Trends Microbiol, 1996 Sep, 4(9), 343 - 7 Cell entry and the pathogenesis of Bartonella infections; Minnick MF et al.; Erythrocyte parasitism, or hemotrophy, is not a common strategy for bacteria . However, Bartonella species are elegantly adapted to parasitize a variety of cell types including red blood cells . Bartonella bacilliformis, a useful model organism for the genus, has been used to study virulence determinants involved in colonization, attachment and invasion of host cells. Cent Eur J Public Health, 1996 Sep, 4(3), 189 - 91 Bacillary tuberculosis in the Czech Republic: developmental trends in the 1981-1993 period; Svandova E et al.; Results of an automated information system on bacillary tuberculosis and mycobacterioses (ISBT) operating in the Czech Republic since 1981 nation-wide have been employed in this study . This system collects and processes data reported by all mycobacteriology laboratories in the country (34 in 1993) on each person disseminating pathogenic and/or opportunistic mycobacteria, on pathological materials examined in these patients and on methods applied for detection and identification of isolated mycobacteria Results of the 1981-1993 period were analyzed in this study . The annual incidence of bacillary tuberculosis cases identified by culture fell down from 2655 (25.8 per 100,000 popul.) in 1981 to 1139 (11.0 per 100,000 popul.) in 1993, i.e . by 57.4% in total, and by 4.8% in average annually . The decrease of annual mean values differed between two periods, from 1981 to 1985 and from 1986 to 1993, being 8.7% in the first and 3.7% in the successive period . The incidence of cases detected by direct microscopy of sputum showed a decline from 615 to 410 cases (5.97 to 3.97 per 100,000 popul.) in the 1981 to 1993 period, i.e . 2.8% annually . The analysis of the development and of the present state of the bacillary tuberculosis is instrumental in estimating the today's burden of the tuberculosis problem in the Czech Republic . Although distinct sings of worsened epidemiological parameters were not shown in this study, some disturbing findings can be considered as alerting: (a) a slowdown of the declining trend of bacillary tuberculosis cases detected by culture techniques seen in a few recent years, and (b) conserving potential tuberculosis pools in patients suffering from serious forms of the disease detectable by direct microscopy. Br J Urol, 1996 Sep, 78(3), 372 - 8 The management of superficial bladder cancer: an interactive seminar; McFarlane JP et al.; OBJECTIVES: To determine the current management of superficial bladder cancer in the United Kingdom . METHODS: An interactive seminar with a series of questions about management and hypothetical clinical scenarios was conducted at the 1995 conference of the British Association of Urological Surgeons . The responses of the audience were recorded electronically and analysed . RESULTS: The results showed that there is a wide variation in practice and some confusion over the place of intravesical treatment using cytotoxic drugs and bacille Calmette-Guerin . CONCLUSIONS: The management of superficial bladder cancer could be improved by a more widespread use of intravesical therapy, along with the introduction of local protocols and national guidelines. Br J Urol, 1996 Sep, 78(3), 369 - 71 Quality of life in patients undergoing bacille Calmette-Guérin therapy for superficial bladder cancer; Mack D et al.; OBJECTIVE: To determine the impact of post-operative education about bladder cancer and topical immunotherapy on the physical, psychological and social well-being of patients with superficial bladder cancer . PATIENTS AND METHODS: Eighty-five patients (mean age 59 years, range 26-85, 64 men and 21 women) receiving topical immunotherapy were questioned during the initial cycle of treatment and during maintenance therapy with bacille Calmette-Guerin (BCG) . Patients completed a questionnaire on their quality of life when they commenced treatment and twice more during maintenance therapy . RESULTS: Psychological distress and physical symptoms were intense when the diagnosis of bladder cancer was revealed to the patients, despite the knowledge that this cancer is usually curable . The overall quality of life, condition of health and sexual activity were mostly only moderate, were poor during initial therapy and better during 3-monthly maintenance therapy . CONCLUSION: In general, the quality of life of these patients is characterized by the disruption of their lifestyle and marked by change in their circumstances . It is the responsibility of the urologist to take the necessary time to educate, comfort and motivate such patients with superficial bladder cancer. Neuroscience, 1996 Sep, 74(2), 599 - 608 The potential role of dendritic cells in immune-mediated inflammatory diseases in the central nervous system; Matyszak MK et al.; Dendritic cells of the rat were studied immunohistochemically with MRC OX62 monoclonal antibody and using electron microscopy . In normal CNS, a small number of OX62+ cells was detected in the choroid plexus and meninges . These cells were absent from other CNS and peripheral nervous system sites studied . Dendritic cells were also studied in two models of immune-mediated inflammatory conditions in the CNS . These were: acute experimental allergic encephalomyelitis and aberrant delayed-type hypersensitivity lesions induced as a response to heat-killed bacillus Calmette-Guerin sequestrated behind the blood-brain barrier . In addition, a group of animals with a delayed-type hypersensitivity response was treated with dexamethasone to assess the effect of steroid treatment on T-cells and OX62+ cells in CNS lesions . Dendritic cells were present in many but not all lesions in acute experimental allergic encephalomyelitis and their numbers were small . In experimental allergic encephalomyelitis lesions, dendritic cells were found predominantly in perivascular cuffs, where they constituted approximately 2% of the total number of major histocompatibility complex class II+ cells . Some of these cells were also detected in the CNS parenchyma, close to the perivascular cuff . In contrast, dendritic cells were present in all delayed-type hypersensitivity lesions studied . Their number in delayed-type hypersensitivity lesions was significantly higher than in experimental allergic encephalomyelitis lesions . Numerous OX62+ cells were found, even in three-month-old lesions . Electron microscopy studies revealed that these cells were often in close contact with lymphocytes . There was no significant change in the density of OX62+ cells, IL2R+ cells and OX19+ T-cells in delayed-type hypersensitivity lesions after seven-day treatment with dexamethasone, although there was a considerable reduction in the number of CD45RA+ T-cells . The high numbers of dendritic cells found in the delayed-type hypersensitivity lesions may be important in contributing to the chronicity of the response . They may also initiate autoimmune responses to CNS antigens uncovered during bystander tissue damage which occurs as a consequence of aberrant delayed-type hypersensitivity responses. Int J Lepr Other Mycobact Dis, 1996 Sep, 64(3), 306 - 10 Silent iritis in treated bacillary negative leprosy; Thompson K et al.; Iridectomy specimens from 59 leprosy patients who had adequate medical records of whom 33 belong to the lepromatous (LL) leprosy variety and 16 normal controls were studied histopathologically . All patients were bacteriologically negative and had received dapsone followed by multidrug therapy (MDT), or MDT only, or only dapsone for varying periods . It was found that leprosy, particularly lepromatous disease, did not significantly decrease the age of formation of cataract . Of the 33 LL patients studied 60.6% had silent iritis . The duration of treatment had no obvious influence on the persistence of iritis . Treatment with only 2 years of MDT for LL patients did not significantly increase the prevalence of persistent silent iritis compared to those who received other types of antileprosy therapy for long periods . It is pointed out that chronic iritis is a serious complication that continues even after the patient is declared clinically and bacteriologically cured, especially in patients who had a history of chronic iritis clinically. Lett Appl Microbiol, 1996 Sep, 23(3), 187 - 91 Thermal resistance of Bacillus stearothermophilus spores in different heating systems containing some approved food additives; Lopez M et al.; The effects of different heating systems on the heat resistance of Bacillus stearothermophilus spores (ATCC 7953, 12980, 15951 and 15952) were investigated . Spores were heated in distilled water, Sorensen buffer (0.18 mol 1-1), McIlvaine buffer (0.0025-0.18 mol 1-1), and several solutions containing sodium chloride (0.06-12%), sodium nitrite (125 ppm), potassium sorbate (0.1%) and sodium benzoate (0.1%) over a wide range of temperatures (115-140 degrees C) . D-values obtained for McIlvaine and Sorensen buffers, at the same molarities, were not significantly different (P > 0.05), but decimal reduction times increased as phosphate concentrations in the solutions decreased . The concentrations, in which statistically significant differences (P < 0.05) were obtained, varied among strains . Among the additives assayed, only sodium chloride reduced heat resistance, being effective at concentrations as low as 0.06% . The z-values calculated in this study ranged from 6.99 to 8.40 with a mean value of 7.60 +/- 0.45 . Although z-values observed for salt and buffers (180 mol 1-1) were slightly higher than obtained in the other conditions assayed, the difference was not statistically significant (P > 0.05). Lett Appl Microbiol, 1996 Sep, 23(3), 146 - 50 Enterotoxin-producing strains of Bacillus thuringiensis isolated from food; Damgaard PH et al.; Strains of Bacillus thuringiensis were isolated from various food items (pasta, pitta bread and milk) and were found to belong to either H-serotype kurstaki