activity profile so that the activity was reduced to 25% of wild-type alpha-amylase at 60 degrees C but was over twofold greater at 5 degrees C . This effect could be ascribed to a decrease in the activation enthalpy by 32% . The mutation to Asp or Lys altered the pH/activity profile concomitant with possible changes in the ionization state of the groups introduced . These results show the feasibility of altering and possibly improving the enzyme activity by mutagenesis of residues near the catalytic groups.

Eur J Biochem, 1993 Feb 1, 211(3), 851 - 9
Molecular cloning and nucleotide sequence of the gene for pyruvate kinase of Bacillus stearothermophilus and the production of the enzyme in Escherichia coli . Evidence that the genes for phosphofructokinase and pyruvate kinase constitute an operon; Sakai H et al.; Pyruvate kinase from Bacillus stearothermophilus is an allosteric enzyme activated by AMP or ribose 5-phosphate but not by fructose 1,6-bisphosphate . The gene for the enzyme was cloned in Escherichia coli and its entire nucleotide sequence was determined . The deduced amino acid sequence consisted of 587 residues and the molecular mass was calculated to be 62 317 Da . The sequence was highly similar to other pyruvate kinases, indicating that they have the same evolutional origin . Similarly to the E . coli enzymes, the enzyme does not contain an N-terminal domain, in contrast to the eukaryotic pyruvate kinases . However, the Bacillus stearothermophilus enzyme had an extra C-terminal sequence consisting of about 110 amino acid residues . A phosphoenolpyruvate-binding motif, which is observed in pyruvate phosphate dikinase, phosphoenolpyruvate: sugar phosphotransferase system enzyme I and phosphoenolpyruvate synthase, was present in the extra C-terminal sequence . There was an open reading frame upstream of the pyruvate kinase gene . The homology of the sequence showed that the gene encodes phosphofructokinase . Both phosphofructokinase and pyruvate kinase were expressed in E . coli cells, and the evidence suggesting that both genes constitute an operon is presented.

Appl Environ Microbiol, 1993 Feb, 59(2), 648 - 51
The antimicrobial effect of a structural variant of subtilin against outgrowing Bacillus cereus T spores and vegetative cells occurs by different mechanisms; Liu W et al.; Subtilin is a ribosomally synthesized antimicrobial peptide that contains several unusual amino acids as a result of posttranslational modifications . Site-directed mutagenesis was employed to construct a structural variant of subtilin in which the unusual dehydroalanine (Dha) residue at position 5 was changed to alanine . Proton nuclear magnetic resonance spectroscopy, amino acid composition, and N-terminal sequence analysis established that the mutation did not disrupt posttranslational processing of the precursor peptide . This mutant subtilin was devoid of antimicrobial activity as assessed by its lack of inhibitory effects on outgrowth of Bacillus cereus T spores . However, this same mutant subtilin was fully active with respect to its ability to induce lysis of vegetative B . cereus T cells . Because an intact Dha-5 residue is required in the one instance but not in the other, it was concluded that the molecular mechanism by which subtilin inhibits (without lysis) spore outgrowth is not the same as the mechanism by which it inhibits (with lysis) vegetative cells.

Appl Environ Microbiol, 1993 Feb, 59(2), 523 - 7
Multiplex polymerase chain reaction for detection and differentiation of the microbial insecticide Bacillus thuringiensis; Bourque SN et al.; A rapid identification of Bacillus thuringiensis strains was established by using multiplex polymerase chain reaction (PCR) . Primers of high homology specific to regions within genes encoding three major classes of B . thuringiensis crystal proteins were used to generate a PCR product profile characteristic of each strain of B . thuringiensis subsp . kurstaki . Differentiation among these strains was made on the basis of the electrophoretic pattern of the PCR products . Known B . thuringiensis subsp . kurstaki strains as well as unidentified strains isolated from insect cadavers were analyzed by PCR . Small amounts of crude sample lysates were assayed in a two-step PCR containing five primers capable of distinguishing between the strains giving products of 1,500, 858, and 653 bp for the CryIA(a) CryIA(b), and CryIA(c) genes, respectively . The method can be applied to rapidly detect the strains of B . thuringiensis subsp . kurstaki in commercial formulations and in the field.

J Bacteriol, 1993 Feb, 175(4), 1053 - 60
A complete physical map of a Bacillus thuringiensis chromosome; Carlson CR et al.; Bacillus thuringiensis is the source of the most widely used biological pesticide, through its production of insecticidal toxins . The toxin genes are often localized on plasmids . We have constructed a physical map of a Bacillus thuringiensis chromosome by aligning 16 fragments obtained by digestion with the restriction enzyme NotI . The fragments ranged from 15 to 1,350 kb . The size of the chromosome was 5.4 Mb . The NotI DNA fingerprint patterns of 12 different B . thuringiensis strains showed marked variation . The cryIA-type toxin gene was present on the chromosome in four strains, was extrachromosomal in four strains, and was both chromosomal and extrachromosomal in two strains . A Tn4430 transposon probe hybridized to 5 of the 10 cryIA-positive chromosomal fragments, while cryIA and the transposon often hybridized to different extrachromosomal bands . Ten of the strains were hemolytic when grown on agar plates containing human erythrocytes . Nine of the strains were positive when assayed for the presence of Bacillus cereus enterotoxin . We conclude that B . thuringiensis is very closely related to B . cereus and that the distinction between B . cereus and B . thuringiensis should be reconsidered.

Pediatr Infect Dis J, 1993 Feb, 12(2), 136 - 9
Evaluation of oral erythromycin and local isoniazid instillation therapy in infants with Bacillus Calmette-Guérin lymphadenitis and abscesses; Noah PK et al.; A randomized placebo-controlled prospective trial was conducted to evaluate the efficacy of erythromycin therapy in 69 patients affected with Bacillus Calmette-Guerin lymphadenitis . When patients who developed subsequent regional abscesses were excluded, erythromycin caused significantly earlier resolution of lymphadenitis (5.1 months vs . 5.7 months for placebo; P < 0.01) compared with placebo . There was no significant difference in the proportion of patients who developed subsequent regional abscesses between the 2 groups (47% for erythromycin, 60% for placebo, P = 0.14) . When the entire group of 69 patients was evaluated for "duration to heal" (regardless of subsequent abscess formation), erythromycin therapy (4.1 +/- 1.5 SD months) did not differ significantly from the placebo group (3.5 +/- 1.3 months, P = not significant) . Patients who developed subsequent abscess (n = 36) along with those with B . Calmette-Guerin regional abscesses at presentation (n = 27) were further studied to compare oral erythromycin therapy with that of single dose 50-mg intranodal isoniazid instillation . Local isoniazid therapy caused significantly earlier resolution of the abscesses (3.9 months) compared with erythromycin therapy (5.2 months; P < 0.001).

J Urol, 1993 Feb, 149(2), 268 - 71
Fibronectin expression on surgical specimens correlated with the response to intravesical bacillus Calmette-Guerin therapy; Fleischmann JD et al.; Attachment of bacillus Calmette-Guerin (BCG) organisms to the bladder during intravesical therapy is thought to be mediated exclusively by the glycoprotein fibronectin, which is expressed variably on epithelial surfaces and on basement membranes . We examined the relationship between the degree of fibronectin expressed on surgical specimens obtained from 50 candidates for BCG therapy and the subsequent clinical response . Immunoperoxidase staining for fibronectin was performed on tumor, nonadjacent normal mucosa and basement membrane tissues, and the intensity of the staining was scored on a scale of 0 to 3+ (control 2+) . In the absence of recurrence at quarterly surveillance cystoscopy, a course of Tice BCG therapy consisted of 6 weekly and 12 monthly instillations . Recurrence of noninvasive tumor prompted a second BCG course . Followup ranged from 24 to 66 months (median 40 months) . Of the 50 patients (11 with carcinoma in situ) disease progression occurred in 9 (none with carcinoma in situ) . Compared to the results for tumors or for basement membranes, the degree of fibronectin expression on normal mucosa was well correlated with the clinical response (r = 0.59, p < 0.001 by Kendall Tau B) . Routine assessment of fibronectin expression on the normal mucosa associated with superficial bladder cancer may be useful for predicting the clinical response to BCG therapy.

J Infect Dis, 1993 Feb, 167(2), 378 - 84
Therapeutic trial of lipid X in a canine model of septic shock; Danner RL et al.; Three groups of dogs were given lipid X (0, 1, or 10 mg/kg) every 8 h for for seven doses, starting simultaneously with the intraperitoneal placement of Escherichia coli-containing fibrin clots . All animals developed bacteremia, hypotension, and a pattern of decreased left ventricular ejection fraction characteristic of septic shock (P = .01) . Survival rates and survival times were not significantly different between treatment groups (P > .2) . In a similar experiment, higher doses of lipid X resulted in a significantly decreased survival time compared with concurrent controls (P = .04) . Animals receiving lipid X did not differ from controls in serial determinations of temperature, hemodynamic measurements, or laboratory parameters (except serum total protein) . Although lipid X has antiendotoxin effects, no benefit could be demonstrated in this antibiotic-treated, gram-negative bacillary-infected model of septic shock . These data do not support a therapeutic role for lipid X in the treatment of gram-negative sepsis.

Glycoconj J, 1993 Feb, 10(1), 64 - 71
Human erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface; Chiarini A et al.; Treatment of human erythrocyte ghosts with phosphatidylinositol-phospholipase C (PIPLC) from Bacillus cereus liberated the ghost-linked sialidase . Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37 degrees C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the beginning of incubation and every 15 min (four subsequent additions) . Liberated sialidase was fully resistant to at least four cycles of rapid freezing and thawing and to storage at 4 degrees C for at least 48 h . The liberated enzyme had an optimal activity at pH 4.2, degraded ganglioside GD1a better than methylumbelliferyl N-acetylneuraminic acid (about fourfold), and gave a Km value of 2.56 x 10(-4) M and an apparent Vmax of 2.22 mU per mg protein on GD1a . Treatment of intact erythrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused liberation of 10-12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the erythrocyte membrane . It is concluded that the erythrocyte membrane sialidase is anchored by a glycosylphosphatidylinositol structure sensitive to PIPLC action, and is partly located on the outer surface.

Comp Biochem Physiol B, 1993 Feb, 104(2), 375 - 9
A rapid and sensitive in vitro assay for the activity of Bacillus thuringiensis delta-endotoxins; Parenti P et al.; 1 . Activated delta-endotoxins from Bacillus thuringiensis strains toxic to lepidopteran larvae inhibit K(+)-dependent accumulation of amino acids into brush border membrane vesicles (BBMV) from the midgut of the susceptible species Bombyx mori . 2 . The activated toxins interfere with the K(+)-dependent uptake of histidine into BBMV only if they are effectively active in vivo . 3 . To calculate IC50 values (the toxin concentration which determines 50% of the effect), dose-response curves were performed for each toxin . The values obtained correlate well with the LD50 determined by bioassay . 4 . This amino acid inhibition test could represent a rapid (3-6 hr, compared to 3-4 days for bioassay) and sensitive method for the screening of larvicidal activity of known or new recombinant delta-endotoxins.

FEMS Microbiol Lett, 1993 Feb 1, 106(3), 275 - 80
Respective role of the 42- and 51-kDa components of the Bacillus sphaericus toxin overexpressed in Bacillus thuringiensis; Nicolas L et al.; The 42- and 51-kDa protein genes of Bacillus sphaericus 1593 have been subcloned independently downstream from the cytA gene promoter of Bacillus thuringiensis serovar israelensis and introduced into a non-mosquitocidal strain of Bacillus thuringiensis . Consequently, each protein was overproduced and accumulated as inclusion bodies which were purified . For the first time, the 42-kDa protein inclusions alone were found to be toxic to Culex pipiens larvae (LC50 at 48 h 300 ng ml-1); in contrast, the 51-kDa protein inclusions were not . Moreover, a synergistic effect between these two components was observed.

FEBS Lett, 1993 Feb 1, 316(3), 264 - 8
Identification and partial purification of a Bacillus thuringiensis CryIC delta-endotoxin binding protein from Spodoptera littoralis gut membranes; Sanchis V et al.; Immunoblotting experiments were performed using CryIC and CryIA(c) Bacillus thuringiensis delta-endotoxins to detect the presence of specific toxin binding proteins on Spodoptera littoralis brush border membrane vesicles . The CryIC toxin binds two proteins of 40 and 65 kDa and the CryIA(c) binds a protein of 40 kDa . The CryIA(c) toxin also binds faintly to a 120 kDa protein on S . littoralis brush border membrane vesicles as does a polyclonal antiserum raised against a putative CryIA(c) 120 kDa binding protein from Manduca sexta . The 40 kDa CryIC binding protein was partially purified by affinity chromatography and is therefore a strong candidate for in vivo S . littoralis CryIC toxin receptor.

Aust N Z J Surg, 1993 Feb, 63(2), 127 - 30
The adverse effect of fibrin-clot inhibiting drugs on intravesical bacillus Calmette-Guérin efficacy for superficial bladder cancer; P'ng KB et al.; Bacillus Calmette-Guerin (BCG) is currently thought to act as a biological immune modifier in effecting antitumour activity . Recent evidence suggests that BCG binding to fibronectin (FN), a tissue glycoprotein, may be a prerequisite step in initiating this response . Drugs inhibiting the availability of exposed FN in the bladder after urothelial disruption may adversely affect the efficacy of BCG . Data are presented of 45 patients with tumour limited to mucosa (pTa) or carcinoma in situ (CIS) given intravesical BCG therapy, with (group 1) or without (group 2) fibrin clot-inhibiting drugs concurrently during treatment . The success rate of 11.1% for group 1 (1/9) patients was significantly less than that of 69.4% for group 2 (25/36), (chi 2 = 7.79, P < 0.01 Fisher's exact test) supporting the suggestion that the concurrent administration of fibrin-clot inhibiting drugs may adversely affect the outcome of BCG therapy.

Rev Rhum Ed Fr, 1993 Feb, 60(2), 162 - 6
{Polyarthritis in 4 patients treated with intravesical BCG-therapy for carcinoma of the bladder}; Belmatoug N et al.; Intravesical administration of Calmette-Guerin bacillus (BCG) is effective in the treatment of superficial bladder carcinoma . Transient arthritis or migratory arthralgia has been reported in 0.5% of cases . The authors report on four men (mean age 65 years), who developed an oligoarthritis in three cases after the 2nd, 5th, and 6th weekly instillation of Pasteur BCG, respectively, and symmetrical polyarthritis in one after 48 BCG instillations over a three-year period . Joints involved were the knees (3/4), ankles (3/4), shoulders (1/4), wrists, and hands (1/4) . Features included morning stiffness and local evidence of inflammation . All four patients had an elevated erythrocyte sedimentation rate and negative tests for rheumatoid factor . Neutrophils were the main cell type in synovial fluid . Synovial biopsy performed in two cases revealed non-specific inflammation . Erosions of the metatarsophalangeal joints were observed in one patient . Locoregional or systemic symptoms included transient fever (3/4), cystitis (4/4), urethritis (1/4), epididymoorchitis (2/4), conjunctivitis (1/4), and pleural effusion with pericarditis (1/4) . The BCG was recovered from an epididymal specimen in one patient . Two patients were positive for the HLA B27 antigen . Outcome was favorable in every case (range: 15 days-6 months) after discontinuation of BCG therapy and administration of isoniazid, rifampin, and prednisone (3 patients) or NSAID (1 patient) . Various pathogenic hypotheses for BCG-induced arthritis are discussed, including infection, immune responses to heat shock proteins, and reactive arthritis.

Boll Soc Ital Biol Sper, 1993 Feb, 69(2), 115 - 9
{Effect of microwaves on the vegetative and spore forms of Bacillus stearothermophilus}; Salvatorelli G et al.; Microwave irradiation provides a rapid and effective method for sterilization of stainless steel scalpel blades or cover glasses contaminated by B . stearothermophilus . A study by SEM of vegetative forms showed that microwave irradiation induce a progressive series of alterations and finally the complete destruction of the microorganism . On the contrary there were no significant morphological variations of the spores after lethal irradiation by microwaves.

J Pediatr, 1993 Feb, 122(2), 198 - 203
Immunopathology and cytokine detection in the skin lesions of patients with Kawasaki disease; Sato N et al.; To investigate the pathogenesis of Kawasaki disease, we examined biopsy specimens from the skin lesions of 10 Japanese patients at the site of the polymorphous exanthem on the hip and at the site of an inoculation with BCG (bacillus Calmette-Gruerin) vaccine of two of them . Ten normal patients were also examined for control purposes . The histopathologic features of the skin lesions were characterized by extensive edema with dilation of the small blood vessels in the papillary dermis; the site of BCG vaccine inoculation showed much greater inflammatory change; neutrophil emigration was weak and most of the infiltrates were CD4+ T lymphocytes and CD13+ macrophages . The expression of the DR locus of human leukocyte antigen was detected not only on the epidermal keratinocyte surface but also on the walls of the small blood vessels and the infiltrating cells around these blood vessels . We also detected cytokines in the skin lesions: (1) interleukin-1 alpha and tumor necrosis factor alpha were strongly positive in all patients with acute Kawasaki disease, (2) interleukin-2 and interferon gamma were weakly or partially positive, (3) no cytokines were detected in the convalescent phase, and (4) the amounts of cytokines at the site of BCG vaccine inoculations were larger than those at the site of the polymorphous exanthem . These findings suggest that CD4+ T lymphocytes and CD13+ macrophages are activated and interleukin-1 alpha and tumor necrosis factor alpha may be involved in the pathogenesis of the inflammation of acute Kawasaki disease.