or neoleonensis . The strains were bioassayed against Pieris brassicae and insecticidal activity of strains was found to correspond to the presence of the cry1A-gene . All strains, except one, were found to express cytotoxic effects on Vero cells as an indicator of enterotoxin activity . Further, the B . thuringiensis strains HD-1 (serotype kurstaki), NB-125 (serotype tenebrionis) and HD-567 (serotype israelensis ) which are used commercially for insect pest management, were also found to have cytotoxic effects on Vero cells. J Neuroimmunol, 1996 Sep, 69(1-2), 141 - 9 Delayed-type hypersensitivity lesions in the central nervous system are prevented by inhibitors of matrix metalloproteinases; Matyszak MK et al.; We have studied the effect of an inhibitor of matrix metalloproleinases, BB-1101, on a delayed-type hypersensitivity (DTH) response in the CNS . We used a recently described model in which heat-killed bacillus Calmette-Guerin (BCG) sequestered behind the blood-brain barrier (BBB) is targeted by a T-cell mediated response after subcutaneous injection of BCG (Matyszak and Perry, 1995) . The DTH lesions are characterised by breakdown of the BBB, macrophage and lymphocyte infiltration and tissue damage including myelin loss . Treatment with BB-1101, which is not only a potent inhibitor of matrix metalloproteinases but also strongly inhibits TNF-alpha release, dramatically attenuated the CNS lesions . Breakdown of the BBB and the recruitment of T-cells into the site of the lesion were significantly reduced . There were many fewer inflammatory macrophages in DTH lesions than in comparable lesions from untreated animals . There was also significantly less myelin damage (assessed by staining with anti-MBP antibody) . The DTH response in animals treated with dexamethasone was also reduced, but to a lesser degree . No significant effect was seen after administration of pentoxifylline, a phosphodiesterase inhibitor with effects including the inhibition of TNF-alpha production . Our results suggest that inhibitors of matrix metalloproteinases may be of considerable therapeutic benefit in neuroinflammatory diseases. Plant Physiol, 1996 Sep, 112(1), 121 - 9 Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis cryIAc gene; Stewart CN Jr et al.; Somatic embryos of jack, a Glycine max (L.) Merrill cultivar, were transformed using microprojectile bombardment with a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) driven by the 35S promoter and linked to the HPH gene . Approximately 10 g of tissue was bombarded, and three transgenic lines were selected on hygromycin-containing media and converted into plants . The recovered lines contained the HPH gene, but the Bt gene was lost in one line . The plasmid was rearranged in the second line, and the third line had two copies, one of which was rear-ranged . The CryIAc protein accumulated up to 46 ng mg-1 extractable protein . In detached-leaf bioassays, plants with an intact copy of the Bt gene, and to a lesser extent those with the rearranged copy, were protected from damage from corn earworm (Helicoverpa zea), soybean looper (Pseudoplusia includens), tobacco budworm (Heliothis virescens), and velvetbean caterpillar (Anticarsia gemmatalis) . Corn earworm produced less than 3% defoliation on transgenic plants, compared with 20% on the lepidopteran-resistant breeding line GatIR81-296, and more than 40% on susceptible cultivars . Unlike previous reports of soybean transformation using this technique, all plants were fertile . To our knowledge, this is the first report of a soybean transgenic for a highly expressed insecticidal gene. J Med Microbiol, 1996 Sep, 45(3), 192 - 9 Identification of Bartonella henselae and B . quintana 16s rDNA sequences by branch-, genus- and species-specific amplification; Dauga C et al.; Given the controversy surrounding the aetiology of cat scratch disease and the association of both Bartonella henselae and B . quintana with bacillary angiomatosis, a method for the direct detection in clinical samples of 16S rRNA from the Proteobacteria alpha subgroup was developed . The primary structure of amplified 16S rDNA was determined by cloning and sequencing . Three sequences were identified: one corresponded exactly to GenBank accession number M73229 (B . henselae); the second was related to, but distinct from, GenBank accession number Z11684 (referred to as 'B . henselae variant'); and a third sequence was identical with GenBank accession number M73228 (B . quintana) . No sequence corresponding to Afipia spp . was found . To speed identification and reduce the cost of analysis, a nested amplification method for B . henselae and B . quintana was devised . These techniques were applied to DNA extracted from 30 unfixed lymph node biopsies, two liver biopsies and 36 node pus samples from patients with suspected cat scratch disease, and from 17 skin biopsies from AIDS patients with suspected bacillary angiomatosis . B . henselae or B . henselae variant sequences were found in 42 (62%) of 68 samples from suspected cat scratch disease . B . quintana was not associated with cat scratch disease, but a B . quintana sequence was found in seven (41%) of 17 samples from suspected bacillary angiomatosis patients . B . henselae 16S rDNA sequences were not found in bacillary angiomatosis specimens. Br J Rheumatol, 1996 Sep, 35(9), 901 - 4 Bone bacillary angiomatosis in an HIV-infected patient; Olive A et al.; Bacillary angiomatosis (BA) is a recently discovered multisystem bacterial infectious disease seen in the setting of immune suppression due to the human immunodeficiency virus (HIV) . A case of an HIV-infected patient with osteolytic bone involvement is reported. J Comput Assist Tomogr, 1996 Sep-Oct, 20(5), 766 - 9 Tuberculous abscess in retromammary region: CT findings; Chung SY et al.; PURPOSE: Our goal was to evaluate CT findings of tuberculous abscess in the retromammary region of the breast . METHOD: Four patients with tuberculosis extending from the retromammary region to the pleura were examined by CT and the findings were evaluated . All cases were also examined with mammography and two cases were evaluated with sonography . Diagnosis was confirmed by acid-fast bacillus stain, culture, and histologic examination . RESULTS: Mammography showed relatively smoothly marginated, round mass density in two cases, nodular density in one, and focal bulging of the pectoral wall in one . A sonogram demonstrated in two cases a fistulous connection from the heterogeneous, fluid-containing lesion with floating internal debris in the retromammary region to the thoracic cavity . In all four cases, CT showed relatively smoothly marginated, inhomogeneous, hypodense lesions with surrounding rims of the cold abscess type . A direct fistulous connection from the retromammary lesion through the thoracic wall into the pleura was seen in two cases . Destroyed rib fragments within the abscess were noted in two cases . CONCLUSION: A tuberculous abscess in the retromammary region usually showed on CT a focal, smoothly marginated, inhomogeneous, hypodense lesion with a surrounding enhancing rim . A direct fistulous connection with the pleura or a destroyed rib fragment in the abscess as revealed by CT can be helpful in the differential diagnosis of other infectious types of retromammary abscess. Appl Environ Microbiol, 1996 Sep, 62(9), 3140 - 5 Restriction map of the 125-kilobase plasmid of Bacillus thuringiensis subsp . israelensis carrying the genes that encode delta-endotoxins active against mosquito larvae; Ben-Dov E et al.; A large plasmid containing all delta-endotoxin genes was isolated from Bacillus thuringiensis subsp . israelensis; restricted by BamHI, EcoRI, HindIII, KpnI, PstI, SacI, and SalI; and cloned as appropriate libraries in Escherichia coli . The libraries were screened for inserts containing recognition sites for BamHI, SacI, and SalI . Each was labeled with 32P and hybridized to Southern blots of gels with fragments generated by cleaving the plasmid with several restriction endonucleases, to align at least two fragments of the relevant enzymes . All nine BamHI fragments and all eight SacI fragments were mapped in two overlapping linkage groups (with total sizes of about 76 and 56 kb, respectively) . The homology observed between some fragments is apparently a consequence of the presence of transposons and repeated insertion sequences . Four delta-endotoxin genes (cryIVB-D and cytA) and two genes for regulatory polypeptides (of 19 and 20 kDa) were localized on a 21-kb stretch of the plasmid; without cytA, they are placed on a single BamHI fragment . This convergence enables subcloning of delta-endotoxin genes (excluding cryIVA, localized on the other linkage group) as an intact natural fragment. J Bacteriol, 1996 Sep, 178(17), 5330 - 2 A germination-specific spore cortex-lytic enzyme from Bacillus cereus spores: cloning and sequencing of the gene and molecular characterization of the enzyme; Moriyama R et al.; A gene (sleB) encoding a 24-kDa germination-specific spore cortex-lytic enzyme, probably an N-acetylmuramyl-L-alanine amidase, was cloned from Bacillus cereus, and its nucleotide sequence was determined . It was indicated that the enzyme is produced as a 259-residue protein with a signal sequence of 32 residues and is present in dormant spores in its active form . Sulfhydryl reagents inactivated the enzyme, but mutation of a single cysteine of the protein, Cys-258, to Gly did not cause complete inactivation of the enzyme, suggesting that the residue does not function as the catalytic center of enzyme. Mol Biol Evol, 1996 Sep, 13(7), 1032 - 8 Higher ribosomal RNA substitution rates in Bacillariophyceae and Dasycladales than in Mollusca, Echinodermata, and Actinistia-Tetrapoda; Sorhannus U; Molecular evolutionary rates within two protistan and three metazoan taxa were estimated using divergence times derived from fossil records . The results indicate that the small-subunit rRNA sequences within Dasycladales (Chlorophyta) and Bacillariophyceae evolved at a rate approximately two to three times faster than that estimated within Echinodermata, Mollusca, and Actinistia-Tetrapoda . It was concluded that this twofold discrepancy demonstrates actual taxonomic differences in the fixation rate of mutations in the small-subunit rRNA. Infect Immun, 1996 Sep, 64(9), 3934 - 6 Urease activity does not contribute dramatically to persistence of Mycobacterium bovis bacillus Calmette-Guérin; Reyrat JM et al.; Multiplication of BCGure-, an isogenic urease-negative mutant of Mycobacterium bovis BCG constructed by allelic exchange (J . M . Reyrat, F . X . Berthet, and B . Gicquel, Proc . Natl . Acad . Sci . USA 92:8768-8772, 1995), was examined in human macrophages and mice . Although ureolytic activity was not essential to BCGure-growth, a slight decrease in the multiplication and persistence of the mutated strain compared with wild-type BCG was observed in lungs of infected mice. J Urol, 1996 Sep, 156(3), 967 - 71 Clinicopathological evaluation of repeated courses of intravesical bacillus Calmette-Guerin instillation for preventing recurrence of initially resistant superficial bladder cancer; Okamura T et al.; PURPOSE: The efficacy of repeated courses of intravesical bacillus Calmette-Guerin (BCG) for superficial bladder cancer was assessed with particular attention to initially resistant cases . MATERIALS AND METHODS: A total of 75 patients with stages Ta to T1b superficial transitional cell bladder carcinoma received 6 weekly instillations of 80 mg . Tokyo strain BCG in 40 ml . saline followed by 6 instillations at monthly intervals . If tumors recurred, another course of treatment was given with surgery . RESULTS: Of 17 patients (22.7%) with recurrent tumor at followup periods of up to 84 months 12 received an additional course of BCG instillations according to the same protocol after transurethral resection of bladder tumors, and 10 (83.3%) showed no further recurrence with or without additional surgery and BCG therapy after a median followup of 42.9 months . Thus, the overall success rate with this approach was 90.7% (68 of 75 patients) . Comparison of patients with and without recurrence revealed a significant difference in number of tumors before therapy (p < 0.05), and a pronounced tendency (p = 0.00507) for recurrence after prior systemic chemotherapy or intravesical instillation . CONCLUSIONS: The results suggest that up to 3 courses of repeated intravesical instillation of BCG are effective even for cases that initially did not respond, and that best results may be achieved if no other prior chemotherapy has been attempted. J Urol, 1996 Sep, 156(3), 962 - 6 A randomized multicenter trial of adjuvant therapy in superficial bladder cancer: transurethral resection only versus transurethral resection plus mitomycin C versus transurethral resection plus bacillus Calmette-Guerin . Participating Clinics; Krege S et al.; PURPOSE: A randomized multicenter trial was done to compare transurethral resection only to transurethral resection plus adjuvant mitomycin C and bacillus Calmette Guerin (BCG) instillation for treatment of superficial bladder cancer (stage pTa/1 grades 1 to 3 except primary stage pTa grade 1) . MATERIALS AND METHODS: Included in the study were 337 patients with superficial stage pTa/1 grades 1 to 3 bladder cancer except primary stage pTa grade 1 tumors . One group underwent transurethral resection alone . Mitomycin C (20 mg./50 ml . sodium chloride) was given every 2 weeks during year 1 and once a month during year 2 . BCG (120 mg/50 ml . sodium chloride was instilled once a week for 6 weeks and once a month for 4 months . RESULTS: At a median followup of 20.2 months, a decrease in recurrence rate was noted for both drug instillations compared to transurethral resection only . The relative risk of recurrence was 0.508 after mitomycin C and 0.618 after BCG instillation compared to transurethral resection alone . There was no significant difference between the mitomycin C and BCG instillations . The progression rate was comparable in all 3 therapy groups, with an estimated common progression rate of 4.22% per year . Side effects occurred most frequently during or after BCG instillation, most often consisting of cystitis . One patient required cystectomy because of ulcerating cystitis and a prostatic abscess subsequent to unsuccessful tuberculostatic therapy . There were no systemic complications . CONCLUSIONS: Our study showed a positive effect of adjuvant chemotherapy and immunotherapy on decreasing tumor recurrence rate . No influence was observed concerning progression rate, which was low overall. J Urol, 1996 Sep, 156(3), 1189 - 93 Mycobacterium cell wall: an alternative to intravesical bacillus Calmette Guerin (BCG) therapy in orthotopic murine bladder cancer; Chin JL et al.; PURPOSE: The antitumor effect of intravesical mycobacterium cell wall (MCW) therapy on orthotopic and heterotopic bladder tumors in the mouse was assessed with magnetic resonance imaging (MRI) . MATERIALS AND METHODS: The live bacillus Calmette Guerin (BCG) organism was replaced with a cell wall extract derived from the outer capsule of Mycobacterium phlei . This alternative form of intravesical therapy was used with the aim of reducing the toxicity associated with the live mycobacterium organism without compromising efficacy . Response to multiple doses of intravesical MCW and BCG was assessed in mice with established MBT-2 tumors after transurethral tumor implantation . RESULTS: Serial MRI of BCG-treated mice revealed significant tumor regression . The MR images correlated well with the corresponding histology of the whole mount bladder sections . Treatment with MCW also resulted in significant inhibition of tumor growth compared with control untreated animals (p < 0.05) although the antitumor effect was less pronounced than that of live BCG . Treatment was well tolerated in the MCW group with no apparent ill effects . Flow cytometric (FCM) analysis of bladder washings with phenotype-specific monoclonal antibodies revealed predominantly a CD3+ T cell infiltrate in the control and BCG-treated as well as the MCW-treated mice . The CD4+ (helper/inducer) subset of T cells predominated over the CD8+ (suppressor/cytotoxic) subset in both the BCG- and MCW-treated animals, and the CD4+/CD8+ ratio in both of the treated groups differed significantly from that of the control untreated groups . CONCLUSION: Intravesical MCW appears to invoke a similar inflammatory response in the mouse bladder mucosa as the live BCG organism and retains an antitumor action . It deserves further evaluation as a potential antitumor agent against bladder cancer . A Phase II clinical trial is now underway. J Mol Biol, 1996 Aug 30, 261(4), 550 - 67 Interaction of the Bacillus stearothermophilus ribosomal protein S15 with 16 S rRNA: II . Specificity determinants of RNA-protein recognition; Batey RT et al.; S15 is a primary ribosomal protein that interacts specifically with a three-way junction in the central domain of 16 S rRNA, whose binding induces a conformational change in the RNA . In the accompanying paper, we demonstrated that S15 binds with high affinity to a 61 nucleotide RNA corresponding to the minimal rRNA binding site . Here, the sequence and structural determinants for the RNA in the Bacillus stearothermophilus S15-rRNA interaction have been probed using site-directed mutagenesis, chemical modification interference, and iodine footprinting of phosphorothioate RNA . Mutations and RNA modifications that interfere with protein binding cluster in two distinct regions, one containing an internal loop and the other containing a three-way junction . The internal loop, defined by two A.G base-pairs and a bulged guanosine, is not important for the specific interaction, however, BS15 interacts with a phylogenetically conserved G.U base-pair above this internal loop . Near the three-way junction in helix 22, a bulged adenosine and two base-pairs adjacent to the junction also provide important determinants for BS15 binding . Chemical modification interference also suggests that four highly phylogenetically conserved nucleotides in the three-way junction may form non-canonical G.G and U.A base-pairs that are required for the BS15-rRNA interaction . Ethylation modification interference suggests that BS15 binding is accompanied by a conformational change in the RNA involving orientation of helices 20 and 22 at an acute angle with respect to one another . Projection of the data provided by mutagenesis, chemical modification interference analysis, and iodine footprinting onto a three-dimensional model illustrates that BS15 is likely to interact with the minor groove along an extended face of helix 22. J Mol Biol, 1996 Aug 30, 261(4), 536 - 49 Interaction of the Bacillus stearothermophilus ribosomal protein S15 with 16 S rRNA: I . Defining the minimal RNA site; Batey RT et al.; The ribosomal RNA binding site of Bacillus stearothermophilus ribosomal protein S15 (BS15) was analyzed using synthetic RNA oligonucleotides derived from the 16 S rRNA central domain . Native gel electrophoresis mobility shift assays demonstrate that BS15 can specifically interact with an RNA oligonucleotide containing nucleotides 585 to 756 (helices 20 to 23) of 16 S rRNA with an apparent dissociation constant of 35 nM . A series of deletion mutants of the rRNA fragment that contains the BS15 specific binding site was tested for their capacity to bind protein using a competition binding assay . The major determinant of the BS15-rRNA interaction is a three-way junction between helices 20, 21, and 22, while helix 23 (nucleotides 673 to 733 of 16 S rRNA) was dispensable for high affinity binding . Helix 22 contains BS15 binding determinants in an internal loop containing two phylogenetically conserved purine-purine base-pairs . In contrast, only small segments of helices 20 and 21 are required to maintain the integrity of the junction . Kinetic measurements of the dissociation and