J Ind Microbiol, 1993 Feb, 11(2), 83 - 8
Production of a fungal protein, Taka-amylase A, by protein-producing Bacillus brevis HPD31; Ebisu S et al.; An expression-secretion vector, pMK300, was constructed to express the Aspergillus oryzae Taka-amylase A (Taa) cDNA . The promoter and signal peptide regions of the HWP (a major cell wall protein of Bacillus brevis HPD31) gene on pMK300 were efficiently utilized in B . brevis HPD31 and a large amount of Taa (22 mg/l) was secreted into the medium . The HWP signal peptide utilized for secretion of Taa was correctly processed during the protein transport across the membrane . The enzymatic properties of Taa produced by B . brevis HPD31 were the same as those of the Aspergillus oryzae Taa in several respects; specific activity, thermal and pH stabilities, and temperature and pH optima . These results, in combination with previous results, indicate that B . brevis HPD31 could be used to produce extracellularly foreign proteins of diverse origins as functional proteins.

J Membr Biol, 1993 Feb, 132(1), 53 - 62
Lepidopteran-specific crystal toxins from Bacillus thuringiensis form cation- and anion-selective channels in planar lipid bilayers; Schwartz JL et al.; Previous studies in our laboratory have shown that CryIC, a lepidopteran-specific toxin from Bacillus thuringiensis, triggers calcium and chloride channel activity in SF-9 cells (Spodoptera frugiperda, fall armyworm) . Chloride currents were also observed in SF-9 membrane patches upon addition of CryIC toxin to the cytoplasmic side of the membrane . In the present study the ability of activated CryIC toxin to form channels was investigated in a receptor-free, artificial phospholipid membrane system . We demonstrate that this toxin can partition in planar lipid bilayers and form ion-selective channels with a large range of conductances . These channels display complex activity patterns, often possess subconducting states and are selective to either anions or cations . These properties appeared to be pH dependent . At pH 9.5, cation-selective channels of 100 to 200 pS were most frequently observed . Among the channels recorded at pH 6.0, a 25-35 pS anion-selective channel was often seen at pH 6.0, with permeation and kinetic properties similar to those of the channels previously observed in cultured lepidopteran cells under comparable pH environment and for the same CryIC toxin doses . We conclude that insertion of CryIC toxin in SF-9 cell native membranes and in artificial planar phospholipid bilayers may result from an identical lipid-protein interaction mechanism.

Cancer Res, 1993 Feb 1, 53(3), 456 - 9
Delayed-type hypersensitivity reactions to tumor-associated antigens in colon carcinoma patients immunized with an autologous tumor cell/Bacillus Calmette-Guérin vaccine; Bloemena E et al.; Five different colon tumor-associated antigens (CTAA) were tested for their ability to induce an immune response in vivo and in vitro in ten colon carcinoma patients immunized with an irradiated autologous tumor cell/Bacillus Calmette-Guerin vaccine (active specific immunization) after resection of the primary tumor . The CTAA were defined by two different human monoclonal antibodies (MCA 1688 and MCA 28A32) derived by immortalization of peripheral blood B-lymphocytes from an active specific immunization patient . Delayed-type cutaneous hypersensitivity responses against a mixture of CTAA 28A32-50K and -32K were positive in seven of ten patients tested . In vitro T-cell responses upon stimulation with CTAA 28A32-32K were found to be positive in seven of ten patients and correlated with delayed-type cutaneous hypersensitivity responses to the antigen mixture . These data suggest that CTAA 28A32-32K might contain an important tumor-related T-cell epitope . Moreover, this method is suitable to define potential future candidates for antitumor vaccine development.

Mutat Res, 1993 Feb, 301(2), 73 - 7
Random mutagenesis of a plasmid-borne glycosidase gene and phenotypic selection of mutants in Escherichia coli; Lopez-Camacho C et al.; The bglA gene from Bacillus polymyxa encodes a beta-glucosidase able to hydrolyze p-nitrophenyl-beta-D-glucopyranoside (PNPG), and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal), chromogenic substrates for beta-glucosidases and beta-galactosidases respectively . A plasmid carrying the blgA gene inserted in vector pUC18 was mutagenized in vitro with hydroxylamine, and subsequently used to transform E . coli selecting for the ampicillin resistance conferred by the cloning vector . The transformants were screened on petri dishes for mutations causing inability to hydrolyze either one of the two substrates, and for mutations increasing resistance of the enzyme to thermal inactivation . The isolation of several mutants with such characteristics suggests that the simple procedure used here can be applied to generate modifications of enzymatic properties that fit specific industrial requirements.

J Mol Biol, 1993 Jan 20, 229(2), 319 - 27
Cloning and characterization of a novel Bacillus thuringiensis cytolytic delta-endotoxin; Koni PA et al.; A gene encoding a major entomocidal polypeptide from Bacillus thuringiensis subspecies kyushuensis delta-endotoxin crystals (CytB) was cloned into Escherichia coli and sequenced . The deduced amino acid sequence gave a predicted molecular mass of 29,236 Da and showed 39% identity and 70% similarity with the 27,371 Da CytA protein from Bacillus thuringiensis subspecies israelensis . The larger size of CytB compared to CytA appears to be due to additional sequence in CytB after the CytA C-terminus . Unlike CytA, CytB was freely expressed in Escherichia coli and formed cytoplasmic inclusions without the need for a "helper" protein . Electron microscopic observation of CytB inclusions revealed them to be generally amorphous, but examples possessing some lattice structure were seen.

Carbohydr Res, 1993 Jan 15, 238, 75 - 91
Preparation, isolation, and characterization of novel heterogeneous branched cyclomalto-oligosaccharides having beta-D-galactosyl residue(s) on the side chain; Koizumi K et al.; Transgalactosylated products of branched cyclodextrins (glucosyl-alpha CD, -beta CD, -gamma CD, and maltosyl-alpha CD, -beta CD, -gamma CD) were synthesized by beta-D-galactosidases from Bacillus circulans and Penicillium multicolor using lactose as a donor substrate and branched CDs as acceptors . Eighteen beta-D-galactosylated branched CDs were isolated and purified by HPLC . Their structures were elucidated by FABMS and 13C NMR spectroscopies, and methylation analysis . The chromatographic behavior of these novel heterogeneous branched CDs on three HPLC columns of different separation modes was compared.

Eur J Biochem, 1993 Jan 15, 211(1-2), 267 - 80
Mutations that significantly change the stability, flexibility and quaternary structure of the l-lactate dehydrogenase from Bacillus megaterium; Kotik M et al.; In order to investigate the physical basis of protein stability, two mutant L-lactate dehydrogenases (LDH) and the wild-type enzyme from Bacillus megaterium were analyzed for differences in quaternary structure, global protein conformation, thermal stability, stability against guanidine hydrochloride, and polypeptide chain flexibility . One mutant enzyme, ({T29A, S39A}LDH), differing at two positions in the alpha-B helix, exhibited a 20 degrees C increase in thermostability . Hydrogen/deuterium exchange revealed a rigid structure of this enzyme at room temperature . The substitutions Ala37 to Val and Met40 to Leu destabilize the protein . This is observable in a greater susceptibility to thermal denaturation and in an unusual monomer/dimer/tetramer equilibrium in the absence of fructose 1,6-bisphosphate Fru(1,6)P2 . The stability, flexibility and protein-conformation measurements were all performed in the presence of 5 mM Fru(1,6)P2, i.e . under conditions where the three investigated LDH species are stable tetramers . Tryptophan fluorescence was used to monitor the unfolding in guanidine HCl of two local structures in or very close to the beta-sheets at the protein surface . The LDHs form folding intermediates in guanidine HCl that aggregate at elevated temperatures . Pronounced differences between the three investigated enzymes are found in their ability to aggregate . The exchange of Thr29 and Ser39 for Ala leads to significantly less aggregation in guanidine HCl than is observed for wild-type LDH . Using 8-anilinonaphthalene-1-sulfonic acid, the folding intermediates were shown to be in accordance with molten-globule-like structures . We have found, by means of molecular sieve chromatography, that the {T29A, S39A}LDH with its increased thermostability has lower susceptibility to disintegrate into monomers in guanidine HCl at 25 degrees C . Despite the differences in aggregation at low guanidine HCl concentrations and temperatures above 25 degrees C, the molten-globule-like structures of the three investigated LDH species are structurally similar, as shown by molecular-sieve chromatography . Although the thermostabilities of the three LDH species are so different in aqueous buffers, their stabilities in guanidine HCl at 20 degrees C are, surprisingly, almost identical . Some comments are made as to the origin of the observed difference between thermal and guanidine HCl stabilities of the LDH . Near-ultraviolet and far-ultraviolet circular dichroism measurements, as well as differences in the amount of activation by Fru(1,6)P2, point to small global structural rearrangements caused by the mutations . Conformational changes upon Fru(1,6)P2 binding or point mutations in the alpha-B helix show that the Fru(1,6)P2-binding site and the alpha-B helix are structurally linked together.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1993 Jan 12, 32(1), 18 - 26
Folding of subtilisin BPN': characterization of a folding intermediate; Eder J et al.; Subtilisin BPN', an extracellular serine protease from Bacillus amyloliquefaciens, requires a 77 amino acid pro-sequence for correct folding in vivo . We report the observation of a metastable folding intermediate during the refolding of wild-type and a proteolytically inactive mutant subtilisin BPN' that lack the pro-sequence . The addition of the pro-sequence as a separate polypeptide chain results in the folding of the intermediate to the native state . The intermediate state of subtilisin is stable at different temperatures, pH values, and salt concentrations for more than a week and retains its competence for folding . The intermediate state possesses a compactness between that of the native and unfolded states . Although it has native-like secondary structure, it shows no distinct near-UV CD spectrum and has a strongly reduced dispersion in the amide and methyl regions of the 1H NMR spectrum . These indicate considerably less tertiary structure than possessed by the native state . However, the intermediate conformation has regions of stable tertiary structure: it has a high-affinity calcium binding site and, after a first noncooperative transition, unfolds with guanidinium chloride in a cooperative process . These results support a folding mechanism for subtilisin BPN' that comprises a high energy transition state, which is lowered by the interaction with the pro-sequence . The similarity to the folding mechanism of alpha-lytic protease supports the hypothesis that a common folding mechanism has been developed through convergent evolution.

Nucleic Acids Res, 1993 Jan 11, 21(1), 93 - 7
Nucleotide sequence of 5' portion of srfA that contains the region required for competence establishment in Bacillus subtilus; Fuma S et al.; The nucleotide sequence of the 20,535 base pairs of the 5' end of the srfA operon, containing the region required for competence development, was determined . This included the srfA promoter region, the first open reading frame, srfAA, encoding surfactin synthetase I and part of the second open reading frame, srfAB, encoding surfactin synthetase II . Three amino acid-activating domains characteristic of those found in peptide synthetases could be discerned in both srfAA (activating Glu, Leu and D-Leu) and srfAB (activating Val, Asp, and D-Leu) . The presence of a conserved spacer motif in the amino-terminal end of srfAA suggests that the srfAA product may not initiate surfactin synthesis . The portion of srfA that contains the region required for competence is composed of srfAA and the first amino acid-activating domain of srfAB.

J Biol Chem, 1993 Jan 5, 268(1), 349 - 55
Purification and characterization of kumamolysin, a novel thermostable pepstatin-insensitive carboxyl proteinase from Bacillus novosp . MN-32; Murao S et al.; We have found a novel type of thermostable, pepstatin-insensitive carboxyl proteinase in the culture filtrate of Bacillus novosp . MN-32 . The carboxyl proteinase, which was named kumamolysin, was purified about 8,300-fold by column chromatography including DEAE-Sepharose CL-6B, Sephadex G-100, and TSKgel DEAE-5PW . The purified kumamolysin gave a single band corresponding to 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The molecular mass of kumamolysin was estimated to be 40 kDa by gel filtration . The isoelectric point of kumamolysin was estimated to be pH 3.5 by isoelectric focusing . Kumamolysin has maximum proteolytic activity at 70 degrees C and at pH 3.0 . Kumamolysin specifically hydrolyzed the Leu15-Tyr16 peptide bond in oxidized insulin B-chain (Km = 9.0 x 10(-5) M, Kcat = 71 s-1; at pH 3.0, 30 degrees C), and additional cleavage at Phe25-Tyr26 was detected at a considerably lower rate . Kumamolysin is insensitive to the known carboxyl proteinase inhibitors pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)propane . Kumamolysin has no similarity to the thermostable acid protease thermopsin from Sulfolobus acidocaldarius (Lin, X.-L., and Tang, J . (1990) J . Biol . Chem . 265, 1490-1495) . Thus, the substrate specificity, the inhibitor sensitivity, the molecular mass, and the thermostability all suggest that kumamolysin is a novel type of carboxyl proteinase.

Insect Mol Biol, 1993, 2(3), 141 - 7
A centromeric satellite DNA of the unisexual Bacillus atticus (Insecta: Phasmatodea); Mantovani B et al.; In the parthenogenetic Bacillus atticus atticus, widespread over most of the Mediterranean basin, a highly repeated satellite DNA (BaB300) has been detected and analysed . BgI II, Taq I and Alu I restriction enzymes cut 316 bp long repeating units from the BaB300 family . These are non-coding, AT rich and well conserved in the three isolated populations studied . By in situ hybridization, the satellite has been located in the centromeric heterochromatin of a subset of medium- and small-sized chromosomes, including the Xs . Related sequences have been found in the bisexual B . grandii grandii and in its specific hybrid B . whitei (=B . rossius/grandii grandii), while they are absent from B . rossius redtenbacheri . These data support the genetic affinity relationships within the genus Bacillus inferred from morphological, allozymic and karyological data . They also encourage further comparisons among Bacillus hybrids, in order to trace their parental genomes in the hybrid nuclei, and within the B . atticus complex (i.e . additional samples of B . a . atticus and diploid/triploid cytotypes of B . a . carius) to study repetitive DNA turnover in unisexual organisms.

Arch Anat Cytol Pathol, 1993, 41(1), 38 - 44
{Value of cytological study by flow cytometry or analysis of image as a complement to macroscopic study by cytoscopy . Apropos of therapeutic follow-up of a case of tumor of the bladder}; Lizard G et al.; The case described corresponds to a grade G2 urothelial tumor with stage pTa extension (according to the World Health Organisation classification) . After transurethral resection of the tumor and treatment by Bacillus Calmette Guerin (BCG), the efficacy of treatment was evaluated by cystoscopy, standard cytology, flow cytometry or image analysis . According to these various methods it has been shown that a normal cystoscopy may or may not be associated with aneuploidy revealed by flow cytometry . Such a case clearly illustrates the value of combining macroscopic examination and a cytologic analysis in particular by flow cytometry in order to increase the accuracy of diagnosis and to evaluate without ambiguity the efficacy of treatment.

Mol Gen Mikrobiol Virusol, 1993 Jan-Feb, (1), 35 - 8
{Structural and molecular-biological characteristics of a new transducing phage from Bacillus thuringiensis Tt 91}; Koroleva IuV et al.; A new bacteriophage Tt91 of larger size and broad lytical spectrum has been isolated from soil to widen the possibility to study new Bacillus thuringiensis strains . The Tt91 bacteriophage was shown to perform the intravariant transduction and, thus, can be used for genetic mapping . The ultrastructural analysis made it possible to refer bacteriophage Tt91 to morphotype A1 and showed similarity of Tt91 morphological structure to the one of CP-group bacteriophages . The performed comparative analysis of phage-specific proteins has confirmed a possible relation of the bacteriophages and revealed the differences between them . The data on the resistance of Tt91 DNA to majority of site-specific restriction endonucleases (17 out of 18 tested) and on DNA melting in the wide interval of temperatures suggests the presence of possible anomalies in the Tt91 DNA.

Microbiol Immunol, 1993, 37(1), 63 - 7
Inhibitory effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) on germination of Bacillus cereus T spores; Shibata H et al.; Effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), a calcium antagonist, on germination of Bacillus cereus T spores induced by L-alanine and inosine was investigated . TMB-8 had no effect on the germination of heat-activated spores, whereas it inhibited that of nonactivated spores . The TMB-8 inhibitory effect was antagonized competitively by inosine, but not by L-alanine . Addition of Ca2+ reversed the inhibitory effect of TMB-8 in a dose-related fashion . Based on the results, a role of inosine and a site(s) for inhibitory action of TMB-8 in the process leading to the germination of nonactivated spores were discussed.

FEMS Immunol Med Microbiol, 1993 Jan, 6(1), 53 - 61
Inhibition of interleukin-6 release and T-cell proliferation by synthetic mirror pseudo cord factor analogues in human peripheral blood mononuclear cells; Wang MH et al.; The effects of synthetic alkyl ((alkyl 6-deoxy-a-D-gluco-heptopyranosyluronate) 6-deoxy-a-D-gluco-heptopyranoside) uronates, a novel type of mirror pseudo cord factor, on the in vitro modulation of interleukin-6 production and T-cell proliferation in human peripheral blood mononuclear cells, were investigated . Synthetic mirror pseudo cord factors with alkyl chains ranging from C16 to C18 have very weak interleukin-6-inducing capacities and lack mitogenic activities for T-cell proliferation . However, they could inhibit IL-6 release induced by sonicated Bacillus Calmette-Guerin (S-BCG), bacterial endotoxin, and phytohaemagglutinin in a dose-dependent manner . Inhibition was observed not only with mononuclear cells but also with purified monocytes . Furthermore, these synthetic compounds could suppress T-lymphocyte proliferation stimulated by sonicated Mycobacterium tuberculosis H37Rv (S-H37Rv) antigens, S-BCG antigens, as well as by recombinant 65 kDa mycobacterial heat-shock protein . In contrast, these compounds failed to inhibit the phytohaemagglutinin-induced T-cell proliferation . We conclude that the inhibition of cytokine release and T-cell proliferation by synthetic mirror pseudo cord factors was due to direct blocking of the function and/or activity of monocytes or antigen-presenting cells.