association rate of the bimolecular complex between BS15 and various minimal rRNA binding sites demonstrate that the basic properties of this interaction were not altered as a result of the deletions . The minimal binding site is a 61 nucleotide RNA that is a good model for the wild-type BS15-16 S rRNA interaction. Biochim Biophys Acta, 1996 Aug 29, 1291(1), 5 - 15 Determination of enzymatic hydrolysis specificity of partially N-acetylated chitosans; Varum KM et al.; A new method for determining the specificity of hydrolysis of the linear binary heteropolysaccharide chitosan composed of (1-->4)-linked 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc; A-unit) and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN; D-unit) residues is described . The method is based on the assignments of the 13C chemical shifts of the identity (A- or D-units) of the new reducing and non-reducing ends and the variation in their nearest neighbours, using low molecular weight chitosans with known random distribution of A- and D-units as substrate . A highly N-acetylated chitosan with fraction of acetylated units (FA) of 0.68 and a number-average degree of polymerization (DPn) of 30 was hydrolysed with hen egg-white lysozyme, showing that both the new reducing and non-reducing ends consisted exclusively of A-units, indicating a high specificity for A-units in subsites DL and EL on lysozyme . Our data suggests that the preceding unit of the reducing A-units, is invariable, and based on earlier studies, most probably an A-unit, while the unit following the non-reducing A-units can be either an A- or a D-unit . A more detailed study of the specificity of lysozyme at subsite DL was performed by hydrolyzing a more deacetylated chitosan (FA = 0.35 and DPn of 20) to a DPn of 9, showing that even for this chitosan more than 90% of the new reducing ends were acetylated units . Thus, lysozyme depolymerizes partially N-acetylated chitosans by preferentially hydrolyzing sequences of acetylated units bound to site CL, DL and EL of the active cleft, while there is no specificity between acetylated and deacetylated units to site FL . In addition, a moderately N-acetylated chitosan with fraction of acetylated units (FA) of 0.35 and a DPn of 20 was hydrolysed with Bacillus sp . No . 7-M chitosanase, showing that both the new reducing and non-reducing ends consisted exclusively of D-units . Our data suggests that the nearest neigbour to the D-unit at the reducing end is invariable, and based on earlier studies, most probably a D-unit, while the unit following the non-reducing D-units can be either an A- or a D-unit . We conclude that the Bacillus chitosanase hydrolyzes partially N-acetylated chitosan by preferentially attacking sequences of three consecutive deacetylated units, hypothetical subsites CC, DC and EC, where the cleavage occur between sugar units bound to subsites DC and EC . A hypothetical subsite FC on the chitosanase show no specificity with respect to A- and D-units . The new NMR method described herein offers a time and labour-saving alternative to the procedure of extensive hydrolysis of the binary heteropolysaccharide chitosan and subsequent isolation and characterization of the oligosaccharides. FEBS Lett, 1996 Aug 26, 392(2), 105 - 9 Structural characterization of extracellular ribonuclease of Bacillus polymyxa: amino acid sequence determination and spatial structure prediction; Lebedev AA et al.; The primary structure of extracellular Bacillus polymyxa ribonuclease (RNase Bpo) was established by mass spectroscopy analysis and automatic Edman degradation of the individual peptides obtained from protein digestion with Glu-specific protease V8 . RNase Bpo consists of 111 amino acid residues, with a relative molecular weight of 12 607 . RNase Bpo is a close structural homolog of RNases of B . amyloliquefaciens (RNase Ba) and B . intermedius (RNase Bi), the similarity of their primary structures being 68% . Molecular modelling of the structure of the complex of RNase Bpo with substrate analog d(CGAC) was performed and a spatial model based on the known crystal structure of RNase Ba complex with the corresponding nucleotide was constructed using the methods of interactive computer graphics and energy minimization . The differences in the primary and tertiary structures of the enzymes were analyzed in order to understand the substrate specificity of Bacillus RNases. J Mol Biol, 1996 Aug 23, 261(3), 432 - 42 Solution structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii; Berg A et al.; The three-dimensional solution structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined from nuclear magnetic resonance data by using distance geometry and dynamical simulated annealing refinement . The structure determination is based on a total of 580 experimentally derived distance constraints and 65 dihedral angle constraints . The solution structure is represented by an ensemble of 25 structures with an average root-mean-square deviation between the individual structures of the ensemble and the mean coordinates of 0.71 A for backbone atoms and 1.08 A for all heavy atoms . The overall fold of the lipoyl domain is that of a beta-barrel-sandwich hybrid . It consists of two almost parallel four-stranded anti-parallel beta-sheets formed around a well-defined hydrophobic core, with a central position of the single tryptophan 21 . The lipoylation site, lysine 42, is found in a beta-turn at the far end of one of the sheets, and is close in space to a solvent-exposed loop comprising residues 7 to 15 . The lipoyl domain displays a remarkable internal symmetry that projects one beta-sheet onto the other beta-sheet after rotation of approximately 180 degrees about a 2-fold rotational symmetry axis . There is close structural similarity between the structure of this 2-oxoglutarate dehydrogenase complex lipoyl domain and the structures of the lipoyl domains of pyruvate dehydrogenase complexes from Bacillus stearothermophilus and Escherichia coli, and conformational differences occur primarily in a solvent-exposed loop close in space to the lipoylation site . The lipoyl domain structure is discussed in relation to the process of molecular recognition of lipoyl domains by their parent 2-oxo acid dehydrogenase. J Acquir Immune Defic Syndr Hum Retrovirol, 1996 Aug 15, 12(5), 433 - 41 Mycobacterium avium and purified protein derivative-specific cytotoxicity mediated by CD4+ lymphocytes from healthy HIV-seropositive and-seronegative individuals; Ravn P et al.; HIV is the greatest single risk factor for the development of tuberculosis . Diseases caused by M . tuberculosis and mycobacteria are the most common opportunistic infections in HIV-infected persons, which may stem from a functional defect of the CD4+ T-cell-mediated killing of macrophages harboring mycobacteria . Our objective was to investigate the M.tuberculosis-and M . avium-specific cytotoxic capacity of T cells from healthy, bacille Calmette-Guerin-vaccinated, HIV-seropositive individuals . Blood mononuclear cells were obtained from 10 healthy HIV-seropositive and 10 healthy seronegative persons with no history of previous or active mycobacterial infection . Antigen-specific killing of macrophages presenting mycobacterial antigens (purified protein derivative or M . avium culture filtrate) was conducted . The phenotype of the killer cells was determined by a fluorescence-activated cell sorter after antigen stimulation and by using purified CD4+ and CD8+ cell subsets . Substantial, but reduced antigen-specific cytotoxicity was observed in patients with asymptomatic HIV infection . The immunological dysfunction leading to reduced cytotoxic activity in healthy HIV-seropositive subjects could not be explained by a defect in the cytotoxic capacity of the individual CD4+ lymphocyte after antigen stimulation, and it could not be explained by a reduction in the total number of CD4+ cells before antigen stimulation . The antigen-specific cytotoxic activity was, however, closely related to the ability of the CD4+ T cells to respond to mycobacterial antigens . The immunological dysfunction leading to reduced mycobacterial-specific cytotoxic activity in healthy HIV-seropositive subjects is caused either by a reduction in the number of antigen-responsive CD4+ T cells (memory) or by an impairment of their ability to respond to antigenic stimuli. Ann Intern Med, 1996 Aug 15, 125(4), 280 - 3 Self-assessment of tuberculin skin test reactions by New York City firefighters: reliability and cost-effectiveness in an occupational health care setting; Prezant DJ et al.; OBJECTIVE: To determine whether self-assessment of purified protein derivative of tuberculin (PPD) skin test reactions, done using a simple two-choice approach, is an effective screening method for tuberculosis . DESIGN: Double-blind comparison between self-assessments and trained professional readings of PPD skin test reactions, done 72 hours after test administration . SETTING: The New York City Fire Department's Bureau of Health Services . PARTICIPANTS: 2011 New York City firefighters and fire officers were given PPD skin tests during a mandatory retraining course . Thirty-seven persons were excluded because of a history of a positive PPD skin test result or a bacille Calmette-Guerin vaccination . All others agreed to participate in testing and self-assessment done using simple written instructions . Self-assessment results were submitted just before trained professional readings were done . MEASUREMENTS: Self-assessments and trained professional readings of PPD skin test reactions . RESULTS: 1833 participants (91%) interpreted their test reactions as flat . Of these interpretations, 1824 (99.5%) matched the professional reading and 9 (0.5%) did not . One hundred seventy-eight participants (9%) interpreted their test reactions as not flat; 136 of these interpretations (76.4%) matched the professional reading and 42 (23.6%) did not (kappa = 0.828; lower 95% confidence limit = 0.790) . The predictive value of a negative self-assessment reading was 99.5%, and the specificity was 97.7% . CONCLUSION: In this occupational health care setting, we follow (and recommend to others with similar populations) a tuberculin screening program based on self-assessment . Repeated tests with follow-up are required for all persons who do not report their results . All persons with self-assessments of "not flat" should return for readings by trained professionals, counseling, and treatment. Biochemistry, 1996 Aug 6, 35(31), 10110 - 8 Effects of both shortening and lengthening the active site nucleophile of Bacillus circulans xylanase on catalytic activity; Lawson SL et al.; The relative positioning of the two carboxyl groups at the active site of glycosidases is crucial to their function and the mechanism followed . The distance between these two groups in Bacillus circulans xylanase has been modified by mutagenesis of the catalytic nucleophile Glu78 . An increase in the separation (Glu78Asp) results in a large (1600-5000-fold) reduction in the rate of the glycosylation step, but little change in the extent of bond cleavage or proton donation at the transition state . A decrease in the separation was achieved by selective carboxymethylation of the Glu78Cys mutant . This modified mutant was only 16-100-fold less active than wild-type enzyme, and its transition state structure was similarly unchanged . Complete removal of the carboxyl group (Glu78Cys) resulted in a mutant with no measurable catalytic activity . Furthermore, it did not even undergo the first step, glycosylation of the active site thiol . These results confirm the importance of precise positioning of the nucleophile at the active site of these enzymes. Biochemistry, 1996 Aug 6, 35(31), 9958 - 66 The pKa of the general acid/base carboxyl group of a glycosidase cycles during catalysis: a 13C-NMR study of bacillus circulans xylanase; McIntosh LP et al.; The 20 kDa xylanase from Bacillus circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl-enzyme intermediate . In this double-displacement reaction, Glu78 functions as a nucleophile to form the intermediate, while Glu172 acts as a general acid catalyst during glycosylation, protonating the departing aglycone, and then as a general base during deglycosylation, deprotonating the attacking water . The dual role of Glu172 places specific demands upon its ionization states and hence pKa values . 13C-NMR titrations of xylanase, labeled with {delta-13C}glutamic acid, have revealed pKa values of 4.6 and 6.7 for Glu78 and Glu172, respectively . These agree well with the apparent pKa values obtained from a study of the pH dependence of kcat/Km and demonstrate that, at the enzyme's pH optimum of 5.7, the nucleophile Glu78 is deprotonated and the general acid Glu172 initially protonated . Remarkably, the pKa for Glu172 drops to 4.2 in a trapped covalent glycosyl-enzyme intermediate, formed by reaction with 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-beta-xylobioside {Miao et al . (1994) Biochemistry 33, 7027-7032} . A similar pKa is measured for Glu172 when a glutamine is present at position 78 . This large decrease in pKa of approximately 2.5 units is consistent with the role of Glu172 as a general base catalyst in the deglycosylation step and appears to be a consequence of both reduced electrostatic repulsion due to neutralization of Glu78 and a conformational change in the protein . Such "pKa cycling" during catalysis is likely to be a common phenomenon in glycosidases. Indian J Exp Biol, 1996 Aug, 34(8), 794 - 8 Crop improvement and disease suppression by a Bacillus spp . SR2 from pea nut rhizosphere; Kumar BS; An antibiotic producing Bacillus strain SR2, isolated from pea nut rhizosphere, showed in vitro antibiosis against many known plant pathogens . Seed bacterization with this strain showed an enhancement in seed germination, shoot height, root length, fresh and dry weights in four crop plants . A multiple drug resistant strain, SR2+, used to monitor root colonization confirmed the root colonization by the organism . Seed bacterization, reduced the number of chick pea wilted plants in wilt-sick soil, making the organism a potential bio-control agent against chick pea wilt. Br J Ophthalmol, 1996 Aug, 80(8), 755 - 8 Experimental Bacillus cereus post-traumatic endophthalmitis and treatment with ciprofloxacin; Alfaro DV et al.; BACKGROUND: Bacillus species remain an important cause of post-traumatic endophthalmitis, often causing permanent visual loss . METHODS: Twenty two rabbits were used to evaluate the clinical and histological findings of Bacillus cereus experimental post-traumatic endophthalmitis . Eyes that had received a scleral laceration and surgical repair were inoculated with Bacillus cereus . Thirty four other rabbits were used to evaluate the efficacy of intravitreal ciprofloxacin in treating experimental disease . RESULTS: Animals developed a post-traumatic endophthalmitis that closely mimicked human disease, characterised by a rapidly progressive and destructive endophthalmitis . Histological evaluation revealed retinal detachment, retinal necrosis, and the infiltration of inflammatory cells into the subretinal space . Intravitreal ciprofloxacin (100 micrograms) prevented the development of disease when given 1 hour and 6 hours after trauma and inoculation . CONCLUSIONS: Clinical and histological examination of experimental Bacillus cereus post-traumatic endophthalmitis suggests that retinal detachment and retinal necrosis play important roles in visual loss . Ciprofloxacin may be of benefit in the management of certain intraocular infections following penetrating injury. J Biochem (Tokyo), 1996 Aug, 120(2), 425 - 32 Aspartate aminotransferase from an alkalophilic Bacillus contains an additional 20-amino acid extension at its functionally important N-terminus; Battchikova N et al.; Aspartate aminotransferase (AspAT), responsible for a minor part of the total AspAT enzymic activity in alkalophilic Bacillus circulans, was purified, its N-terminal amino acid sequence was determined, and its gene was cloned as two separate fragments . DNA sequencing showed an open reading frame of 432 amino acids (M(r) 47,439) exhibiting moderately low homology with AspATs from other sources . Sequence alignment of the enzyme with chicken mitochondrial, chicken cytoplasmic and Escherichia coli AspATs was performed with the MULTALIN program and further optimized assuming that the three-dimensional structures of the proteins were conserved . The primary structure of the studied AspAT diverged markedly from the others in the catalytically important small domain and in a segment of 31 amino acids in the large domain . The functional N-terminal arm was about two times longer than those of AspATs from other sources . According to the molecular model, the unique regions of B . circulans AspAT are all located together, forming a continuous network of contacts . Additional contacts formed by the elongated N-terminal arm may result in some limitation of domain movements in the alkalophilic enzyme in comparison to in other known AspATs. Immunology, 1996 Aug, 88(4), 479 - 81 The Nramp1 antimicrobial resistance gene segregates independently of resistance to virulent Mycobacterium tuberculosis; Medina E et al.; Nramp1 is a recently cloned gene that is involved in resistance of mice to infection with certain microbial pathogens, including the attenuated bacillus Calmette-Guerin (BCG) strain of Mycobacterium bovis, as well as certain other mycobacteria . With a view to determining whether Nramp1 influences resistance of mice to infection with virulent M . tuberculosis, BALB/c mice homozygous for the susceptibility allele of Nramp1, and DBA/2 mice homozygous for the resistance allele, as well as their F1 and F2 progeny, were typed according to their possession of these alleles using a 'hot start' polymerase chain reaction (PCR) procedure . As assessed by the ability of the mice to survive infection, the results show that Nramp1 plays no discernible role in resistance to tuberculosis. Int J Food Microbiol, 1996 Aug, 31(1-3), 341 - 7 Effect of pH of the recovery medium on the apparent heat resistance of three strains of Bacillus cereus; Gonzalez I et al.; The influence of pH of the recovery medium, in the range 7.6-5.4, on the apparent heat resistance of three strains of Bacillus cereus (ATCC 4342, 7004 and 9818) has been investigated . The highest counts of heat-injured spores were obtained at pH near neutral, decreasing markedly as pH was reduced, especially with longer heating times . When the media were acidified, the apparent D-values tended to decrease, although some exceptions related to the strain and the nature of the medium were observed . z-Values determined were not affected by the pH of the medium. Int J Food Microbiol, 1996 Aug, 31(1-3), 311 - 6 Randomly amplified polymorphic DNA (RAPD) assay for genomic fingerprinting of Bacillus cereus isolates; Stephan R; Randomly amplified polymorphic DNA (RAPD) assay was used for epidemiological subtyping of B . cereus and B . lentus . Within 25 isolates of B . cereus up to 22 strain types could be determined when five primers were used . RAPD patterns, which were found in three B . lentus strains, clearly differed form those of B . cereus . The RAPD technique proved to be an effective tool for the characterization of B . cereus strains. Int J Food Microbiol, 1996 Aug, 31(1-3), 149 - 59 Evaluation of serotyping, biotyping, plasmid banding pattern analysis, and HEp-2 vacuolation factor assay in the epidemiological investigation of Bacillus cereus emetic-syndrome food poisoning; Nishikawa Y et al.; To assess the value of the plasmid banding patterns, the vacuolation factor (VF) assay, biotyping, and serological typing as epidemiological markers for strains of Bacillus cereus causing emetic-syndrome illness, 43 isolates from five outbreaks and an additional 76 strains isolated in food-poisoning outbreaks caused by other enteric pathogens were examined by these