Biofizika, 1993 Jan-Feb, 38(1), 22 - 30
{Ionic pairs in Bacillus intermedius 7P ribonuclease}; Grishina IB et al.; On the basis of X-ray determined coordinates specified with computer graphics and new data on amino acids sequence in the structure of Bacillus intermedius 7P ribonuclease there were analysed potentially charged residues and found ionic pairs binding independently denaturing regions-energetic domains . Five clusters of charged amino acids were found in the protein structure and several ionic bonds between the energetic domains were discovered: Asp7, Asp11-Arg109 and Lys26-Asp53,Glu72 . Breaking of the latter makes their heat denaturation independent . Local surrounding of the ionic bonds is characterized and their role in the structure and cooperative properties of the globule is discussed.

Eur J Cancer, 1993, 29A(5), 708 - 11
Phase II study of vindesine and dacarbazine with or without non-specific stimulation of the immune system in patients with metastatic melanoma; Verschraegen CF et al.; A single dose of dacarbazine (DTIC), followed by a 5-day intravenous infusion of vindesine (VDS) was administered every 3 weeks to 103 patients with metastatic melanoma . One half of the patients were randomised to receive intravenous methanol extraction residue (MER) of bacillus Calmette-Guerin (BCG) in addition to chemotherapy, on days 7 and 14 of each course . 98 patients were evaluable . The response rates in treatment groups were 16 and 17%, respectively (confidence interval 9-24%) . Neither the response rate nor the survival improved when MER was added to chemotherapy . Toxicity was moderate except for a significant granulocytopenia . The combination of DTIC and VDS is not more effective than DTIC alone and has added neurotoxicity.

J Vet Diagn Invest, 1993 Jan, 5(1), 64 - 8
Bacterial agents detected in a 10-year study of bovine abortions and stillbirths; Kirkbride CA; In a 10-year survey started in 1980, specimens from 8,995 bovine abortions and stillbirths were submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory . Of these, 8,962 were suitable for some type of examination . Bacteria were determined to be the cause of 1,299 (14.49%) . The 5 bacteria most commonly associated with bovine abortion or stillbirth were Actinomyces pyogenes, 378 (4.22%); Bacillus spp., 321 (3.58%); Listeria spp., 121 (1.35%); Escherichia coli, 98 (1.09%); and Leptospira interrogans, 79 (0.88%) . Twelve other genera of bacteria were associated with > or = 10 abortions or stillbirths, and 12 more species were associated with < or = 10 abortions or stillbirths.

Schweiz Arch Tierheilkd, 1993, 135(3), 79 - 88
{Infection with Bacillus piliformis (Tyzzer's disease) in foals}; Paar M et al.; Tyzzer's disease, caused by Bacillus piliformis and characterized by a multifocal, necrotizing hepatitis was diagnosed in a 25 days old, male, German warmblood foal . The animal was submitted to the clinic of horses because of colic symptoms and a severe depression . Upon arrival it was comatose, recumbent and showed opisthotonos . Due to a severe, metabolic acidosis and a rapid progression of the disease, the foal was euthanatized . In immediately formalin-fixed tissue samples Bacillus piliformis was detected in the cytoplasma of hepatocytes by different histological techniques (Giemsa stain, silver impregnation technique by Warthin-Starry, immunohistology) . This case is discussed within a literature review.

Urol Res, 1993 Jan, 21(1), 33 - 7
Keyhole limpet hemocyanin immunotherapy of murine bladder cancer; Lamm DL et al.; The current treatment of choice for superficial bladder cancer, bacillus Calmette-Guerin, has significant adverse side effects . We have compared two alternative immunotherapies--crude keyhole limpet hemocyanin (KLH) and Immucothel, a KLH modified for clinical use (Biosyn)--in an intralesional mouse model of bladder cancer (MBT2) . Crude KLH required either immunization before tumor transplant or frequent intralesional therapy after transplantation to be effective . In addition, Immucothel required pre-immunization to be effective, and increasing the frequency and dosage of post-transplant immunization was not effective without pre-immunization . Preliminary investigations into the KLH-induced anti-tumor mechanism(s) suggest that natural killer cell activity may be involved . Both crude KLH and Immucothel appear to be effective immunotherapies of use in the treatment of transitional cell carcinoma.

J Gen Microbiol, 1993 Jan, 139 ( Pt 1), 39 - 47
Molecular cloning and nucleotide sequence of the gene encoding a calcium-dependent exoproteinase from Bacillus megaterium ATCC 14581; Kuhn S et al.; The gene nprM encoding the calcium-dependent extracellular proteinase from Bacillus megaterium ATCC 14581 was cloned in the vector pBR322 and expressed in Escherichia coli HB101 . The DNA sequence of the cloned 3.7 kb fragment revealed only one open reading frame consisting of 1686 bp with a coding capacity of 562 amino acid residues . A predicted Shine-Dalgarno (SD) sequence was observed 9 bp upstream from the presumptive translation start site (ATG) . A possible promoter sequence (TAGACG for the -35 region and TATAAT for the -10 region) was found about 69 bp upstream of the ATG start site . The deduced amino acid sequence exhibited a 24 amino acid residue signal peptide and an additional polypeptide 'pro' sequence of 221 amino acids preceding the putative mature protein of 317 amino acid residues . Amino acid sequence comparison revealed 84.5% homology between the mature protein and that of a thermolabile neutral protease from B . cereus . It also shares 73% homology with the thermostable neutral proteases of B . thermoproteolyticus and B . stearothermophilus . The zinc-binding sites and the catalytic residues are completely conserved in all four proteases . NprM has a temperature optimum of 58 degrees C, a pH optimum of between 6.4 and 7.2, and is stimulated by calcium ions and inhibited by EDTA . These results indicate that the enzyme is a neutral (metallo-) protease.

Klin Padiatr, 1993 Jan-Feb, 205(1), 56 - 8
{Congenital tuberculosis in a premature infant}; Limbach HG et al.; 5 days after birth a premature baby of 1,300 g (31st week of gestation) was examined, because a congenital tuberculosis was suspected . In the stomach secretion mycobacterium tuberculosis could in fact be found . Abdominal symptoms as well as a post-natal anemia could be lead back to the infection . For three months a double parenteral therapy with INH (10 mg/kg/d) and Rifampicin (10 mg/kg/d) was carried through, then a mono therapy with INH for another 6 months . The bacillus had become resistant to streptomycin . At the beginning of the chemotherapy a cholestasis developed . The tuberculosis could be cured.

J Wildl Dis, 1993 Jan, 29(1), 123 - 6
Cilia-associated respiratory bacillus in wild rats in central Iowa; Brogden KA et al.; Twenty-eight wild rats were live-trapped in central Iowa (USA) to estimate the prevalence of the cilia-associated respiratory (CAR) bacillus . Both light and electron microscopy were used to look for the Gram-negative, filamentous bacterium among cilia in tracheal and lung tissue sections . The organism was observed in the trachea of 20 rats with chronic respiratory disease and in the trachea of three of eight normal rats . Therefore, the organism appears to be common among wild rats in central Iowa.

Plasmid, 1993 Jan, 29(1), 63 - 9
The complete nucleotide sequence of a small cryptic plasmid from a rumen bacterium of the genus Butyrivibrio; Hefford MA et al.; The complete nucleotide sequence of a plasmid, designated pRJF1, isolated from a rumen bacterium of the genus Butyrivibrio, has been determined . pRJF1 is a small plasmid (2631 bp) which shows the high AT content (64%) typical of plasmids from gram-positive organisms . Computer analysis of sequence data revealed two major open reading frames encoded on the same strand but in different frames . The smaller, ORF1 (435 bp), is preceded by Shine Dalgarno (SD) and Escherichia coli-10, -35 sequences and is predicted to encode an acidic polypeptide of 145 aa . The putative protein shows little homology to known bacterial proteins except in a small region similar to part of the Pre protein (plasmid recombination enzyme) of Bacillus plasmid pTB913 . Pre proteins of the pTB913 family, however, show conservation of several amino acids in the amino terminal region of the protein that may be related to function . This pattern of amino acid conservation is not observed in ORF1 . The predicted product of ORF2 (849 bp) is a basic protein of 283 as that shows significant similarity to repA gene products of plasmids from gram-negative (but not gram-positive) bacteria . No typical Shine Dalgarno sequence or -10, -35 sequences precede this coding region . While sequence homologous to nick sites of plasmids from gram-positive organisms that replicate via ssDNA intermediates were also detected in pRJF1, the order of the three consecutive inverted repeats typical of nick sites is not conserved . pRJF1, however, does contain an extended region of directly and inversely repeated sequence motifs reminiscent of theta replication origins.

Can J Microbiol, 1993 Jan, 39(1), 52 - 60
Sporocidal properties of peracetic acid and hydrogen peroxide, alone and in combination, in comparison with chlorine and formaldehyde for ultrafiltration membrane disinfection; Alasri A et al.; The sporocidal properties of peracetic acid, hydrogen peroxide, chlorine, and formaldehyde were compared in vitro using a dilution-neutralization micromethod . A combination of peracetic acid and hydrogen peroxide was also tested to assess their interactions . The activities of these agents, which are widely used as disinfectants, were evaluated against Bacillus spore isolates found on stored membranes and collection cultures . Peracetic acid and chlorine exhibited an excellent antimicrobial activity, with a destruction of 10(5) spores/mL after 5 min of contact . Generally the effects of the biocides tested were time dependent . The sporocidal activities of hydrogen peroxide and formaldehyde were the lowest . The combination of peracetic acid and hydrogen peroxide, tested by a checkerboard micromethod, was found to be synergistic . The minimal sporocidal concentration (MSC) was established in terms of time for each biocide . The lowest MSC values for peracetic acid, hydrogen peroxide, chlorine, and formaldehyde were 168-336 ppm (1-2 h of contact), 5625-11250 ppm (5-7 h), 168-336 ppm (2-3 h), and 1875-3750 ppm (5-30 min), respectively . The MSC of a biocide combination of peracetic acid and hydrogen peroxide showed that synergy was maintained with increasing contact time and that the MSC could be reduced by two to eight times when compared with those of the biocides alone . Optimal concentrations and contact times of those chemicals that were promising in vitro were then tested for their ability to disinfect ultrafiltration membranes . The sporocidal activities of peroxide compounds and chlorine were confirmed and the synergism between peracetic acid and hydrogen peroxide was also maintained.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1993 Jan, 59(1), 114 - 9
Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains; Brousseau R et al.; Arbitrary primer polymerase chain reaction technology has been applied to the identification of commercial strains of Bacillus thuringiensis by using total DNAs extracted from single bacterial colonies as templates . Characteristic DNA banding patterns can be readily and reproducibly obtained by agarose gel electrophoresis . This method has been used to distinguish commercial products containing B . thuringiensis serovar kurstaki (3a3b) . When a single primer was used this method was capable of producing discriminating DNA fingerprints for 33 known serovars . Differentiation from the closely related species Bacillus cereus is also readily achieved . This technique should prove to be a powerful tool for identification and discrimination of individual B . thuringiensis strains.

Protein Eng, 1993 Jan, 6(1), 11 - 8
A catalytically-impaired class A beta-lactamase: 2 A crystal structure and kinetics of the Bacillus licheniformis E166A mutant; Knox JR et al.; In the beta-lactamase (penicillinase) of Bacillus licheniformis 749/C, site specific mutation of Glu166 to Ala caused a million-fold reduction of catalytic activity towards both penicillin and cephalosporin substrates and resulted in the stoichiometric accumulation of the acyl enzyme . The rate of deacylation generally slowed by as much as 10(-7) compared to the wild type . The acyl enzyme intermediate was observed by HPLC, but not by X-ray diffraction . The mutant was crystallized from methoxyPEG 5000 at pH 6.2 in space group P2(1) with Z = 4 . Molecular replacement based on the wild type structure followed by refinement produced an R factor of 17.2% for 25,800 3 sigma data from 10 to 2 A . Deviations from bond and angle ideals are 0.005 A and 1.5 degrees respectively . The mutant differs very little from the wild type structure, with only 0.25 A (r.m.s.) differences in backbone atoms; the CD spectra and thermal stabilities of the two enzymes are identical . Changes in the positions of the reactive Ser70 and conserved Lys73 are not significant, suggesting that the proposed salt linkage to Glu166 in the wild type enzyme is weak or non-existent . The calculated solvent exposure of Ser70 and Lys73 increases slightly and a buried water molecule is now positioned near Lys73 . The hydrolytic water seen in the native active site shifts markedly by 1.6 A, but is held in the active site by Asn170, which possibly becomes an ineffective substitute for Glu166 in activating the water for deacylation.

J Clin Invest, 1993 Jan, 91(1), 69 - 76
Characterization of the internalization of bacillus Calmette-Guerin by human bladder tumor cells; Kuroda K et al.; Adjuvant intravesical Mycobacterium bovis BCG is the treatment of choice for recurrent superficial bladder cancer . Fibronectin (FN) was previously demonstrated to be necessary for the retention of BCG within the bladder and for the expression of antitumor activity . Recent studies have demonstrated that BCG attach and are ingested by bladder epithelial cells, suggesting the existence of a second bacterial attachment mechanism . We report the characterization of the molecules involved in BCG attachment and internalization by the human bladder transitional cell carcinoma cell line T-24 . Pretreatment of T-24 cells with monoclonal antibodies to either alpha 5 or beta 1 integrin subunits significantly inhibited both BCG attachment and ingestion . Exogenous FN was observed to enhance both attachment and ingestion of BCG, and anti-FN was observed to inhibit both phenomena . Latex beads precoated with either FN or laminin (LN) but not BSA were ingested by T-24 cells, but only FN-coated beads inhibited BCG attachment and ingestion . Pretreatment of BCG with FN augmented both attachment and ingestion . The role of bacterial FN binding proteins was evaluated . A monoclonal antibody to a 55-kD FN-binding protein was observed to abrogate attachment and ingestion . These results demonstrate that attachment and ingestion of BCG are mediated in part by the alpha 5 beta 1 integrin receptor and are dependent on FN . These studies demonstrate a mechanism of entrance of mycobacteria into epithelial cells and suggest a second role for FN in the adjuvant antitumor effect of BCG.

J Bacteriol, 1993 Jan, 175(2), 510 - 8
Genetic determinants of host ranges of Bacillus sphaericus mosquito larvicidal toxins; Berry C et al.; The 51.4-kDa-41.9-kDa binary toxin produced by different strains of Bacillus sphaericus shows differential activity toward Culex quinquefasciatus, Aedes atropalpus, and Aedes aegypti mosquito larvae . The patterns of larvicidal activity toward all three mosquito species and growth retardation in A . aegypti have been shown to be due to the 41.9-kDa protein . By using mutant toxins expressed in Escherichia coli, insecticidal activity and growth retardation correlated with amino acids centered around position 100 of the 41.9-kDa protein . In its response to these toxins, A . atropalpus resembled C . quinquefasciatus rather than its congener, A . aegypti.

Clin Exp Immunol, 1993 Jan, 91(1), 68 - 72
Antigen-dependent in vitro culture of protective T cells from BCG-primed mice; D'Souza S et al.; Induction of protective immunity against pathogenic mycobacteria depends on vaccination with live organisms such as Bacille Calmette-Guerin (BCG) . However, it is not known how many and which antigens are involved in the protective host response . In this study, we developed a system of antigen-dependent in vitro culture which is suitable for the analysis of protective subunits, presented in a soluble form . Spleen cells from Mycobacterium bovis BCG-immune mice, enriched for T cells and depleted of adherent cells on a column of G-10 Sephadex, were cultured for periods varying between 3 and 14 days before transfer and challenge with M . tuberculosis in irradiated hosts . Following 10 days in culture, immune T cells sustained their capacity to transfer protection to tuberculous infection when incubated in the presence of either live BCG or a soluble extract from M . tuberculosis, but lost this ability when cultured in the absence of antigen, or in the presence of the polyclonal mitogen concanavalin A . One immunodominant antigen, represented by the recombinant 38-kD antigen, failed to sustain the adoptive protection, despite pronounced stimulation of lymphoproliferation in culture . Antigenic in vitro stimulation of protective T cells was accompanied by enhanced responsiveness to exogenous IL-2 . The experimental system described may be generally suitable to test in vitro the protective potentials of soluble molecular subunits of mycobacteria.

J Urol, 1993 Jan, 149(1), 179 - 82
Antitumor effects of bacillus Calmette-Guerin in a syngeneic rat bladder tumor model system, RBT323; Cornel EB et al.; We have studied the antitumor effects of Bacillus Calmette-Guerin (RIVM strain) in the syngeneic rat bladder tumor model RBT323 . In an immunohistochemical infiltrate study we compared the antitumor effects of BCG with the immunopathological findings in order to get more insight into the possible effector mechanisms of BCG . The antitumor effects of BCG appeared not to be dose-dependent, in the dose range tested . In rechallenge experiments no difference in growth of control tumors was seen between rats pretreated- or not pretreated with BCG . There was, however, a significant increase in antitumor effect of BCG after pretreatment with BCG . Immunohistological examination of BCG treated tumors revealed infiltrates consisting of macrophages, B-cells and T-cells . These results imply that whereas no specific response against the RBT323 cells is generated by BCG treatment, possibly BCG induced antigens do serve as immunogens in this nonimmunogenic syngeneic rat bladder tumor model system.

World J Urol, 1993, 11(3), 165 - 8
Mechanisms of action of bacillus Calmette-Guérin in the treatment of superficial bladder cancer; Patard JJ et al.; Local immunotherapy with bacillus Calmette-Guerin (BCG) is an effective treatment to prevent recurrence and progression of superficial bladder cancer, but the antitumor mechanism of action of BCG remains unclear . There are some experimental and clinical data suggesting that BCG antigens are processed not only by immunocompetent cells but also by urothelial cells and tumor cells . The foreign antigen may be presented at the cell surface by major histocompatibility complex (MHC) class II molecules and recognized by CD4 cells . The cytotoxic effect could result from the direct activity of CD4 cells or from the cytotoxic effect of released cytokines and the activation of other cytotoxic cells {cytolytic T-lymphocytes CTLs; CD8 cells), macrophages, natural killer (NK) or lymphokine-activated killer (LAK) cells} . These mechanisms are also involved in tumor rejection, and the identification of some specific tumor rejection antigens presented to specific CTLs could provide new therapeutic approaches.

World J Urol, 1993, 11(3), 148 - 52
Prognostic factors in superficial bladder cancer; Chopin DK et al.; A large body of evidence suggests that intravesical prophylaxis using bacillus Calmette-Guerin can favorably alter the natural history of superficial bladder cancer . The search for prognostic factors impacting the clinical alternative between conservative and radical treatment has been the subject of numerous efforts and remains a major objective in improving the carcinological results and the quality of life patients with superficial bladder cancer . In this regard, information is available on three levels: clinicopathological parameters, quantitative analysis, and biological markers . Recent advances in as well as the limitations and pitfalls of the use of three kinds of information in prognostication are reviewed . The proper use of prognostic factors will modify our attitude toward superficial bladder cancer in the near future.

Int Arch Allergy Immunol, 1993, 101(4), 352 - 8
Analysis of perchloric-acid-soluble Mycobacterium bovis bacille bilié de Calmette-Guérin antigen; Kim DS et al.; The perchloric-acid-soluble Bacille bilie de Calmette-Guerin (BCG) antigen (BCG-P) was investigated for the diagnosis of tuberculosis . Monoclonal antibodies against BCG-P were produced . After SDS-PAGE, silver staining and immunoblotting were done . Pathological specimens of pulmonary tuberculosis, BCG organism itself and atypical mycobacterium were stained using the indirect immunofluorescent technique with the strongest one among these monoclonal antibodies . Intradermal skin test and ELISA were also done with BCG-P . BCG-P was composed of a major 65-kD protein and a minor 61-kD protein . Fourteen monoclonal antibodies were produced which were all IgM . These antibodies reacted with the 65-kD protein which also showed cross-reactivity with Mycobacterium tuberculosis in pathologic tissues and atypical mycobacterium . BCG-P did not react with the sera of patients with active pulmonary tuberculosis . However, intradermal skin test with BCG-P showed positive reactions in the patients with a positive purified protein derivative of tuberculin skin test . This antigen could be a good antigen to evaluate the success of BCG vaccination.

Pathologica, 1993 Jan-Feb, 85(1095), 1 - 15
New entities in cutaneous soft tissue tumours; Calonje E et al.; Connective tissue tumours represent a significant group among biopsied skin lesions and manifest a wide range of morphological patterns . While the majority are easily diagnosed, notably most fibrous histiocytomas and neural lesions, a proportion pose considerable diagnostic difficulty . With the increasing recognition and characterisation of previously undescribed entities within the family of soft tissue tumours in recent years, the number of unclassifiable lesions should diminish . The ultimate aim of accurate recognition and classification is to provide appropriate clinical care based on reasoned prediction of a given lesion's behaviour . With this in mind, we wish to briefly review herein some of the more recently characterised dermal connective tissue lesions, including plexiform fibrohistiocytic tumour, cellular fibrous histiocytoma, spindle celled atypical fibroxanthoma, adult myofibroma, dermatomyofibroma, so-called cellular neurothekeoma, ectopic meningothelial hamartoma, tufted angioma, sinusoidal haemangioma and bacillary angiomatosis.

Mol Gen Mikrobiol Virusol, 1993 Jan-Feb, (1), 20 - 4
{Study of the nature of determinants for synthesis of delta-endotoxin and beta-exotoxin in Bacillus thuringiensis subsp . thuringiensis strains--bitoxybacillin producers}; Kuzina LV et al.; The growth of Bacillus thuringiensis subsp . thuringiensis strains (BT) 202, 1140, 98 producing bitoxybacillin in the presence of novobiocin, mitomycin C and at high temperature 43 degrees C resulted in obtaining of the mutants for the synthesis of crystalline protein (Cry) and exotoxin (Exo) . Analysis of the plasmid content of the mutants has shown the Cry Exo phenotyre to correlate with the loss of 55 MD plasmid in the strain BT202 and the loss of 60MD plasmid in BT1140 . The transfer of these plasmids into Bacillus cereus leads to the transfer of endo- and exotoxin production properties . The synthesis of Cry in BT98 is controlled by the 56MD plasmid, while the synthesis of Exo is encoded by the chromosome or plasmid that cannot be eliminated or transferred into other strains . Localization of Cry on the 55 and 56 MD plasmids in BT202 and BT98 is confirmed by the hybridization of the plasmid DNA with the DNA-probe.

Folia Microbiol (Praha), 1993, 38(1), 10 - 4
Netropsin inhibits the increase of intracellular Ca(2+)-dependent serine proteinase activity in sporulating Bacillus megaterium; Kucerova H et al.; Netropsin suppressed the increase of intracellular proteolytic activity when added to B . megaterium incubated in a sporulation medium . The inhibited enzyme was a Ca(2+)-dependent serine proteinase . Sporulation and protein turnover in later sporulation phases were inhibited as well . Different concentrations of netropsin affected various aspects of protein catabolism differently.

Bioorg Khim, 1993 Jan, 19(1), 133 - 8
{Nucleotide sequence of phospholipase C and sphingomyelinase genes from Bacillus cereus BKM-B164}; Gavrilenko IV et al.; A BamHI-EcoRV DNA fragment, containing tandemly arranged genes for the Bacillus cereus VKM-B164 phosphatidylcholine-specific phospholipase C and sphingomyelinase, has been cloned and sequenced in pUC19 plasmid (EMBL accession No X64141) . The obtained gene sequences have been compared with those the B . cereus strains SE-1, IAM-1208 and GP-4 reported previously . Both phospholipase C and sphingomyelinase gene sequences of the VKM-B164 strain were found to be identical to those of the strain SE-1 and to differ from the sequences of GP-4 and IAM-1208 strains, containing another version of the gene sequences.

Antonie Van Leeuwenhoek, 1993 Jan, 63(1), 1 - 16
The relation of proton motive force, adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction in Bacillus licheniformis under aerobic growth conditions; Bulthuis BA et al.; The magnitude of the proton motive force (delta p) and its constituents, the electrical (delta psi) and chemical potential (-Z delta pH), were established for chemostat cultures of a protease-producing, relaxed (rel-) variant and a not protease-producing, stringent (rel+) variant of an industrial strain of Bacillus licheniformis (respectively referred to as the A- and the B-type) . For both types, an inverse relation of delta p with the specific growth rate mu was found . The calculated intracellular pH (pHin) was not constant but inversely related to mu . This change in pHin might be related to regulatory functions of metabolism but a regulatory role for pHin itself could not be envisaged . Measurement of the adenylate energy charge (EC) showed a direct relation with mu for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low mu and an increased value at higher mu . For both limitations, the ATP/ADP ratio was directly related to mu . The phosphorylation potential (delta G'p) was invariant with mu . From the values for delta G'p and delta p, a variable -->H+/ATP-stoichiometry was inferred: -->H+/ATP = 1.83 +/- 0.52 mu, so that at a given -->H+/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for mu = 0 to mu max (we discuss how more than half of this decline will be independent of any change in internal cell-volume) . We propose that the constancy of delta G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing mu are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible mu with a minimum loss of growth-efficiency . Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.

J Gen Microbiol, 1993 Jan, 139 ( Pt 1), 21 - 30
Identification of two expressed flagellin genes in the insect pathogen Bacillus thuringiensis subsp . alesti; Lovgren A et al.; Flagellin from Bacillus thuringiensis subspecies alesti strain Bt75 was isolated both from the culture medium and from flagella . Two protein forms with molecular masses close to 32 kDa were obtained from flagella; one form was identical to the flagellin purified from the culture medium . The N-terminal amino acid sequences were identical for both forms . Two genes coding for flagellin have been identified in B . thuringiensis subsp . alesti . The flaB gene was cloned and sequenced in its entire length . The clone containing the flaA gene was incomplete . Both genes were expressed in the mid-exponential growth phase . The flaB gene was flanked by long (355 bp) direct repeats protruding into the coding region in both the N- and C-terminal parts of the gene . DNA sequences related to the flaB gene were found in most other B . thuringiensis subspecies, and in two of them, subsp . kurstaki and subsp . entomocidus, such sequences were present in multiple copies.

Int J Clin Pharmacol Res, 1993, 13(2), 65 - 8
Lack of activities of amikacin and sulphamethoxazole against Mycobacterium avium-intracellulare; Yew WW et al.; Twenty clinical isolates of Mycobacterium avium-intracellulare were tested with amikacin and sulphamethoxazole for in-vitro susceptibilities . The MICs and MBCs of the former drug ranged from 8 to > 64 mg/L (median MIC: 64 mg/L, median MBC: > 64 mg/L) . The MICs and MBCs of the latter drug were found to be > 256 mg/L . Each of eight patients with invasive pulmonary disease due to these organisms was treated with amikacin for six months and with cotrimoxazole (sulphamethoxazole-trimethoprim) for one year . Only one patient had sustained bacteriological conversion . Three patients showed a transient reduction of bacillary load during the period of amikacin administration . The rest all failed to show response . Thus sulphamethoxazole was found to have no activity against Mycobacterium avium-intracellulare, and amikacin has doubtful activity when used alone in treatment of M . avium-intracellulare infection.

Bull Soc Pathol Exot, 1993, 86(2), 144 - 7
{Initial resistance to streptomycin, isoniazid, rifampicin and ethambutol in bacillus-laden tuberculosis patients at the National Center of Pneumo-physiology at Cotonou (Benin)}; Anagonou SY et al.; The authors are reporting results from a study about BK sensitivity among principal antituberculosis drugs in Benin Republic: 81.5% of them are susceptible for all specific antibiotics, 8.5% are offering resistance to INH and Streptomycin together . In this preliminary study no primitive resistance for RMP has been found.

Urol Int, 1993, 51(2), 67 - 72
Use of maintenance intravesical bacillus Calmette-Guérin (BCG), with or without intradermal BCG, in patients with recurrent superficial bladder cancer . Long-term follow-up of a randomized phase 2 study; Witjes JA et al.; To study maintenance Bacillus Calmette-Guerin (BCG) therapy we treated 49 patients with recurrent superficial bladder cancer pTa and/or carcinoma in situ, failing previous intravesical chemotherapy . They received 20 intravesical instillations in 1 year, with or without 6 intradermal inoculations . Side effects were frequent, causing treatment stop in 14 patients (29%) . Drug-induced cystitis was the most frequent side effect, seen in 29 patients (59%) . Forty-two patients were evaluable for response . During the first 2 years of the study 14 patients (33%) had one (n = 8) or more (n = 6) recurrences . A total of 18 patients (43%) remained recurrence free with a median follow-up of 47 months . Progression was seen in 2 patients from pTa to pT1, another patient died of metastatic bladder cancer after 47 months . PPD skin test conversion was of no prognostic value . The use of intradermal inoculations caused no significant increase of toxicity and had no effect on efficacy, however, both groups were small . It caused no local side effects . We conclude that maintenance BCG therapy can be useful for patients with recurrent superficial bladder cancer failing previous intravesical chemotherapy.

Pneumonol Alergol Pol, 1993, 61(5-6), 248 - 53
{Drug sensitivity of bacillus strains M . avium-intracellulare (MAIC), M . kansasii cultured from patients with mycobacteriosis before treatment}; Zwolska-Kwiek Z et al.; The aim of study was to evaluate a drug sensitivity of M . avium-intracellulare, M . xenopi and M . kansasii cultured from 55 patients with mycobacterioses . The identification of strains was performed with morphological and biochemical tests and thin-layer chromatography . Resistance tests were done on egg L-J and agar media for selected drugs . It was documented that MAIC strains were non-sensitive on isoniazid and rifampicin, and other ones . The most active drug was cycloserine inhibiting growth of 80% but rifabutine--50% of strains . M . xenopi strains were sensitive for tested drugs including isoniazid and rifampicin (about 30%) . M . kansasii strains were in 100% sensitive for cyclosporine, rifabutine, Davercin and ofloxacin and partly for isoniazid, streptomycin and Augmentin.

Eur J Cancer, 1993, 29A(9), 1237 - 42
Long-term adjuvant immunotherapy in stage I high risk malignant melanoma, comparing two BCG preparations versus non-treatment in a randomised multicentre study (EORTC Protocol 18781); Czarnetzki BM et al.; The present study reports the results of a multicentre adjuvant trial with BCG (Bacillus Calmette-Guerin) in high risk patients (Breslow thickness > or = 1.5 mm, Clark level > or = III) with malignant melanoma, after surgical removal of their primary tumour . The trial was specifically designed in order to resolve the controversy and to provide some definite answers regarding the value of adjuvant BCG treatment in stage I malignant melanoma . Patients were randomised to either BCG RIV (108 patients) or BCG Pasteur (109 patients) for 3 years or to follow-up only (110 patients) . The two vaccines used had greatly divergent properties regarding their mode of preparation, their composition and their immunomodulating activities . Of the 353 randomised patients, 23 were ineligible, 3 refused participation after randomisation and 327 were evaluable for final analysis . Median follow-up time was 6 years (range 0-10 years) . The log-rank test comparison showed no statistical difference between the three arms regarding time to progression (P = 0.55) and duration of survival (P = 0.82) . Treatment was generally well tolerated, with no major adverse events in either treatment arm . These findings confirm data with different BCG preparations and with stage II melanoma which also demonstrated no benefit regarding patient survival and time to relapse.

Med Parazitol (Mosk), 1993 Jan-Feb, (1), 8 - 10
{The effect of water temperature on the action of bacterial insecticides against mosquito larvae}; Rasnitsyn SP et al.; The LD50 logarithm of Bacillus thuringiensis and B . sphaericus toxins effects on the larvae of Aedes aegypti and Culex pipiens is in inverse correlation with the water temperature . The influence of the temperature on the toxins effect on C . pipiens larvae is higher than that for A . aegypti larvae . Similarly, B . sphaericus toxin effect depends on the water temperature to a greater measure than that of B . thuringiensis toxin . The authors predict a high efficacy of B . sphaericus-based insecticides in hot climate countries.

Bull World Health Organ, 1993, 71(3-4), 367 - 75
{Campaign against Culex quinquefasciatus using Bacillus sphaericus: results of a pilot project in a large urban area of equatorial Africa}; Hougard JM et al.; Culex quinquefasciatus, which is sometimes the vector of Bancroft's filariasis, is a harmful mosquito, the immature stages of which live in collections of waste water resulting from human activity . Larval control, the most appropriate method, is at present carried out with chemical insecticides . But the toxicity of these compounds, together with phenomena of resistance, and the cost of substitute insecticides have turned research towards products of biological origin, and one of the most promising is a liquid concentrate of Bacillus sphaericus strain 2362 . This was applied experimentally over an area of 200 hectares in a large city in the south of Cameroon, characterized by a short dry season during which mosquito density is at its highest . Spraying was carried out every three months for a year in a concentration of 10 g/m2 and its efficacy evaluated at the level of adult mosquitos through an indirect system of capture on human baits . The results of this study show, in essence, a reduction by 52.7% in the number of females captured in the overall study area treated, and that the impact of treatment is greater during the period of high mosquito density (55.1%) than in the low density period (40.7%) . After analysis of the full set of results, the authors conclude that the pattern of rainfall, the conditions in which the insecticide is applied and reinvasions of mosquitos from untreated areas were the principal factors limiting the efficacy of this control campaign.(ABSTRACT TRUNCATED AT 250 WORDS)

Int J Syst Bacteriol, 1993 Jan, 43(1), 150 - 6
Bacillus cohnii sp . nov., a new, obligately alkaliphilic, oval-spore-forming Bacillus species with ornithine and aspartic acid instead of diaminopimelic acid in the cell wall; Spanka R et al.; A group of 20 alkaliphilic Bacillus strains in which all strains revealed the same unique comination of properties--obligate alkaliphily, oval spores distending the sporangium, and ornithine and aspartic acid instead of diaminopimelic acid in the cell wall--was examined . Most of the strains had been isolated by a five-step enrichment and isolation procedure . The G+C content was determined to span a range from 33.5 to 35.0 mol% . Unsaturated fatty acids amounted to 17 to 28% of the total cellular fatty acids . Through DNA-DNA hybridization experiments 11 strains could be grouped in one species . Low homology values with the type strains of validly published Bacillus species with similar G+C contents suggest that these strains belong to a hitherto undescribed species for which the name Bacillus cohnii is proposed . The type strain of the new species is strain RSH (= DSM 6307).