techniques, and the results were compared . Thirty-eight (88%) of the 43 outbreak strains produced vacuolation responses in HEp-2 cells and were all starch-hydrolysis negative . The other 76 strains associated with outbreaks caused by other food-poisoning bacteria gave all negative VF production results except four strains, and 56 (74%) of these strains produced positive reactions in starch hydrolysis tests . Starch hydrolysis emerged as a convenient screen for VF production, because no starch hydrolysis-positive strains produced VF . With the exception of one isolate, all 38 VF-positive isolates from emtic-syndrome outbreaks were serotype H.1 . Isolates from four of the five outbreaks revealed identical plasmid banding patterns in each outbreak, whereas only three of eight serotype H.1 strains from the fifth outbreak exhibited indistinguishable plasmid banding patterns . These results suggest that the plasmid banding pattern analysis may be of value in discriminating between isolates of the same serotype, and establishing if an outbreak arises from a common food source . In conclusion, the vacuolation factor assay combined with the plasmid banding patterns proved to be a valuable tool for the epidemiological investigation of emetic-syndrome outbreaks caused by B . cereus . Moreover, these methods are particularly useful for laboratories that do not have ready access to serotyping facilities. Bioorg Med Chem, 1996 Aug, 4(8), 1197 - 201 Properties of diacetyl (acetoin) reductase from Bacillus stearothermophilus; Giovannini PP et al.; The cells of Bacillus stearothermophilus contain an NADH-dependent diacetyl (acetoin) reductase . The enzyme was easily purified to homogeneity, partially characterised, and found to be composed of two subunits with the same molecular weight . In the presence of NADH, it catalyses the stereospecific reduction of diacetyl first to (3S)-acetoin and then to (2S,3S)-butanediol; in the presence of NAD+, it catalyses the oxidation of (2S,3S)- and meso-butanediol, respectively, to (3S)-acetoin and to (3R)-acetoin, but is unable to oxidise these compounds to diacetyl . The enzyme is also able to catalyse redox reactions involving some endo-bicyclic octen- and heptenols and the related ketones, and its use is suggested also for the recycling of NAD+ and NADH in enzymatic redox reactions useful in organic syntheses. Mol Microbiol, 1996 Aug, 21(4), 739 - 49 Catabolite repression mediated by the catabolite control protein CcpA in Staphylococcus xylosus; Egeter O et al.; The gene ccpA encoding the catabolite control protein CcpA of Staphylococcus xylosus has been cloned and characterized . The CcpA protein belongs to the Lacl/GaiR family of bacterial regulators and is comprised of 329 amino acids, with a molecular mass of 36.3 kDa . It shows 56% identity with the CcpA proteins of Bacillus subtills and Bacillus megaterium . Inactivation of the ccpA gene in the genome of S . xylosus relieved the activities of three enzymes, alpha-glucosidase, beta-glucuronidase, and beta-galactosidase, from cataboilte repression by several carbohydrates . Concomitantly, transcription initiation of the maltose-utilization operon malRA, including the alpha-glucosidase gene malA, was no longer subject to glucose-specific control . Carbon source-dependent malRA regulation was also lost upon deletion of a palindromic sequence in the malRA promoter region resembling the catabolite-responsive elements essential for CcpA-dependent catabolite repression in Bacillus . These results strongly suggest that S . xylosus CcpA controls transcription of catabolite-repressible genes and operons by binding to catabolite-responsive operators when rapidly metabolizable carbohydrates are available . Accordingly, the cloned S . xylosus ccpA gene could complement the ccpA mutation in B . subtilis . The ccpA gene of S . xylosus is transcribed from two promoters, one of which is subject to autogenous repression by CcpA . Autoregulation results in a slight reduction of CcpA protein in glucose-grown cells . The characterization of the role of CcpA in carbon catabolite repression in S . xylosus demonstrates that a regulatory mechanism originally detected in Bacillus applies to another Gram-positive bacterium with low GC content. Biochem Mol Biol Int, 1996 Aug, 39(6), 1185 - 92 Asporogenic Bacillus megaterium mutant 27-36 degrades intrinsically short-lived proteins but fails to convert most of other proteins to a short-lived fraction; Chaloupka J et al.; Asporogenic mutant blocked in the 0-II sporulation stage degraded pulse-labelled proteins in the sporulation medium at the same rate as the parental strain for the first two hours . The degraded fraction was mostly composed of intrinsically short-lived proteins which were degraded even after enriching the medium with amino acids and growth resumption . Proteins accessible to degradation because of nutritional shift down formed a lesser proportion of this fraction . The acceleration of protein turnover in the parent strain during the irreversible sporulation phase was not developed in the mutant . A first order kinetic model of protein degradation was used for parameter estimation . Ca(2+)-dependent intracellular serine proteinase was synthesized in an inactive form, which was activated by increasing Ca2+ concentration to 30 mM. Immunol Cell Biol, 1996 Aug, 74(4), 346 - 8 The effect of BCG, zymosan and Coxiella burnetti extract on Eimeria infections; Smith NC et al.; Infection of animals with species of Eimeria induces a hyper-reactivity to endotoxin as manifest by a greatly increased capacity of infected animals to produce TNF in response to LPS in vivo compared with uninfected animals . This finding indicates priming for hyperactivation of macrophages by Eimeria infection and raises the possibility that non-specific triggering of macrophages by agents such as Bacille Calmette-Guerin (BCG), zymosan or Coxiella burnetti extract may be a simple means of control for coccidiosis . However, all of these agents enhanced oocyst excretion in mice, rats or chickens infected with Eimeria vermiformis, Eimeria nieschulzi or Eimeria tenella, respectively, without affecting the patent period. Am J Infect Control, 1996 Aug, 24(4), 254 - 61 Challenges associated with assessment of risk for tuberculosis in a dental school setting; Murphy DC et al.; BACKGROUND: Reported risk among health care workers, related to tuberculosis exposure, motivated us to accurately assess this occupational risk at our College of Dentistry . METHODS: Our sample population included all dental students entering their junior (third) year (beginning of maximum patient exposure) . Screening for tuberculosis infection, with the standardized Mantoux test (purified protein derivative {PPD}), was conducted by the authors; several variables, previously associated with inaccurate test results in the literature, were controlled for during the study . RESULTS: Among the study population of 158 students, the PPD conversion rate, as determined by one-step testing, after one academic year was 10.6% . To further investigate factors (other than possible workplace exposure) contributing to PPD conversion in the study population, the authors also examined PPD results from previous employee and student screenings and conducted a retrospective chart review of selected patients registered at the college for the same period . CONCLUSIONS: We found that being born outside of the United States and having previously received bacille Calmette-Guerin vaccine are associated with positive PPD test results . In addition, PPD conversion among the study group may not be associated with occupational exposure at the College but, in fact, may be related to other factors, including community- and hospital-based clinical exposure . Finally, we recommend further research that examines the possible systemic effects of periodic testing with PPD on test subjects. Artif Organs, 1996 Aug, 20(8), 832 - 5 15-Deoxyspergualin: a newly developed immunosuppressive agent and its mechanism of action and clinical effect: a review . Japan Collaborative Transplant Study Group for NKT-01; Amemiya H; 15-Deoxyspargualin (DSG) is a synthetic analogue of spergualin isolated from the culture filtrate of Bacillus laterosporus . It shows a strong immunosuppressive effect by antiproliferating action inhibiting the IL-2-stimulated maturation of T cells from the G0/G1 phases to the S and G2/M phases . Hsc70, a constitutive member of Hsp70 was identified as the immunophilin of DSG by Nadlar et al . In allogeneic transplantation of rat heart and dog kidney, DSG was definitely proved to prevent rejection and to rescue ongoing rejection . Our multicenter clinical trials on DSG showed that the percent efficacy to reverse acute rejection was 70-80%, and 600 days graft survival was 90% in the cases effectively treated with DSG at acute rejections . The combined use of DSG with methylprednisolone to treat acute rejection and the use of DSG to treat rejections in chronic phases were reported as the beneficial uses of DSG. Microb Pathog, 1996 Aug, 21(2), 97 - 109 Pathobiological significance of colony morphology in Mycobacterium avium complex; Reddy VM et al.; Mycobacterium avium complex (MAC) strains are known to exhibit variation in colony morphology . In addition to the smooth transparent (ST), smooth opaque (SO) and rough opaque (RO), which are the most common morphological forms, intermediate (IM) and pin point (PP) forms were also occasionally observed . In order to understand the pathobiological significance of these different colony forms, we investigated their virulence in beige mice, ability to bind to plastic and epithelial cells, differences in the lipids, and modulation of macrophage functions by the bacillary extracts . ST variants, the most common form seen in AIDS patients, were more virulent with increased multiplication in lungs, livers and spleens of beige mice and showed increased adherence to plastic and epithelial cells . SO, RO, PP colonial forms did not