Microbiol Immunol, 1993, 37(3), 187 - 94
Role of calcium in biphasic germination of Bacillus cereus T spores sensitized to lysozyme; Shibata H et al.; Biphasic germination induced by inosine in the presence of Ca2+ was examined using Bacillus cereus T spores treated with sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) at pH 10 . The first phase of the germination was stimulated by Ca2+ in the concentration-dependent manner, showing the optimal concentration at 0.5-1.0 mM . The second phase appeared to be insensitive to the cation . The optimal temperatures for the first and the second phase were 25 C and 40 C, respectively; the optimal pHs for the two phases were 7-9 and around 7.5, respectively . Heat resistance and dipicolinic acid of the SDS-DTT-treated spores were lost mostly during the first phase . A Ca(2+)-specific chelator, glycoletherdiamine-N,N,N',N'-tetraacetic acid (GEDTA), inhibited the first phase evoked by Ca2+, while it had no inhibitory effect on the second phase . In contrast, the divalent cations examined, except Mg2+ and Sr2+, affected not only the first phase but also the second phase . The order of inhibitory effect on the first phase was Hg2+ > Zn2+ > Ba2+, Co2+, Cu2+ > Mn2+; on the second phase, it was Hg2+ > Cu2+ > Zn2+ > Co2+ > Mn2+ > Ba2+.

Nucleic Acids Symp Ser, 1993, (29), 139 - 40
Cloning of a gene required for the alkaliphily of alkaliphilic Bacillus sp . strain C-125; Aono R et al.; An alkali-sensitive mutant was obtained from alkaliphilic Bacillus sp . strain C-125 . A DNA fragment required for the alkaliphily was cloned from the chromosomal DNA of strain C-125 by using the alkali-sensitive mutant as a host . The nucleotide sequence of the cloned DNA fragment indicated the occurrence of several ORFs . Comparison of these ORFs with sequences in the available databases revealed no closely related sequences.

Mem Inst Oswaldo Cruz, 1993 Jan-Mar, 88(1), 131 - 4
Partial isolation and some properties of enterotoxin produced by Bacillus cereus strains; Guaycurus TV et al.; Extracellular proteins produced by Bacillus cereus AL-42 and AL-15 were fractioned by chromatography on QAE-Sephadex and Sephadex G75 . This last chromatographic process resulted in three peaks . The major peak showed vascular permeability activity to rabbits, lethality to mice, and cytotoxicity to Vero and Hela cells . The analysis by SDS-PAGE after ultrafiltration confirm recent findings that the enterotoxin is a compound with molecular mass > 30,000.

Proc Natl Sci Counc Repub China B, 1993 Jan, 17(1), 7 - 14
Complete nucleotide sequence and identification of a putative promoter region for the expression in Escherichia coli of the cryIA(b) gene from Bacillus thuringiensis var . aizawai HD133; Chak KF et al.; The sequence of a cry gene from Bacillus thuringiensis var . aizawai HD133 was determined . This cry gene encodes a protein of 1155 amino acids, the molecular weight of which is 130622 Da . When the nucleotide sequence of this cry gene was compared with the nucleotide sequence from B.t . var . aizawai IPL7 and var . berliner 1715, only five nucleotide changes were found . Therefore, this cry gene should be grouped into the cryIA(b) gene type . A 72 nucleotide sequence upstream of the open reading frame of the cry gene was enough to conduct the transcription of the cry gene in E . coli . Using a promoter probe vector system, it was demonstrated that any DNA fragment containing this 72 nt sequence exhibited promoter activity in vivo . It is likely that a putative promoter sequence may be present within this 72 nucleotides region for the expression of this cry gene in E . coli.

Bull Pan Am Health Organ, 1993, 27(3), 297 - 310
Association between HIV and tuberculosis: technical guide; Anti-PGL1 levels in leprosy patients and their contacts; Departamento de Clinica Medica, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, Brasil1 . We determined the anti-PGL1 levels of 402 individuals from the Ribeirao Preto region since the phenolic glycolipid (PGL1) is a specific Mycobacterium leprae antigen . This group consisted of 47 leprosy patients (26 with the lepromatous form, 16 with the tuberculoid form and 5 with the borderline form), 12 tuberculosis patients, 19 leprosy contacts, and 324 healthy blood donors from the Hemocenter of the University Hospital, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo . Anti-PGL1 levels were detected by ELISA . 2 . Anti-PGL1 levels were normal in patients with tuberculoid and borderline leprosy, in tuberculosis patients and in almost all of the healthy blood donors . Patients with untreated lepromatous leprosy had elevated anti-PGL1 levels while most patients under treatment (9/16) had normal anti-PGL1 levels . Only 3% of blood donors (10/324) had elevated anti-PGL1 levels, but when these individuals were submitted to clinical and bacilloscopic examination no signs of disease were found . To complete the clinical investigation, these 10 subjects were submitted to the Mitsuda reaction which was negative in 3 of them . All of these 10 subjects are being monitored, since they may be at risk to develop leprosy . 3 . On the basis of the present data, it seems that ELISA is a potentially important assay for the detection and chemotherapy of subclinical leprosy, permitting the control of epidemic centers of the disease.

Abdom Imaging, 1993 Fall, 18(4), 336 - 8
Hepatic bacillary angiomatosis in a patient with AIDS; Wyatt SH et al.; We report a case of bacillary angiomatosis involving the liver in addition to multiple cutaneous sites in a patient with AIDS . Abdominal CT demonstrated multiple, small low-attenuation lesions throughout the hepatic parenchyma . Consideration of this entity in the proper clinical setting is essential since it is a readily treatable infection.

Pol J Occup Med Environ Health, 1993, 6(1), 19 - 25
Experimental study of subacute oral, dermal and inhalation toxicity of bulmoscide preparation; Halkova Z et al.; The purpose of this study is to give the toxicological characteristics of subacute oral, dermal and inhalation exposure of microbial preparation Bulmoscide, which is based on bacterium Bacillus thuringiensis, serotype H-14 . The study was performed on sexually mature Wistar rats of both sexes . No significant changes in non-specific, clinico-laboratory and biochemical parameters as well as morphological examinations in any tested groups compared to the control groups were revealed . The doses 110 mg.kg-1 (1.32 x 10(9) sp.kg-1) at oral administration in duration of 90 days, 3000 mg.kg-1 (3.6 x 10(10) sp.kg-1) in 21 day dermal application and maximum attainable concentration of dust aerosol 18 mg.kg-3 (2.2 x 10(8) sp.m-3) during a 30 day inhalation exposure are "no toxic effect" levels . On the basis of the data, the selective bacterial insecticide Bulmoscide has been determined to be of low toxic and low hazardous preparation in compliance with the Hygiene Classification of pesticides.

AIDS Clin Rev . 1993-94;:43-60.
Bacillary angiomatosis; Berger TG et al.; The manifestations of bacillary angiomatosis include diverse cutaneous lesions, visceral parenchymal bacillary peliosis of the spleen and liver, and involvement of single or multiple organ systems . The organisms causing BA are R . henselae and R . quintana, and these organisms have now been cultured from the spleen and cutaneous lesions of BA as well as the blood of patients with visceral and cutaneous BA . The antibiotic regimen of choice for HIV-infected patients is erythromycin 500 mg 4 x a day for 2-4 months, but relapse may necessitate lifelong suppressive therapy . We are maintaining a registry of patients with bacillary angiomatosis and request that we be contacted (415-206-8680, UCSF) if you diagnose a case, preferably before therapy is instituted.

Pathobiology, 1993, 61(3-4), 230 - 5
Inhibitory role of alveolar macrophages in colony-stimulating factor production by the lung tissue from bacillus Calmette-Guérin-treated animals; Goliaei B et al.; Production of colony-stimulating factors (CSF) by lung tissue from rats injected by bacillus Calmette-Guerin (BCG) and the role of alveolar macrophages (AM) in this process was studied . Injection of BCG at 10, 100, or 1,000 mg/kg changed the CSF production by the lung in a time-dependent manner . Maximum stimulation was observed at 10 mg/kg and 3 days of interval between BCG injection and animal sacrifice . Longer periods or higher concentrations had no effect or actually depressed CSF production . BCG injection also changed the number of AM in a time- and dose-dependent manner . When AM from BCG-treated animals were lavaged out and the AM-depleted lung tissues were cultured, an increase in CSF production with respect to controls was observed at 10 mg/kg of BCG . On the other hand, in control animals removal of AM had no effect on CSF production by the lung . The results suggest that BCG treatment alters the number of AM and CSF production by the lung . AM, in BCG-injected animals, inhibit CSF production by the lung tissue while in non-injected animals they do not play any role in this process.

Arch Tierernahr, 1993, 44(2), 111 - 21
{The nutritive efficiency of Bacillus cereus as a probiotic in the raising of piglets . 1 . Effect on the growth parameters and gastrointestinal environment}; Kirchgessner M et al.; In an experiment with 4 x 12 piglets the probiotic effect of the Bacillus cereus strain FH 1457 S on the performance and changes in the gastrointestinal tract was studied . Beside a negative control the feed was supplemented with 10(7), 10(8) and 10(9) CFU Bacillus cereus/kg . The experiment included two feeding periods, whereby in the first a prestarter and in the second a piglet rearing feed was administrated ad libitum with the animals kept individually in cages . Over the whole feeding trial the piglets with the additions of 10(7), 10(8) und 10(9) CFU Bacillus cereus/kg feed showed higher daily weight gains of 9.5%, 11.1% and 8.6% with an increased feed consumption of 11.1%, 11.0% and 7.9% . The feed conversion rate was not influenced by the additions . In the first period daily weight gains were increased by 8.9%, 14.7% and 7.8%, the feed consumption in this period was 10.2%, 12.1% and 7.2% higher than in the control group . The addition of 10(8) CFU Bacillus cereus had a significantly higher daily weight gain in the first period compared to the control and the feed conversion rate was improved by 2.4% . In the second period the daily weight gains were increased by 9.9%, 8.9% and 9.1%, the feed consumption by 11.4%, 10.5% and 8.8% because of Bacillus cereus addition to the feed . The frequency of diarrhea was not influenced by the treatments . In each Bacillus cereus dosage the concentrations of ammonia, lactate and volatile fatty acids in small intestine contents were mostly diminished . In caecum contents the concentrations of acetic and propionic acid were decreased in the groups supplemented with Bacillus cereus.

Biochimie, 1993, 75(12), 1099 - 108
Discrimination between transfer-RNAs by tyrosyl-tRNA synthetase; Bedouelle H et al.; We have constructed a model of the complex between tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and tRNA(Tyr) by successive cycles of predictions, mutagenesis of TyrRS and molecular modeling . We confront this model with data obtained independently, compare it to the crystal structures of other complexes and review recent data on the discrimination between tRNAs by TyrRS . Comparison of the crystal structures of TyrRS and GlnRS, both of which are class I synthetases, and comparison of the identity elements of tRNA(Tyr) and tRNA(Gln) indicate that the two synthetases bind their cognate tRNAs differently . The mutagenesis data on tRNA(Tyr) confirm the model of the TyrRS:tRNA(Tyr) complex on the following points . TyrRS approaches tRNA(Tyr) on the side of the variable loop . The bases of the first three pairs of the acceptor stem are not recognized . The presence of the NH2 group in position C6 and the absence of a bulky group in position C2 are important for the recognition of the discriminator base A73 by TyrRS, which is fully realized only in the transition state for the acyl transfer . The anticodon is the major identity element of tRNA(Tyr) . We have set up an in vivo approach to study the effects of synthetase mutations on the discrimination between tRNAs . Using this approach, we have shown that residue Glu152 of TyrRS acts as a purely negative discriminant towards non-cognate tRNAs, by electrostatic and steric repulsions . The overproductions of the wild type TyrRSs from E coli and B stearothermophilus are toxic to E coli, due to the mischarging or the non-productive binding of tRNAs . The construction of a family of hybrids between the TyrRSs from E coli and B stearothermophilus has shown that their sequences and structures have remained locally compatible through evolution, for folding and function, in particular for the specific recognition and charging of tRNA(Tyr).

Eur J Cancer B Oral Oncol, 1993 Jan, 29B(1), 1 - 2
Irradiation mucositis: a reappraisal; Martin MV; Irradiation mucositis is a complication of anticancer therapy . It is regarded as an unavoidable consequence of treatment . Recent studies have shown that this condition is probably a consequence of abnormal Gram negative bacillary carriage in the oral cavity . If this abnormal carriage is avoided then prevention or amelioration of irradiation mucositis may be possible.

Ann Soc Belg Med Trop, 1993, 73 Suppl 1, 25 - 34
Identification of the leprosy bacillus and related mycobacteria by analysis of mycocerosate profiles; Minnikin DE et al.; Members of the phthiocerol dimycocerosate family of waxes were extracted from Mycobacterium bovis BCG, Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans and a skin biopsy from a leprosy patient . The waxes were degraded by alkaline hydrolysis and the mycocerosic acids converted to pentafluorobenzyl ester . Profiles of the esters, recorded using electron-capture gas-chromatography, gave characteristic profiles for the mycocerosates from M . leprae but those from M . bovis, M . tuberculosis and M . kansasii were superficially similar . The mycocerosate profiles from M . marinum and M . ulcerans were similar, but distinct from the others . Selected ion monitoring negative ion-chemical ionisation gas chromatography-mass spectrometry of of the pentafluorobenzyl esters allowed the analysis of mycocerosate isomers not revealed on gas chromatography alone . M . bovis and M . tuberculosis had similar profiles of C29, C30 and C32 mycocerosates; and additional C33 component was also present in M . kansasii . The mycocerosates from M . marinum and M . ulcerans were C27, C29 and C30 and those from M . leprae were distinct in having C29, C30, C32, C33 and C34 components . These methods have excellent potential for use in the detection of mycobacterial disease by direct analysis of infected tissue without prior cultivation of the causative agent.

Probl Tuberk, 1993, (4), 42 - 4
{Effect of immozymase inhalation on the detection of bacterial discharge in pulmonary tuberculosis}; Krasnov VA et al.; Administration of immosimase, a proteolytic enzyme complex developed in Russia, provoked active Tb discharge when the latter is occult in cases of destructive tuberculosis . Inhalation of the preparation enriches the patients' sputum with the bacilla . This permitted active tuberculosis diagnosis in additional 28% of the examinees.

J Biochem (Tokyo), 1993 Jan, 113(1), 67 - 73
Pyroglutamyl peptidase gene from Bacillus amyloliquefaciens: cloning, sequencing, expression, and crystallization of the expressed enzyme; Yoshimoto T et al.; The pyroglutamyl peptidase {EC 3.4.11.8} gene from Bacillus amyloliquefaciens was cloned and expressed in Escherichia coli DH1 . The transformant of E . coli DH1 harboring plasmid pBPG 1 with a 2.1 kb chromosomal DNA fragment showed 80-fold higher activity than B . amyloliquefaciens . The nucleotide sequence of a 0.9 kb fragment that contains the promoter and the mature protein coding region was determined by the dideoxy chain-termination method . An open reading frame of 648 bp starting with an ATG methionine codon was found, which encodes a protein of 215 amino acid residues with a deduced molecular weight of 23,286 . The enzyme has two cysteine residues (Cys68 and Cys144) per subunit molecule . Substitution of Cys144 with Ser by site-directed mutagenesis resulted in a complete loss of the activity, while that of Cys68 with Ser did not affect the activity at all . This result and titration with DTNB suggest that Cys144 is concerned in the catalytic action and Cys68 is located inside the enzyme . The expressed enzyme was purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with an activity recovery of 42.7% . The enzyme was most active at pH 6.5 and stable at pH 7.0-9.0 . Its molecular weight was estimated to be 51,000 by gel filtration, suggesting it to be a dimer . Big crystals of the wild and PCMB-modified enzymes were obtained by the hanging drop method.

Antonie Van Leeuwenhoek, 1993-94, 64(2), 137 - 43
Protein secretion in Bacillus brevis; Udaka S et al.; Many strains of Bacillus brevis were isolated from nature as very efficient producers of extracellular proteins . Strains identified as B . brevis including these protein-hyperproducers were reclassified into at least 6 species according to numerical analysis, DNA base composition, and DNA-DNA hybridization . We developed a host-vector system using appropriate strains of these Bacillus brevis as a host, which is excellent for the secretion of heterologous proteins . Utilizing the powerful promoters and signal peptide-coding regions of the cell wall protein gene, various expression-secretion vectors were constructed . The cell wall protein genes of these B . brevis are transcribed from multiple and tandemly arranged promoters . Transcription from P2, one of the major promoters among them, was enhanced at the early stationary phase of growth, when divalent cations in the medium was depleted and the cell wall protein layers started to be shed . Translation of the cell wall protein gene transcripts starts at the two sites located tandemly in the same reading frame . The two forms of secretory precursors, translation products from the two sites, are cleaved at the same position giving rise to the same mature proteins . The nucleotide sequence from the promoter to the translation start site is highly conserved in protein-hyperproducing B . brevis . For the efficient secretion of some heterologous proteins, protein-hypersecreting mutants had to be selected . The engineering of the signal peptide was also often necessary to obtain a good secretion of heterologous proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurochirurgie, 1993, 39(5), 326 - 9
{Tuberculous abscess of the cerebellum}; Dechambenoit G et al.; Cerebral tuberculous abscess is exceptional . We report on the case of a H.I.V . positive 29 year-old man suffering from high intracranial pressure together with a cerebellar syndrome . The CT scan revealed a voluminous abscess of the left cerebellar hemisphere . Anatomical and pathological examination of the extracted mass showed a tuberculous abscess . 16 months after surgery and antituberculous treatment, the patient's symptoms have disappeared and he has resumed his professional activities . Though controversial, a pathogenic continuum between a tuberculoma and a tuberculous abscess may be assumed to exist . The A.I.D.S . pandemic reactivates the tuberculous foci, increases the risks of contagion reduced the effectiveness of treatment, and justifies a systematic search for the bacillus of Koch, after puncture, in any cerebral abscess.

Chin J Biotechnol, 1993, 9(4), 219 - 27
Insect tolerance of transgenic Populus nigra plants transformed with Bacillus thuringiensis toxin gene; Tian Y et al.; Leaves and stem segments of Populus nigra were transformed with A . tumefaciens LBA4404 harboring a binary vector containing chimeric genes of NPT and 35S-omega-B.t . toxin-Nos . Nineteen regenerated kanamycin resistant plants were analyzed by DNA hybridization, out of which 10 were shown to be the candidates of transgenic plants . Insect tolerance tests showed that the transgenic plants were toxic to two lepidopteran pests, Lymantria dispar L . and Apocheimia cinerarius Erschoff . Based on the results of Southern blot of PCR products and the cluster analysis of their growth and insect resistance, three independent transgenic plants were selected, which were then propagated in the nursery field . This field test is currently in progress.

Rocz Panstw Zakl Hig, 1993, 44(2-3), 227 - 30
{Laboratory studies of the usefulness of biopreparation Dipel in reducing the german cockroach (Blattella germanica L.) population}; Zukowski K; In the studies conducted for finding of biological factors effectively controlling insects of hygienic importance German cockroaches were subjected to the action of an insecticide containing spores of Bacillus thuringiensis var . kurstaki . One gram of the insecticide contained 25 miliard (US billion) spores with protein endotoxin crystals . The insects were given drinking water and food containing 1.0%, 0.5%, 0.25%, 0.125% of the initial preparation . The experiment was carried out on 1800 cockroaches . Half of them (600 mature insects differentiated for sex and age and 300 larvae in stage 1st of development) were treated with the preparation, the remaining insects served for control . The insects were kept at about 28 degrees C . After 9 days o the experiment in the group receiving 1% Dipel on food 76.4% mature insects and 83.0% larvae in 1st stage of development died . After 14 days all males died, and after 17 days died all females . In the control group died in that time 2.3% and 3.4% respectively . In case of lower per cent of the preparation in food the time of dying was prolonged, and at the lowest per cent it was 22 days . The study showed that Dipel effectively reduced the population of German cockroaches, and although in initial period its action was slower than that of chemical agents, it could be used for the control of these insects, especially as it in non-toxic for humans, warm-blooded animals and useful insects.

Southeast Asian J Trop Med Public Health, 1993, 24 Suppl 2, 64 - 8
Recent advances in vector control in filariasis; Sucharit S; Three factors are involved when vector control is incorporated into an integrated control program for filariasis: (1) the vector species or species complex of each filarial worm; (2) type of control measures employed; (3) pesticide application . Recent advances in vector control in filariasis include the use of bed nets, environmental manipulation, insecticides and biological control . Three promising advances in vectrol control are the use of pathogenic bacteria such as Bacillus thuringiensis H-14 and B . sphaericus, juvenile hormone mimics or insect growth regulator (IGR) and predatory crustaceans.

Rocz Panstw Zakl Hig, 1993, 44(4), 387 - 94
{Model studies of milk containing inhibitory substances . I . Sensitivity of microbiological tests for detection of inhibitory substances and studies of turbidity changes in milk}; Wieczorek J et al.; In the present studies use was made of raw milk derived from a model farm in the Olsztyn voivodeship . Washing and washing-disinfecting agents: Mirax, Bis, Radion, Iosan CP, Rudlak and Ixi, as well as crystalline penicillin were added to milk . Their lowest concentrations inhibiting growth of indicator strain (MIC): Bacillus stearothermophilus var . calidolactis C 953, were determined by two microbiological tests . Moreover, the changes in light diffusion by milk, caused by the applied inhibitory substances, were examined . It was found that the microbiological tests for the determination of the lowest inhibitory concentration of the sanitization agents and of penicillin were much more sensitive in the case of milk with a 0.05% fat content, as compared with whole milk.

Probl Khig, 1993, 18, 114 - 24
{The acute toxicity of a bacterial insecticidal preparation containing a beta-exotoxin}; Khalkova Zh et al.; The acute toxicity of a new Bulgarian bioinsecticide preparation is determined, produced on the basis of Bacillus thuringiensis containing 0.1% beta-exotoxin by oral, dermal, inhalational and intraperitoneal introduction in experimental animals . The evaluation of the toxicity is performed by a complex of toxicometric, integral, haematologic and pathologicoanatomic methods according to the WHO and USA criteria for studying the safety of the microbic agents for plant protection . It is already established that the preparation introduced orally in dose 10,000 mg.kg-1 (1.6.10(11) cell kg.-1 10 micrograms.kg-1 beta exotoxin), applied dermally in dose 6000 mg.kg-1 (9.6.10(10), 6 micrograms.kg-1 beta exotoxin) and in concentration 300 mg.m-3 (4.8.10(10) cell m-3, 300 micrograms.m-3 beta exotoxin) provokes no lethality, intoxication and changes in the integral, haematologic and pathologicoanatomic studies of test animals . LD50 at intraperitoneal introduction in white rats is 387.20 mg (6.2.10(10), kg-1, 387.2 micrograms kg-1 beta exotoxin) for male and 364.0 mg kg-1 (0.58.10(9) kg-1 364.0 micrograms kg-1 beta exotoxin) for female animals . The investigations point out that according to rate of acute toxicity the bacterial preparation containing 0.1% beta exotoxin is referred to the low toxic substances and reveals no danger for acute oral, dermal and inhalational poisonings when the regulations for production and use are observed.

Vestn Rentgenol Radiol, 1993 Jan-Feb, (1), 15 - 8
{Effectiveness of fluorography in the prevention and diagnosis of pulmonary tuberculosis and cancer}; Shteintsaig AI et al.; Annual fluorographic examinations, involving about 80% of adult population, helped detect 81.6 +/- 0.6% of patients with pulmonary tuberculosis and 37 +/- 0.5% of those with pulmonary carcinoma . Unjustified reduction of the number of prophylactic phorofluorographies resulted in a reduction of the level of diagnosis of these conditions: the share of detected tuberculosis patients reduced to 62.3% . The incidence of bacterial discharge in newly detected cases increased by 19.7%, the disintegration phase increased by 16% . Decrease of the bacillary nucleus parallelled by an almost twofold growth in the number of lethal cases of tuberculosis evidence poor detection of the patients . Reduction in the number of cases of pulmonary carcinoma detected by prophylactic photofluorography resulted in the reduction of operable patients with this condition from 23.5 to 18% . A retrospective analysis of photofluorograms of newly detected patients with destructive tuberculous processes demonstrated the necessity of annual photofluorographic check-ups of the adult population aimed at the detection of pulmonary tuberculosis . The authors emphasize that the intervals between prophylactic check-ups of subjects at risk of developing pulmonary tuberculosis should by no means be prolonged.

Biosci Biotechnol Biochem, 1993 Jan, 57(1), 56 - 60
Enzymatic synthesis of a novel trisaccharide, glucosyl lactoside; Shibuya T et al.; Cyclomaltodextrin glucanotransferase from Bacillus stearothermophilus produced a series of oligosaccharides by the transglycosylation reaction with cyclomaltohexaose as a glycosyl donor and D-lactose as its acceptor . The content of oligosaccharide A as the main transfer product was 22.1% of the total sugar . The content was increased to 34.1% by hydrolysis with glucoamylase . Oligosaccharide A was isolated by column chromatography and crystallized . By chemical and enzymatic analyses, oligosaccharide A was found to be a novel non-reducing trisaccharide; O-beta-D-galactopyranosyl-(1-->4)-O- beta-D-glucopyranosyl alpha-D-glucopyranoside.

Appl Microbiol Biotechnol, 1993 Jan, 38(4), 507 - 13
Characterization, cloning and sequencing of a thermostable endo-(1,3-1,4) beta-glucanase-encoding gene from an alkalophilic Bacillus brevis; Louw ME et al.; A Bacillus brevis gene coding for an endo-(1,3-1,4)-beta-glucanase was cloned in Escherichia coli and sequenced . The open reading frame contains a sequence of 759 nucleotides encoding a polypeptide of 252 amino acid residues . The amino acid sequence of the beta-glucanase gene showed only a 50% similarity to previously published data for Bacillus endo-(1,3-1,4)-beta-glucanases . The optimum temperature and pH for enzyme activity were 65-70 degrees C and 8-10, respectively . When held at 75 degrees C for 1 h, 75% residual activity was measured . The molecular mass was estimated to be about 29 kDa on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and the enzyme was found to be resistant to SDS.

J Cell Biochem Suppl, 1993, 17F, 104 - 17
Hamster bronchial carcinogenesis induced by carcinogen-containing sustained release implants placed endobronchially: a clinically relevant model; Hammond WG et al.; For several widely appreciated reasons, the Syrian hamster has been the most frequently used experimental animal in investigations of conducting airway carcinogenesis . To develop a model where bronchogenic cancer arises focally at a predetermined site, we used the Laskin-Kuschner self-retaining intrabronchial pellet principle, employing a carcinogen-containing silastic polymer sustained release implant (SRI) . The SRI is placed in the right lower lobe bronchus via tracheostomy; when modified, the SRI can be removed without loss of the animal . Special preparation of the SRI implant site after fixation but prior to paraffin embedding allows for full histopathological examination of the carcinogen-affected target tissue . Logistic regression analysis of histological findings provides valid quantitative inter-regimen comparisons of histomorphic classifications suitable for determining modulation of carcinogenesis by external influences . Using this model, we demonstrated that the sequential progression of carcinogenesis (SPC) in hamster bronchus is similar to that which occurs in humans and in dogs, including both the ploidy increases that are progressive during the SPC, and the histological patterns of the induced cancers . We have shown genetic variation in susceptibility to carcinogenesis among inbred hamster strains, and have assessed effects of Bacillus Calmette-Guerin (BCG) immunostimulation on the SPC . Time/dose response studies were performed, as were comparisons between four polycyclic aromatic hydrocarbon carcinogens . Systemic administration of 5-azacytidine (AZC) soon after SRI placement was found to inhibit the SPC, to alter the ploidy changes during the SPC and in the eventual cancers, and to affect the degree of differentiation of the cancers . Studies using removable SRIs have assessed the duration of carcinogen exposure required to induce a neoplastic transformation that proceeds to cancer without further carcinogen exposure . Serial syngeneic transplantation of cancers arising in inbred animals has shown that the degree of tumor differentiation is affected by the extent of host immunocompetence, and has also led to development of models for study of the processes of metastasis.

Acta Pathol Jpn, 1993 Jan-Feb, 43(1-2), 11 - 7
Abscess-forming granulomatous lymphadenitis: histological typing of suppurative granulomas and clinicopathological findings with special reference to cat scratch disease; Kojima M et al.; In order to clarify the histological and immunohistochemical characteristics of suppurative granuloma in abscess-forming granulomatous lymphadenitis (AGL), and the relation between AGL and cat scratch disease (CSD), 36 cases of AGL were studied . The combined results showed that there were two types of suppurative granulomas . The suppurative granulomas histologically revealed small lymphocytes of predominantly T cell phenotype distributed among the epithelioid histiocytes bordering central necrotic areas in the suppurative granulomas . These suppurative granulomas could be further subdivided into two groups, mainly those with and without the intermingling of large transformed cells of B-cell phenotypes: Type B granuloma with large transformed B cells and Type A without large transformed B cells . Both types of granulomas were observed in a varying degree in most cases . According to the predominant type of granulomas, 36 patients with AGL were further classified into two groups: Group I of Type A dominance and Group II of Type B dominance . Warthin-Starry (WS) silver stain positive bacteria, which are said to be a causative agent of CSD, were present in about 50% of both groups . No Brown-Hopps' Gram-positive bacteria, fungus, toxoplasma, Chlamydia or Bacillus Calmette-Guerin antigen were found in any case . Clinically, there was no significant difference between these two groups . On the other hand, the detection of WS-positive bacteria seemed to have some relationship with the duration of disease and the history of exposure to cats, and 70% of AGL cases occurred in autumn without a single concurrent epidemic.

Biol Res, 1993, 26(3), 357 - 64
Evaluation of penicillin acylase production by two strains of Bacillus megaterium; Illanes A et al.; Penicillin acylase is a key enzyme for the production of semisynthetic beta-lactam antibiotics . The intracellular enzyme from Escherichia coli has been thoroughly studied and characterized . The extracellular enzyme from Bacillus megaterium, despite its potential advantages, has received less attention in the recent scientific literature . A comparative study is presented for the production of penicillin acylase with two strains of Bacillus megaterium in batch fermentation in previously optimized complex and defined media . The enzyme produced by the selected strain has been recovered, partially purified and its kinetic behaviour determined.

J Biol Chem, 1992 Dec 25, 267(36), 25625 - 7
Protein control of iron-sulfur cluster redox potentials; Langen R et al.; The relationship between the three-dimensional structures of iron-sulfur proteins and the redox potentials of their iron-sulfur clusters is of fundamental importance . We report calculations of the redox potentials of the {Fe4S4(S-cys)4}-2/-3 couple in four crystallographically characterized proteins: Azotobacter vinelandii ferredoxin I, Peptococcus aerogenes ferredoxin, Bacillus thermoproteolyticus ferredoxin, and Chromatium vinosum high potential iron protein (HiPIP) . Our calculations use the "protein dipoles Langevin dipoles" microscopic electrostatic model, which includes both protein and solvent water . The variations in calculated redox potentials are in excellent agreement with experimental data . In particular, our results confirm the important role of amide groups close to the cluster in separating the potential of C . vinosum HiPIP from those of the other three proteins . However, the potentials of these latter exhibit a substantial range despite extremely similar amide group environments of their clusters . Our results show that the potentials in these proteins are tuned in part by varying the access of solvent water to the neighborhood of the cluster . Our calculations provide the first successful quantitative modeling of the protein control of iron-sulfur cluster redox potentials.

J Biol Chem, 1992 Dec 15, 267(35), 25059 - 66
Identification of glutamic acid 105 at the active site of Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase using epoxide-based inhibitors; Hoj PB et al.; Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) was modified by the mechanism-based, affinity-labeling reagent {14C}(3,4)-epoxybutyl beta-D-cellobioside . Following partial inactivation a completely inactivated enzyme preparation containing 1.1 mol of covalently bound inhibitor/mol of protein was obtained by chromatography on a cellulosic matrix . The inactivated enzyme was digested with endoproteinase Glu-C and radioactive peptides purified by reversed-phase high performance liquid chromatography (HPLC) . The affinity label was esterified exclusively to the gamma-carboxylate of Glu105 in the sequence Gly-Thr-Pro-Trp-Asp-Glu-Ile-Asp-Ile-Glu109 . The sequence motif Glu-(Ile/Leu)-Asp-Ile is found in many glucanases and xylanases and may therefore serve to identify the catalytic nucleophile in beta-glycanases, which otherwise exhibit a low degree of sequence identity . The esterification of Glu105 by the affinity label abolished endoproteinase Glu-C-mediated hydrolysis of the Glu-Ile106 peptide bond . Identification of phenylthiohydantoin-Glu105 during automated sequence analysis was not possible unless the affinity label was liberated by prior base hydrolysis . These observations formed the basis for the development of a highly sensitive approach for the identification of catalytic carboxylates in polysaccharide hydrolases employing non-radioactive inhibitors, comparative HPLC mapping, electrospray mass spectrometry, and Edman degradation.

Tidsskr Nor Laegeforen, 1992 Dec 10, 112(30), 3795 - 8
{Gerhard Henrik Armauer Hansen--still of current interest}; Harboe M; Armauer Hansen described his first observation of Mycobacterium leprae on 28 February 1873 in detail . His discovery of the leprosy bacillus was the result of a logical process in which distinct stages can be clearly seen . The first step was clinical, and established criteria for leprosy as "a specific disease" . The second stage was epidemiological . Contrary to the main view favouring a genetic basis, these observations convinced him of the infectious nature of the disease . The third stage was the search for the bacillus . His basic observations were reproduced, as documented . Armauer Hansen afterwards tried to fulfill Koch's postulates, in order to prove that the bacillus was the causative agent of leprosy . His second main contribution is the work on leprosy control, emphasizing isolation to contain the infection . The subsequent decline of leprosy in Norway differs from the trend in many developing countries, where leprosy has remained an important public health problem . In recent years Norwegian medical research has made significant contributions to leprosy research, in the field of epidemiology in Bergen, and through immunological studies of the disease originating from the Armauer Hansen Research Institute in Addis Ababa, Ethiopia.