show increase in growth in any of the organs over a period of 4 weeks . IM colonial variants showed increased growth in lungs and spleens but not in livers . Thin layer chromatographic (TLC) analysis of lipid extracts showed one specific component in the high polar lipids of the SO variant, while ST variant did not show any specific component in any of the three families of lipids (high, intermediate and low polarity) . The RO variant either expressed low levels or lost many of the components of lipids of high and intermediate polarity, however produced increased levels of lipids of low polarity . One of the components of low polar lipids was specific for RO variant and was produced in large quantity . The isogenic variants differed in the total lipid and sugar contents and also differed in their ability to modulate macrophage functions. AACN Clin Issues, 1996 Aug, 7(3), 378 - 89 Tuberculin testing in patients with human immunodeficiency virus/acquired immune deficiency syndrome; Jones SG; After declining for decades, the incidence of Mycobacterium tuberculosis is increasing . The Mantoux tuberculin skin test, which uses purified protein derivative (PPD) of tuberculin, has been used for years as a screening device to detect the presence of exposure and infection to tuberculosis . However, the advent of human immunodeficiency virus (HIV) has elicited many questions regarding the validity of traditional standards for PPD administration and interpretation . The uncertainties in interpreting tuberculin skin tests in immunocompromised individuals is part of the challenge that is being faced by the health-care profession in this 2nd decade of acquired immune deficiency syndrome . This article will help advanced practice nurses understand the relationship between the immune system, tuberculosis, and the PPD skin test; the problem of anergy with immunocompromised patients, particularly those who are HIV-infected; issues involved in placement and interpretation of the results of the PPD test; new Agency for Health Care Policy and Research (AHCPR) standards for PPD interpretation with HIV-infected persons; the "booster" effect and two-step PPD testing; concerns regarding bacille Calmette-Guerin vaccine; and the use of a critical pathway to aid in rapid identification and isolation of the patient with HIV admitted with a potential diagnosis of tuberculosis versus Pneumocystis carinii pneumonia. J Clin Microbiol, 1996 Aug, 34(8), 1985 - 91 Preparation of mycobacterial DNA from blood culture fluids by simple alkali wash and heat lysis method for PCR detection; Kulski JK et al.; A sodium iodide-isopropanol (NI) method was compared with an alkali wash and heat lysis (AH) procedure for the preparation and extraction of DNA from BACTEC 13A blood culture fluid samples from AIDS patients for use in a PCR for the detection and identification of mycobacteria . The sensitivity and efficiency of the DNA extraction methods were assessed by a multiplex PCR which detected the members of the genus Mycobacterium and differentiated between M . intracellulare, M . tuberculosis, and M . avium isolates with a limit of detection of between 0.28 pg (67 cells) and 120 pg (28,571 cells) of standard mycobacterial DNA . The PCR amplified mycobacterial DNA prepared by the AH procedure from 40 acid-fast bacillus-positive blood cultures with growth index values of > 20 U but not from 48 blood cultures with growth index values of < 21 U . The AH method was about 10 times more sensitive than the NI method for extracting DNA from 13 acid-fast bacillus-positive BACTEC fluid samples for PCR analysis . The study shows that the AH procedure in combination with the multiplex PCR is a simple, specific, and sensitive method which can be used in the routine diagnostic laboratory to detect and identify different members of the genus Mycobacterium in blood culture fluid samples from AIDS patients. J Clin Microbiol, 1996 Aug, 34(8), 1952 - 6 Experimental transmission of Bartonella henselae by the cat flea; Chomel BB et al.; Bartonella henselae is an emerging bacterial pathogen, causing cat scratch disease and bacillary angiomatosis . Cats bacteremic with B . henselae constitute a large reservoir from which humans become infected . Prevention of human infection depends on elucidation of the natural history and means of feline infection . We studied 47 cattery cats in a private home for 12 months to determine the longitudinal prevalence of B . henselae bacteremia, the prevalence of B . henselae in the fleas infesting these cats, and whether B . henselae is transmitted experimentally to cats via fleas . Vector-mediated transmission of B.henselae isolates was evaluated by removing fleas from the naturally bacteremic, flea-infested cattery cats and transferring these fleas to specific-pathogen-free (SPF) kittens housed in a controlled, arthropod-free University Animal Facility . B . henselae bacteremia was detected in 89% of the 47 naturally infected cattery cats . A total of 132 fleas were removed from cats whose blood was simultaneously cultured during different seasons and were tested individually for the presence of B . henselae DNA by PCR . B . henselae DNA was detected in 34% of 132 fleas, with seasonal variation, but without an association between the presence or the level of bacteremia in the corresponding cat . Cat fleas removed from bacteremic cattery cats transmitted B . henselae to five SPF kittens in two separate experiments; however, control SPF kittens housed with highly bacteremic kittens in the absence of fleas did not become infected . These data demonstrate that the cat flea readily transmits B . henselae to cats . Control of feline infestation with this arthropod vector may provide an important strategy for the prevention of infection of both humans and cats. Microbiol Res, 1996 Aug, 151(3), 263 - 71 Larvicidal activity of Bacillus thuringiensis natural isolates; indigenous to Japan, against two nematoceran insect pests occurring in urban sewage environments; Saitoh H et al.; A total of 1449 Bacillus thuringiensis strains, indigenous to Japan, were screened for larvicidal activity against two nematoceran insect pests, the mosquito, Culex pipiens molestus (Culicidae), and the moth-fly, Telmatoscopus albipunctatus (Psychodidae) . Mosquito specific strains were abundant in H serotypes 3abc (serovar kurstaki), 3ade (fukuokaensis), 4ac (kenyae), 7 (aizawai), 11ac (kyushuensis) and 29 (amagiensis), while moth-fly specific strains were predominantly found in H serotype 17 (tohokuensis) . Strains toxic to both insects were most frequently detected in H serotypes 10 (darmstadiensis) and 17/27 . Seven selected B . thuringiensis strains were highly toxic to Culex and/or Telmatoscopus . There was a diversity in SDS-PAGE profiles of inclusion proteins of these strains. Tuber Lung Dis, 1996 Aug, 77(4), 315 - 21 Bacille Calmette Guérin immunization of health care workers exposed to multidrug-resistant tuberculosis: a decision analysis; Stevens JP et al.; SETTING: North American health care workers with exposure to multidrug-resistant tuberculosis . OBJECTIVE: To evaluate the relative utilities of bacille Calmette Guerin (BCG) immunization and post-infection chemoprophylaxis for the protection of health care workers exposed to multidrug-resistant Mycobacterium tuberculosis . DESIGN: Decision analysis using SMLTREE software and published data for probabilities . RESULTS: BCG vaccination was preferred by a small margin over post-infection chemoprophylaxis . Sensitivity analysis revealed that possible changes in probability values used tended to tilt the result towards use of BCG vaccination . The threshold for protective efficacy of BCG vaccination was 26% . CONCLUSIONS: BCG vaccination should be considered for health care workers in environments where there is a substantial risk of exposure to multidrug-resistant tuberculosis. Tuber Lung Dis, 1996 Aug, 77(4), 308 - 14 Patient characteristics associated with failure of tuberculosis prevention; Menzies D et al.; OBJECTIVES: The cost-effectiveness of tuberculin screening may be substantially reduced by non-compliance of patients and physicians . We have examined the association of these problems with the socio-demographic characteristics of tuberculin reactors . METHODS: Community-based tuberculin screening was conducted among students in grades 6 and 10, and in post-secondary health training, as well as young adult workers . A follow-up survey was conducted to determine if tuberculin reactors referred for further evaluation actually reported, if they were prescribed therapy when indicated, and if they took therapy when it was prescribed . Association of reactors' socio-demographic characteristics with these outcomes was analyzed . RESULTS: Canadian-born subjects were less likely to report if they were: older (adjusted and standardized odds ratio: 0.7, 95% confidence interval: {0.5, 0.9}), resident in more affluent neighbourhoods (0.7 {0.6, 0.99}), and from single parent households (0.1 {0, 0.9}) . Even when indicated, physicians were less likely to prescribe treatment for Canadian-born subjects who reported bacille Calmette-Guerin vaccination, but had not actually received this (0.3 {0.1, 0.7}), or who were from single-parent households (0.1 {0, 0.9}) . Physicians were less likely to prescribe treatment for foreign-born who gave a history of BCG vaccination (0.1 {0.1, 0.3}), and were more likely to prescribe treatment for reactors from countries such as Haiti or Vietnam . The only factor significantly associated with compliance was that older Canadian-born subjects were less compliant (0.6 {0.4, 0.97}) . CONCLUSIONS: Failure to report for further medical evaluation and physician non-compliance were associated with a number of socio-demographic characteristics, and substantially reduced the benefit of a tuberculosis screening program. Thorax, 1996 Aug, 51(8), 871 - 2 Pleuropulmonary infection with chest wall infiltration by Eikenella corrodens; Killen JW et al.; Eikenella corrodens is a facultative anaerobic bacillus which is part of the normal flora of the oral cavity and has an unusual antibiotic sensitivity for an anaerobe . The case history is presented of a young man with chest wall infiltration by Eikenella corrodens. Eur J Biochem, 1996 Aug 1, 239(3), 702 - 9 Structure and activity of persicomycins, toxins produced by a Pseudomonas syringae pv . persicae/Prunus persica isolate; Barzic MR et al.; A toxigenic property has been demonstrated in a Pseudomonas syringae pv . persicae/Prunus persica isolate . Several substances, which are named persicomycins, have been purified in variable quantities from cultures . The structures of four of them were established by NMR and chemical ionization mass spectrometry . These compounds are 3-(3'-hydroxy)hydroxy fatty acids and thus represent a new family among the phytobacterial toxins . Other minor substances have also been isolated and have been shown to belong to the same family on the basis of their 7H-NMR spectra . All of them cause necrosis of peach tree tissues, a symptom similar to the one obtained after bacterial infection and antibiosis of microorganisms such as Bacillus thuringiensis . These results provide evidence that necrosis-inducing toxins are not restricted to the pathovar syringae . Furthermore, similar substances were purified from necrosed tissues of inoculated and diseased peach trees . 