Biochemistry, 1992 Dec 8, 31(48), 12169 - 72
Kinetics of Bacillus cereus phosphatidylinositol-specific phospholipase C with thiophosphate and fluorescent analogs of phosphatidylinositol; Hendrickson HS et al.; Thiophosphate analogs (C-S-P bond) of phosphatidylinositol (Cn-thio-PI: racemic hexadecyl-, dodecyl-, and octylthiophosphoryl-1-myo-inositol) and a fluorescent analog (pyrene-PI: rac-4-(1-pyreno)-butylphosphoryl-1-myo-inositol) were all substrates for phosphatidylinositol-specific phospholipase C from Bacillus cereus . Hydrolysis of thio-PI was followed by coupling the production of alkylthiol to a disulfide interchange reaction with dithiobispyridine . Hydrolysis of pyrene-PI was followed using a HPLC-based assay with fluorescence detection . The activity of PI-PLC with thio-PI analogs showed an interfacial effect . C16-Thio-PI, which had a critical micelle concentration (CMC) of 7 microM, gave a hyperbolic activity versus concentration curve between 0 and 2 mM, while C8-thio-PI, which had a CMC above 10 mM, showed very low activity which increased greatly upon introduction of an interface in mixed micelles with hexadecylphosphocholine (HDPC) . Pyrene-PI, which aggregates above 0.3 mM, gave a sigmoidal activity curve with much higher activity above the CMC . All three thio-PI homologs as mixed micelles with HDPC gave hyperbolic activity curves with PI-PLC that were a function of bulk concentration of substrate at constant surface concentration and surface concentration of substrate at constant bulk concentration . The maximal activity of PI-PLC with pure C16-thio-PI micelles was 6.25 mumol min-1 mg-1, while that with pyrene-PI was estimated to be 68 mumol min-1 mg-1 . With pure C16-thio-PI micelles, 0.022 mM substrate gave half Vmax, similar to that in mixed micelles with HDPC.

Med J Aust, 1992 Dec 7-21, 157(11-12), 756 - 9
Ocular injuries caused by magpies; Horsburgh BJ et al.; This paper presents a series of six patients with ocular injuries resulting from magpie attacks . Five cases involved children . In two cases the penetration was overlooked initially . In one case the keratitis was caused by Bacillus cereus . Full ophthalmic examination, including indirect ophthalmoscopy and microbiological studies, must be undertaken initially to identify unrecognised eye injuries and to prevent the possible sight-threatening complications of vitreal fibrosis with subsequent retinal detachment or endophthalmitis.

N Engl J Med, 1992 Dec 3, 327(23), 1625 - 31
Isolation of Rochalimaea species from cutaneous and osseous lesions of bacillary angiomatosis; Koehler JE et al.; BACKGROUND . Bacillary angiomatosis is characterized by vascular lesions, which occur usually in patients infected with the human immunodeficiency virus (HIV) . A newly described gram-negative organism, Rochalimaea henselae, has been associated with cutaneous bacillary angiomatosis, but no organism has been isolated and cultivated directly from cutaneous tissue . METHODS . We used two methods to isolate the infecting bacterium from four HIV-infected patients with cutaneous lesions suggestive of bacillary angiomatosis: cultivation with eukaryotic tissue-culture monolayers and direct plating of homogenized tissue onto agar . The patients' blood was cultured with the lysis-centrifugation method . Isolates recovered from skin and blood were identified by sequencing all or part of the 16S ribosomal RNA gene amplified with the polymerase chain reaction . RESULTS . R . quintana, historically known as the agent of trench fever, was isolated from cutaneous lesions in three patients, after tissue homogenates were cultivated with endothelial-cell monolayers; R . henselae was isolated from a cutaneous lesion in one patient . In two patients, R . quintana was isolated from both cutaneous tissue and blood; in one patient it was also isolated from bone . CONCLUSIONS . In bacillary angiomatosis, either of two species of rochalimaea--R . quintana or R . henselae--can be isolated from cutaneous lesions or blood, providing an additional method of diagnosis.

Immunol Invest, 1992 Dec, 21(7), 613 - 21
The effect of BCG on thoracic duct lymphocytes; Stuhldreher D et al.; Adoptive immunotherapy is becoming increasingly more important in the management of advanced malignancies . This report describes the results of immunomodulation therapy with BCG in the Dunning tumor . In addition it describes new techniques in the harvesting of lymphocytes . Thoracic duct lymphocytes from 34 rats were evaluated for the effect of the presence of the Dunning R-3327 AT-3 tumor as well as for the response to bacillus Calmette Guerin (BCG) . Both tumor and BCG resulted in significant changes in the helper/suppressor T cell ratios.

Antimicrob Agents Chemother, 1992 Dec, 36(12), 2816 - 20
Comparison of two antibiotic regimens (piperacillin plus amikacin versus ceftazidime plus amikacin) as empiric therapy for febrile neutropenic patients with cancer; Feliu J et al.; A total of 170 febrile episodes in neutropenic patients with cancer were randomly assigned to be treated with piperacillin-amikacin or ceftazidime-amikacin . The overall response rates were similar in both groups (68 and 65%, respectively) . Response rates for clinically or microbiologically documented episodes were 54.5% for piperacillin-amikacin and 58.8% for ceftazidime-amikacin . Response rates for gram-negative bacillary infections were 65 and 73%, respectively . There was also no difference for gram-positive infections (31 and 50%, respectively) . The toxicities were also comparable and consisted of skin rashes, hypokalemia, and diarrhea . Vancomycin was added if the fever persisted 72 h after the beginning of therapy; it increased the response rates to 94% when used with piperacillin-amikacin and 92% when used with ceftazidime plus amikacin . Our results suggest that the combinations show similar global efficacies in the treatment of febrile episodes in cancer patients.

Heredity, 1992 Dec, 69 ( Pt 6), 539 - 46
Increase in the resistance to the Bacillus thuringiensis supernatant effect in a Drosophila melanogaster wild type Oregon R line; Paumard-Rigal S et al.; We report here the genetical and X chromosome rDNA molecular study of two Drosophila melanogaster Oregon R lines . These lines differ extensively in the degree of resistance of the females both to the lethal effect of an increased temperature and to that of Bacillus thuringiensis beta-exotoxin, which is an inhibitor of the nucleolar RNA polymerase . The 3B line, whose females are resistant, came from an Oregon R population subjected over several generations to increased temperature, 28 degrees C or over, while the other line is derived from the initial stock . Twofold variation was observed in the total number of ribosomal genes between the two lines . This variation applied to most ribosomal units, including the active ones . Variations among X chromosome rDNA content in a wild type population have thus been revealed using tests of resistance to the Bacillus thuringiensis beta-exotoxin . Additive variations in specific unit types between the two lines indicate that modifications to the rDNA content are not rare events.

Appl Environ Microbiol, 1992 Dec, 58(12), 3921 - 7
Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleoptera species; Donovan WP et al.; Bacillus thuringiensis EG2838 and EG4961 are highly toxic to Colorado potato beetle larvae, and only strain EG4961 is toxic to southern corn rootworm larvae . To investigate the cause of the different insecticidal activities of EG2838 and EG4961, cryIII-type genes toxic to coleopterans were cloned from each strain . The cryIIIB gene, cloned as part of an 8.0-kb EcoRI fragment of EG2838 DNA, encoded a crystal protein (CryIIIB) of 74,237 Da . The cryIIIB2 gene, cloned as part of an 8.3-kb PstI-Asp718 fragment of EG4961 DNA, encoded a crystal protein (CryIIIB2) of 74,393 Da that was 94% identical to CryIIIB . Analysis of the transcriptional start sites showed that cryIIIB and cryIIIB2 were initiated from a conserved region located within 130 nucleotides upstream from the translation start sites of both genes . Although the CryIIIB and CryIIIB2 proteins were similar in sequence, they displayed distinct insecticidal activities: CryIIIB was one-third as toxic as CryIIIB2 to Colorado potato beetle larvae, and CryIIIB2, but not CryIIIB, was toxic to southern corn rootworm larvae . Genes encoding crystal proteins of approximately 32 and 31 kDa were located adjacent to the cryIIIB and cryIIIB2 genes, respectively . The 32- and 31-kDa crystal proteins failed to enhance the insecticidal activities of CryIIIB and CryIIIB2.

J Periodontol, 1992 Dec, 63(12), 941 - 53
Eikenella corrodens in human oral and non-oral infections: a review; Chen CK et al.; There is substantial evidence in support of the existence of distinct clinical forms of human periodontal disease . Moreover, these different forms of periodontal disease may be associated with relatively distinct subgingival microflora, often involving microaerophilic or anaerobic Gram-negative bacterial species . Eikenella corrodens is a facultative Gram-negative bacillus which is a common inhabitant of the oral cavity and the intestinal and genital tracts . Its primary ecologic niche within the oral cavity appears to be dental plaque, both in periodontally healthy individuals and in periodontitis patients . However, E . corrodens is recognized as an infrequent human pathogen capable of causing extraoral infections, either as the sole infectious agent or as part of a mixed infection, its potential role in the etiology of periodontal disease is not well understood . E . corrodens is often present in the supra- and subgingival plaque of periodontally healthy subjects . On the basis of cross-sectional and longitudinal studies, E . corrodens appears to be somewhat more prevalent in subgingival plaque samples of periodontitis subjects than periodontally healthy individuals . However, the percentage of E . corrodens in the total cultivable microflora did not vary between the two groups . Microbiologic studies attempting to define the relationship between E . corrodens and periodontal disease assume that this species is essentially homogeneous and that all strains exhibit comparable pathogenic potential . However, E . corrodens exhibits 1) variable colony morphology, biochemical and serologic reactivity; 2) marked phenotypic diversity with respect to outer membrane protein and lipopolysaccharide structure; and 3) marked diversity in the restriction patterns of total genomic DNA . Thus, it is possible that a limited number of clones of E . corrodens may be associated with periodontal disease and/or extraoral infection, while other strains are relatively harmless commensals . Additional studies, possibly employing strain-specific nucleic acid probes, may be required to define the role of E . corrodens as a human periodontal pathogen.

Cell Biochem Funct, 1992 Dec, 10(4), 237 - 41
Studies on the ATPase of Bacillus cereus; Higuti IH et al.; The membrane ATPase (EC 3.6.1.3) of Bacillus cereus was solubilized by a 'shock-wash' process and purified . The non-specific phosphatase contaminant was separated by glycerol density gradient centrifugation . The optimum temperature was 39.5 degrees C and the pH optimum at 7.5 . On SDS-polyacrylamide gel electrophoresis two classes of subunits were observed in equal proportions with molecular weights of 70 K and 83 K . The effect of various compounds on the enzymatic activity was studied . The enzyme was insensitive to NaN3, oligomycin and to divalent cations, but was inhibited by citrate and oxalate.

Am J Trop Med Hyg, 1992 Dec, 47(6), 721 - 9
Basic epidemiology of tuberculosis in Peru: a prevalence study of tuberculin sensitivity in a Pueblo joven; Getchell WS et al.; Tuberculosis continues to cause significant morbidity and mortality in developing nations . As a first step in defining the magnitude of the problem in Peru, we determined the prevalence of tuberculin sensitivity in an age-stratified, community-based population on the outskirts of Lima in December 1990 . The overall prevalence of 10 mm or more induration in 368 individuals was 34% . When stratified by age, the prevalence was 12% in the 0-1-year-old group, 18% in the 2-4-year-old group, 24% in the 5-14-year-old group, 60% in the 15-24-year-old group, and 68% in the > or = 25-year-old group . Vaccination with bacillus Calmette-Guerin (87% of the study population) caused significant increases in weak (5-9 mm) reactions to purified protein derivative, but did not cause strong (10 mm or more) reactions . The prevalence of tuberculous infection in this population is higher than that previously reported in Peru and in most other high-risk populations . Unfortunately, the current political and economic situation in Peru makes it difficult to implement public health measures to prevent infection and progression of infection to disease.

FEMS Microbiol Immunol, 1992 Dec, 5(5-6), 337 - 45
Effect of blocking TNF-alpha on intracellular BCG (Bacillus Calmette Guerin) growth in human monocyte-derived macrophages; Fazal N et al.; Four agents, thalidomide, oxpentifylline, dexamethasone and a polyclonal anti-TNF-alpha antibody, were all shown by specific Elisa to block endogenous TNF-alpha production by Bacillus Calmette Guerin (BCG)-infected human monocyte-derived macrophages in in vitro culture . There was however no significant enhancement of intracellular BCG growth, over a 7-day incubation, in human monocyte-derived macrophages in the presence of any of the TNF-alpha-blocking agents, as determined by both radiometric and CFU counting methods of assessing bacterial viability and growth . The result suggests that the action of TNF-alpha alone is unlikely to be an important effector mechanism in antimycobacterial immunity within human cells.

Am Heart J, 1992 Dec, 124(6), 1541 - 4
Prospective study of blood culture during transesophageal echocardiography; Shyu KG et al.; To ascertain the incidence and significance of bacteremia associated with transesophageal echocardiography (TEE), 132 consecutive patients (aged 17 to 73 years) free of apparent infection who were undergoing 135 transesophageal echocardiographic procedures from October 1990 to August 1991, were prospectively studied . For each procedure, two sets of blood cultures were obtained for culture 30 to 60 minutes before TEE, immediately after, and 180 to 240 minutes after the procedure . For each blood culture, 10 ml of venous blood was evenly inoculated into aerobic and anaerobic culture bottles and inoculated for 7 days using a radiometric system . A throat swab was obtained immediately before each procedure . Three of 270 preprocedure blood cultures were positive for Bacillus cereus, Staphylococcus simulans, and Peptostreptococcus species, respectively . No blood culture was positive in the immediate postprocedure period . Two of 270 late blood samples grew Staphylococcus epidermidis in the same patient . Nevertheless, the microorganisms isolated from blood cultures were different from those isolated from the throat swab . No patients had fever or evidence of infective endocarditis after TEE during the follow-up period . It is concluded that the incidence of TEE-related bacteremia is extremely low, and a general recommendation for antibiotic prophylaxis during TEE is not warranted.

J Bacteriol, 1992 Dec, 174(24), 7919 - 25
Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans; Kitazono A et al.; The gene coding for proline iminopeptidase in Bacillus coagulans was cloned and expressed in Escherichia coli . Nucleotide sequencing revealed an 861-bp open reading frame with an unusual TTG initiation codon, encoding a 287-amino-acid protein . The calculated molecular weight of the product was 32,415 . The amino acid sequences of the amino-terminal region and those of some peptide fragments obtained by endoproteinase Asp-N digestion of the purified enzyme completely coincided with those deduced from the nucleotide sequence . The rare TTG initiation codon that normally codes for leucine was translated as a formal initiation codon; a methionine residue was found at the amino terminus of the enzyme . By using a vector bearing the strong tac promoter, an expression level as high as 200-fold that of the first clone was achieved . The replacement of the TTG initiation codon with ATG and a simultaneous reduction of the distance to the tac promoter resulted in a further increase of 2.5-fold . The expressed enzyme was easily purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with a recovery of activity of 36% . The molecular weight was found to be 33,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Hi-Load 16/60 Superdex 200 fast protein liquid chromatography column . The expressed enzyme showed the same catalytic and physicochemical properties as those of the wild type, specifically cleaving the N-terminal proline from small substrates.

Eur J Biochem, 1992 Dec 1, 210(2), 585 - 90
Binding of Bacillus sphaericus binary toxin to a specific receptor on midgut brush-border membranes from mosquito larvae; Nielsen-Leroux C et al.; The presence of specific receptors for Bacillus sphaericus binary toxin on brush-border membrane fractions (BBMF) from Culex pipiens larvae midgut cells was demonstrated by an in vitro binding assay . Both activated and radiolabelled polypeptides from the 51-kDa and 42-kDa binary toxin of B . sphaericus 1593 specifically bound to BBMF . Direct binding and homologous competition experiments indicated a single class of B . sphaericus toxin receptors, with a dissociation constant (Kd) of approximately 20 nM and a maximum binding capacity (Bmax) of approximately 7 pmol/mg BBMF protein . The sugars GalNAc, GlcNAc and N-acetyl neuraminic acid had no detectable inhibitory effect on toxin binding to C . pipiens BBMF . Binding experiments with the non-susceptible mosquito species Aedes aegypti failed to detect significant binding of B . sphaericus binary toxin to A . aegypti BBMF.

Infect Immun, 1992 Dec, 60(12), 5126 - 31
The arginine-dependent cytolytic mechanism plays a role in destruction of Naegleria fowleri amoebae by activated macrophages; Fischer-Stenger K et al.; Mouse peritoneal macrophages activated by different immunomodulators (Mycobacterium bovis bacillus Calmette-Guerin or Propionibacterium acnes) destroy Naegleria fowleri amoebae by a contact-dependent process and by soluble cytolytic molecules secreted by macrophages in response to lipopolysaccharide . The goal of this study was to determine whether the arginine-dependent cytolytic mechanism which results in the production of nitric oxide from arginine by activated macrophages destroys the amoebae . Amoebicidal activity of activated macrophages was determined by coculturing macrophages with N . fowleri amoebae radiolabeled with 3H-uridine . The percent specific release of radiolabel was used as an index of cytolysis of the amoebae . The inhibitors NG-monomethyl-L-arginine and arginase were used to determine whether the arginine pathway was a major effector mechanism responsible for amoebicidal activity of activated macrophages . Both the arginine analog NG-monomethyl-L-arginine and arginase, which breaks down arginine, decreased macrophage amoebicidal activity . Addition of arginine to arginine-free medium restores amoebicidal activity to activated macrophage cultures . These results demonstrate that the arginine pathway is an important mechanism for the destruction of susceptible N . fowleri amoebae.

Gene, 1992 Dec 1, 122(1), 181 - 5
Cloning of a gene from Bacillus cereus with homology to the mreB gene from Escherichia coli; Narahara A et al.; We have cloned and sequenced a gene coding for a putative shape-determining protein (MreB) highly homologous to the mreB gene product of Escherichia coli . The amino acid (aa) identity was 53% and the similarity 72% . The gene is expressed early in the logarithmic phase . The aa sequence comparison showed that the protein, like the E . coli MreB, has structural similarity to actin and heat-shock protein Hsc70 encoded by a new super-gene family.

Am J Gastroenterol, 1992 Dec, 87(12), 1839 - 45
Listeria monocytogenes peritonitis: case report and literature review; Sivalingam JJ et al.; Listeria monocytogenes is a Gram-positive bacillus that is pathogenic in both the normal and compromised host . We describe Listeria peritonitis and cerebritis in a patient with cirrhosis due to non-A, non-B hepatitis, and review the 11 other cases of Listeria peritonitis reported in the English-language literature . Listeria is a rare cause of peritonitis in debilitated, older patients, with two-thirds of the cases occurring in patients with chronic liver disease . Listeria peritonitis may also occur in patients undergoing peritoneal dialysis, or in those with malignancy . Peritonitis due to Listeria is clinically similar to spontaneous bacterial peritonitis, and is associated with fever, variable abdominal pain, and neutrocytic ascites; bacteremia commonly accompanies Listeria peritonitis . This syndrome can be successfully treated with antimicrobial drugs, although the third-generation cephalosporins commonly used in the therapy of spontaneous bacterial peritonitis are not recommended . Ampicillin may be the drug of choice, with combination therapy with an aminoglycoside reserved for cases that do not respond to ampicillin alone.

J Urol, 1992 Dec, 148(6), 1894 - 7
Clinical evidence of systemic persistence of bacillus Calmette-Guerin: long-term pulmonary bacillus Calmette-Guerin infection after intravesical therapy for bladder cancer and subsequent cystectomy; Bohle A et al.; A patient with urothelial bladder carcinoma is reported who suffered from culture proved pulmonary bacillus Calmette-Guerin (BCG) infection 14 months after a single course of intravesical BCG and 11 months after subsequent radical cystectomy for progressive cancer . This unusual case raises the question of the ultimate fate of intravesically instilled BCG and the possible persistence of these mycobacteria in remote organs . Systemic spread and dormant survival at least in some cases are suggested, and therapeutic and diagnostic consequences are discussed.

J Urol, 1992 Dec, 148(6), 1892 - 3
Complications of intravesical bacillus Calmette-Guerin: a case report; Gonzalez JA et al.; Bacillus Calmette-Guerin (BCG) is the most effective agent currently available to treat superficial bladder cancer . However, this form of therapy is not without potential serious or fatal complications . In addition to the potentially toxic systemic side effects attributed to hematogenous absorption of the bacillus, direct upper tract seeding may occur in patients with vesicoureteral reflux . We report on a patient treated with intravesical BCG for bladder cancer in whom unilateral necrotizing granulomatous pyelonephritis developed . Although severe, this complication is rare and we conclude that reflux is not a contraindication for intravesical BCG therapy.

J Egypt Soc Parasitol, 1992 Dec, 22(3), 643 - 52
Histopathological and histochemical studies on the sigmoidal and rectal tissues of hepatosplenic children with gastro-intestinal troubles; Mangoud AM et al.; Hepatosplenomegaly accompanied with different intestinal troubles is more or less a marked clinical manifestations in children . The histopathological and histochemical changes were studied in biopsied materials taken from the sigmoidal and rectal tissues of 49 children . These children had schistosomiasis mansoni (26), schistosomiasis and amoebiasis (4), schistosomiasis and tuberculosis (TB.) (2), amoebiasis (4), thalassemia (6), acute myeloid leukaemia (AML) (1), mucopolysaccharidosis (1) and bacillary dysentery (5) . The pathological changes were erosion ulceration hyperplasia, atrophy, crypt-abscess and fibrosis (mucosa) and oedema, congestion, cellular infiltration (Lamina propria) . The chemical changes were the mucin secretion, deposition of collagen and fibrin and activity of the argentaffin cells . Not all the disease agent had the same effect, but changes were marked mainly in children with S . mansoni and/or E . histolytica.

Biotechnology (N Y), 1992 Dec, 10(12), 1579 - 83
Hyperthermostable variants of a highly thermostable alpha-amylase; Joyet P et al.; Genetic screening at temperatures between 70-80 degrees C far exceeds the range of growth of most bacteria, and is not applicable to isolate easily thermostable protein variants . We describe a temperature shift protocol and an in vivo screening method which allowed us to identify a hyperthermostable variant of the thermostable alpha-amylase from Bacillus licheniformis . Our strategy was to select, after hydroxylamine mutagenesis, an intragenic suppressor mutation which overcomes a mutation leading to a thermolabile enzyme . Sequence analysis of the mutated gene revealed only one change in the amino acid sequence, substituting a valine for alanine at position 209 . This single amino acid replacement increased the half-life of the protein at 90 degrees C by a factor of two to three relative to the wild-type enzyme . When this substitution was combined with another stabilizing substitution (H133Y) we described previously, the stabilizing effects were additive . The half-life of the new protein was about 12 hours at 90 degrees C, corresponding to a nine to ten-fold increase over the wild-type enzyme and the industrial Bacillus licheniformis alpha-amylase Termamyl . These mutations are located in a predicted folding domain of the protein which appears crucial in determining thermal stability.

J Am Mosq Control Assoc, 1992 Dec, 8(4), 376 - 80
Efficacy of Bacillus sphaericus against the malaria vector Anopheles gambiae and other mosquitoes in swamps and rice fields in Zaire; Karch S et al.; The microbial control of Anopheles gambiae and other mosquitoes with a granular formulation of Bacillus sphaericus (Vectolex) was evaluated in rice fields and swamps, located around the suburban region of Kingabwa-village in Kinshasa, Zaire . Ten treatment cycles with 15-day intervals were carried out with the same application rate, 10 kg/ha, during the dry season (May to September 1991) . The treatments reduced larval populations of An . gambiae by 98% after 48 h, but repetitive applications were required every 15 days to maintain control . The persistence of B . sphaericus spores was more apparent in rice fields than in swamps . A significant reduction in nuisance biting by Culex quinquefasciatus and Mansonia uniformis was observed . For An . gambiae, a decrease of 13.6% in human biting was noted during the post-treatment period . The entomological inoculation rate was reduced from 0.238 to 0.143 . The efficacy of B . sphaericus does not appear to offer outstanding potential for control of An . gambiae in rice fields and swamps and seems to be limited due to different factors tied to ecology and natural conditions in the fields.

Wei Sheng Wu Xue Bao, 1992 Dec, 32(6), 387 - 93
{Morphology and delta-endotoxin proteins of Bacillus thuringiensis from soils and their toxicities to insects}; Li R et al.; 94 strains of Bacillus thuringiensis were isolated from soils in southwest and northwest of China . The morphology of cells, spores and parasporal crystals of these strains was investigated under transmission and scanning electro-microscope . Proteins of delta-endotoxins from all strains were analysed by rapid SDS-PAGE . 9 species of insects in Lepidoptera, Coleoptera and Diptera were tested for assay of delta-endotoxins . Some kinds of parasporal crystals were quite different in form and in composition of protein from those reported before . Most of strains were nontoxic to all of 9 species used in bio-assay . Some strains were very effective in species of Coleoptera or Noctuidae.

Rev Rhum Mal Osteoartic, 1992 Dec, 59(12), 836 - 8
{BCG osteoarthritis on knee prosthesis after intra-vesical BCG therapy}; Chazerain P et al.; A case of Calmette-Guerin bacillus (BCG) infection of a knee implant during intravesical BCG-therapy is reported . The course was favorable after replacement of the implant and administration of antituberculous agents . This case of septic osteoarthritis due to the BCG is different from cases of reactive polyarthritis reported after intravesical instillations of BCG . It probably resulted from diffusion of the BCG via the bloodstream.

Appl Biochem Biotechnol, 1992 Dec, 37(3), 283 - 94
Purification and characterization of a milk clotting protease from Mucor bacilliformis; Areces LB et al.; An acid protease having milk clotting activity has been isolated from Mucor bacilliformis cultures . The enzyme was basically purified by ionic exchange chromatography . An average yield of 29 mg purified product was obtained from 100 mL crude extract . As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed . The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus . The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases . As expected, its clotting activity was drastically inhibited by pepstatin A action . On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin.

Ecotoxicol Environ Saf, 1992 Dec, 24(3), 338 - 46
The effects of agitation, sediment, and competition on the persistence and efficacy of Bacillus thuringiensis var . israelensis (Bti); Sheeran W et al.; The persistence and efficacy of Bacillus thuringiensis var . israelensis (Bti) were examined in aquatic microcosms under varying conditions of agitation, sediment content, and microbial competition . Agitation was found to be the most important factor in maintaining persistence of Bti cells and the bioavailability of Bti toxin . Sediment acted to decrease efficacy by increasing settling of the toxic particles, but did not decrease the persistence of Bti cells were found, but efficacy of the Bti endotoxin was adversely affected.

Biochim Biophys Acta, 1992 Nov 23, 1112(1), 83 - 8
Glycolipid-anchored acetylcholinesterases from rabbit lymphocytes and erythrocytes differ in their sensitivity to phosphatidylinositol-specific phospholipase C; Richier P et al.; The type of membrane association of acetylcholinesterase (AChE, EC 3.1.1.7) was studied in rabbit lymphocytes and erythrocytes . In both cases, the unique AChE molecular form was an amphiphilic dimer (referred to as G2a) anchored in the membrane by a glycosylphosphatidylinositol . In lymphocytes, G2a AChE was directly converted into its hydrophilic G2h counterpart by a treatment with Bacillus thuringiensis phosphatidylinositol-phospholipase C (PI-PLC, EC 3.1.4.10) . In erythrocytes, AChE was resistant to PI-PLC but was rendered sensitive by a prior deacylation with alkaline hydroxylamine . This observation suggests that, as previously reported for human erythrocyte AChE, an acylation of the inositol ring in the glycolipid anchor of rabbit erythrocyte AChE (that does not occur in lymphocytes) prevents the cleavage.

J Mol Biol, 1992 Nov 20, 228(2), 580 - 95
The crystal structure of the Bacillus lentus alkaline protease, subtilisin BL, at 1.4 A resolution; Goddette DW et al.; The crystal structure of subtilisin BL, an alkaline protease from Bacillus lentus with activity at pH 11, has been determined to 1.4 A resolution . The structure was solved by molecular replacement starting with the 2.1 A structure of subtilisin BPN' followed by molecular dynamics refinement using X-PLOR . A final crystallographic R-factor of 19% overall was obtained . The enzyme possesses stability at high pH, which is a result of the high pI of the protein . Almost all of the acidic side-chains are involved in some type of electrostatic interaction (ion pairs, calcium binding, etc.) . Furthermore, three of seven tyrosine residues have potential partners for forming salt bridges . All of the potential partners are arginine with a pK around 12 . Lysine would not function well in a salt bridge with tyrosine as it deprotonates at around the same pH as tyrosine ionizes . Stability at high pH is acquired in part from the pI of the protein, but also from the formation of salt bridges (which would affect the pI) . The overall structure of the enzyme is very similar to other subtilisins and shows that the subtilisin fold is more highly conserved than would be expected from the differences in amino acid sequence . The amino acid side-chains in the hydrophobic core are not conserved, though the inter-residue interactions are . Finally, one third of the serine side-chains in the protein have multiple conformations . This presents an opportunity to correlate computer simulations with observed occupancies in the crystal structure.

Biochem J, 1992 Nov 15, 288 ( Pt 1), 117 - 21
Site-directed mutagenesis at aspartate and glutamate residues of xylanase from Bacillus pumilus; Ko EP et al.; To elucidate the reaction mechanism of xylanase, the identification of amino acids essential for its catalysis is of importance . Studies have indicated the possibility that the reaction mechanism of xylanase is similar to that of hen's egg lysozyme, which involves acidic amino acid residues . On the basis of this assumption, together with the three-dimensional structure of Bacillus pumilus xylanase and its amino acid sequence similarity to other xylanases of different origins, three acidic amino acids, namely Asp-21, Glu-93 and Glu-182, were selected for site-directed mutagenesis . The Asp residue was altered to either Ser or Glu, and the Glu residues to Ser or Asp . The purified mutant xylanases D21E, D21S, E93D, E93S, E182D and E182S showed single protein bands of about 26 kDa on SDS/PAGE . C.d . spectra of these mutant enzymes show no effect on the secondary structure of xylanase, except that of D21E, which shows a little variation . Furthermore, mutations of Glu-93 and Glu-182 resulted in a drastic decrease in the specific activity of xylanase as compared with mutation of Asp-21 . On the basis of these results we propose that Glu-93 and Glu-182 are the best candidates for the essential catalytic residues of xylanase.

Biochemistry, 1992 Nov 10, 31(44), 10893 - 900
Amphiphilic forms of butyrylcholinesterase in mucosal cells of rat intestine; Sine JP et al.; The properties of a cholinesterase from mucosal cells of rat intestine have been characterized . The enzyme was identified as butyrylcholinesterase because it was more sensitive to iso-OMPA (IC50 = 1.0 x 10(-6) M) than to BW284C51 (IC50 = 5.5 x 10(-5) M) and was not inhibited by substrate excess . It displayed a higher affinity for acetylthiocholine than for butyrylthiocholine . A major molecular form was observed sedimenting at 5.9 S . Two other minor molecular forms were identified as a hydrophilic tetramer (G4, sedimenting at 10.5 S) and a monomer (G1, sedimenting at 4.3 S) . The 5.9 S component was referred to as "G" form (G for globular) and not "G2" as usual dimers for the following reasons: (i) the G form was unaffected by the reducing agents, beta-mercaptoethanol and dithiothreitol, which converted disulfide-linked dimers of acetylcholinesterase into monomers, (ii) the G form was shifted from 5.9 to 3.4 S when the sucrose gradient contained Triton X-100 . This value of 3.4 S (in Triton X-100) appeared too low for a typical G2 form . The shift in the S value was partly reversible: the 3.4 S form resedimented at 5.2 S in the absence of detergent . The behavior of the G form in sucrose gradients indicated that it was amphiphilic . This was confirmed in nondenaturing electrophoreses and also by quantitative binding of the G form to octyl-Sepharose . The hydrophobic domain of the G form was not a glycolipid, as shown by its insensitivity to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and its nonaggregating properties in the absence of nondenaturing detergent.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1992 Nov 5, 228(1), 265 - 76
Key residues in the allosteric transition of Bacillus stearothermophilus pyruvate kinase identified by site-directed mutagenesis; Walker D et al.; The structural gene for pyruvate kinase from Bacillus stearothermophilus has been cloned in Escherichia coli and sequenced . The open reading frame from the ATG start codon to the TAG stop codon is 1482 base-pairs and encodes a peptide of relative molecular mass 52,967 . In the expression vector pKK223-3, containing the synthetic tac promoter, the gene is overexpressed in E . coli cells to an estimated level of 30% total soluble cell protein . A purification procedure for the overexpressed protein has been established . The construction and characterization of a pair of mutant proteins has given insight into the structural basis of allosteric regulation in the tetrameric enzyme . Substituting tryptophan for tyrosine at position 466 (mutant Trp466-->Tyr) resulted in an activated form of the enzyme, having a reduced K1/2 for the substrate phosphoenolpyruvate . We propose that the characteristics of this mutant might be the result of bulk removal releasing steric inhibition to the formation of an interdomain salt bridge between Asp356 and Arg444 . The regulatory behaviour of the double mutant produced by making the additional substitution aspartate for glutamate at position 356 (Trp466-->Tyr/Asp356-->Glu) corroborates this . The position of the salt bridge is such that it might be pivotal to the conformation of a pocket that is proposed to open up when the active R-conformation is adopted . We suggest that the mechanism of activation of B . stearothermophilus pyruvate kinase by ribose-5-phosphate might hinge on an interaction with, or indirectly through, residue Trp466, removing it from the vicinity of the potential salt bridge between Asp356 and Arg444 and thus effecting a closing together of the protein structure concomitant with an opening up of the pocket region.

J Mol Biol, 1992 Nov 5, 228(1), 108 - 17
X-ray structure determination and comparison of two crystal forms of a variant (Asn115Arg) of the alkaline protease from Bacillus alcalophilus refined at 1.85 A resolution; Sobek H et al.; The X-ray structure determination, refinement and comparison of two crystal forms of a variant (Asn115Arg) of the alkaline protease from Bacillus alcalophilus is described . Under identical conditions crystals were obtained in the orthorhombic space group P2(1)2(1)2(1) (form I) and the rhombohedral space group R32 (form II) . For both space groups the structures of the protease were solved by molecular replacement and refined at 1.85 A resolution . The final R-factors are 17.9% and 17.1% for form I and form II, respectively . The root-mean-square deviation between the two forms is 0.48 A and 0.86 A for main-chain and side-chain atoms, respectively . Due to differences in crystal lattice contacts and packing, the structures of the two crystal forms differ in intermolecular interaction affecting the local conformation of three flexible polypeptide sequences (Ser50-Glu55, Ser99-Gly102, Gly258-Ser259) at the surface of the protein . While the two overall structures are very similar, the differences are significantly larger than the errors inherent in the structure determination . As expected, the differences in the temperature factors in form I and II are correlated with the solvent accessibility of the corresponding amino acid residues . In form II, two symmetry-related substrate binding sites face each other, forming a tight intermolecular interaction . Some residues contributing to this intermolecular interaction are also found to be involved in the formation of the complex between subtilisin Carlsberg and the proteinaceous inhibitor eglin C . This demonstrates that the two symmetry-related molecules interact with each other at the same molecular surface area that is used for binding of substrates and inhibitors.

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