3-(3'-Hydroxydecanoyloxy)hexadecenoic acid was isolated from both such tissues and from cultures, which strongly suggests a similar toxigenesis in vivo and in vitro . The involvement of persicomycins in the die-back disease of peach trees is now clearly established, which demonstrates that the toxigenic property of the bacterium participates in the disease . The phytotoxicity of the persicomycins is discussed in comparison with the lipodepsipeptide necrotic toxins of the syringae pathovar. FEMS Microbiol Lett, 1996 Aug 1, 141(2-3), 261 - 4 Influence of the 20-kDa protein from Bacillus thuringiensis ssp . israelensis on the rate of production of truncated Cry1C proteins; Rang C et al.; The potential role of a molecular chaperone on the rate of production of extensively altered Bacillus thuringiensis Cry1C proteins was investigated . Analysis of the proteins produced by the recombinant B . thuringiensis strains showed that the truncated proteins were produced at a low rate . Expression of the 20-kDa protein gene from B . thuringiensis ssp . israelensis in tandem with the truncated-cry1C genes led to the production of a greater amount of proteins . The formation of inclusion bodies, however, did not occur even when the 20-kDa protein gene was expressed. FEMS Microbiol Lett, 1996 Aug 1, 141(2-3), 163 - 7 The chromosome map of Bacillus thuringiensis subsp . canadensis HD224 is highly similar to that of the Bacillus cereus type strain ATCC 14579; Carlson CR et al.; A physical map of the Bacillus thuringiensis subsp . canadensis HD224 chromosome based on AscI, NotI, and SfiI restriction sites has been established . The chromosome map of 4.3 Mb was similar to a revised map of the chromosome of the B . cereus type strain ATCC 14579, except that the B . thuringiensis subsp . canadensis HD224 chromosome lacked a NotI site and had two additional AscI sites . The positions of 27 probes were identical in the common macromap . A probe for the insecticidal toxin gene, cryIA, hybridized only to the B . thuringiensis subsp . canadensis HD224 chromosome . The BssHII ribotype patterns were almost identical confirming the similarity between the two strains. FEMS Microbiol Lett, 1996 Aug 1, 141(2-3), 151 - 6 Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak; Lund T et al.; Three enterotoxic components have been isolated from a strain of Bacillus cereus which was involved in a large food poisoning outbreak in Norway in 1995 . The components were purified by chromatography on three different columns . Three proteins of 39, 45 and 105 kDa, respectively, were found to be necessary for maximum cytotoxicity . The amino acid N-terminal sequences of the 39 and 45 kDa proteins were determined . The 45 kDa component was the same protein as the main antigen detected in the Bacillus Diarrhoeal Enterotoxin Visual Immunoassay (Tecra) . The 39 kDa protein showed some similarity to the L1 protein of haemolysin BL from B . cereus . Furthermore, the three toxic components were all recognised by a polyclonal antiserum reported to detect enterotoxin from B . cereus . The proteins were different from the B- and L2-components of haemolysin BL, previously suggested to be a primary virulence factor, and had no detectable haemolytic activity. FEMS Microbiol Lett, 1996 Aug 1, 141(2-3), 145 - 9 Evidence for a further enterotoxin complex produced by Bacillus cereus; Granum PE et al.; Out of 321 strains of Bacillus cereus from several sources and isolated in four different countries, 239 (74%) produced cytotoxins . Only 127 (53%) of the cytotoxic strains were positive for the B-component gene of the haemolysin BL (enterotoxin) by polymerase chain reaction (PCR) . Western blots using antiserum produced against enterotoxin(s) gave positive results for 199 (83%) of the cytotoxic B . cereus strains . On closer examination of seven of the strains, involved in food poisoning, we found that two strains completely lacked the L2- and B-components (of the haemolysin BL), and two strains were negative for the B-component gene by PCR, but were positive for the L2-component . From our experiments we concluded that there is at least one enterotoxin complex in addition to the haemolysin BL enterotoxin and enterotoxin T. Photochem Photobiol, 1996 Aug, 64(2), 259 - 66 Mechanism of UVB-induced suppression of the immune response to Mycobacterium bovis bacillus Calmette-Guerin: role of cytokines on macrophage function; Jeevan A et al.; Previously we demonstrated that treatment of mice with either UVB radiation or supernatants derived from UVB-irradiated PAM 212 keratinocytes decreased the induction of the delayed-type hypersensitivity (DTH) response to Mycobacterium bovis bacillus Calmette-Guerin (BCG), impaired the clearance of bacteria from their lymphoid organs and also altered macrophage functions . In order to characterize the cytokines involved in these phenomena, UV-irradiated mice were injected with antibodies to interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-beta 1), or tumor necrosis factor-alpha (TNF-alpha) . Injection of UVB-irradiated mice with anti-IL-10 immediately after UV irradiation restored the DTH response and reversed the UV-induced inhibition of bacterial clearance . Injection of UV-irradiated mice with anti-TGF-beta only partially restored the DTH response although it allowed a better clearance of BCG than injection of mice with the control antibody . In contrast, injection of anti-TNF-alpha did not affect the UVB-induced suppression of DTH or impaired bacterial clearance . Similarly, the ability of macrophages to phagocytose BCG and kill the intracellular organisms was restored to almost normal levels after injecting UV-irradiated mice with antibodies specific for IL-10 or TGF-beta . Injection of mice with either recombinant IL-10 or TGF-beta mimicked the effect of whole-body UV irradiation on immune function . These results suggest that IL-10 has a major role in UV-induced suppression of both DTH to BCG and impairment in the clearance of bacteria and that TGF-beta has a more significant role in blocking bacterial clearance . Furthermore, these cytokines seem to modulate immune responses by altering macrophage functions in UVB-irradiated mice. J Egypt Soc Parasitol, 1996 Aug, 26(2), 525 - 37 Assignment of the crystal toxin genes of the mosquitocidal bacterium, Bacillus thuringiensis israelensis to a specific plasmid; Rady MH et al.; Bacillus thuringiensis israelensis 4Q1-WT and the prepared mutants, 4Q1-72 and 4Q1-81 were bioassayed against Aedes caspius larvae . The strain -4Q1-WT, which contains all plasmid arrys and the strain 4Q1-72 which contains the 108 kb plasmid gave 100% mortality, while stain 4Q1-81 which contains no plasmids gave 0% mortality . Crystals from all tested strains were isolated, solubilized and characterized using PAGE to detect any homology or difference in crystal production, properties, and the relatedness to the plasmid profile. J Egypt Soc Parasitol, 1996 Aug, 26(2), 423 - 32 Isolation and curing of plasmid DNA from Bacillus thuringiensis israelensis strains showing variable insecticidal activities; Rady MH et al.; Bacillus thuringiensis israelensis 4Q1-WT (B.t.i.) have 9 plasmids . Fourteen mutates of this wild type were prepared with cured plasmids . The wild bacterial strain the mutants and fourteen industrial strains were compared according to the number, size of plasmids and their potential to induce mortality to Aedes caspius larvae . The 108 Kb plasmid is essential for B.t israelensis to induce insect kill. Nat Med, 1996 Aug, 2(8), 893 - 8 Immunogenicity and protective efficacy of a tuberculosis DNA vaccine; Huygen K et al.; Tuberculosis is the most widespread and lethal infectious disease affecting humans . Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell-mediated immune responses and conferred significant protection against challenge with live M . tuberculosis and M . bovis bacille Calmette-Guerin (BCG) . These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M . tuberculosis, leading to the development of a more effective vaccine. Nat Med, 1996 Aug, 2(8), 888 - 92 Vaccination against tuberculosis by DNA injection; Tascon RE et al.; There are 3 million deaths per annum worldwide due to tuberculosis, and AIDS is compounding the problem . A better vaccine than the live mycobacterium currently in use, bacillus Calmette-Guerin (BCG), is needed . When mice were injected with plasmid DNA encoding a single mycobacterial antigen (65-kDa heat shock protein, hsp65) they made specific cellular and humoral responses to the protein and became immune to subsequent challenge with Mycobacterium tuberculosis . Protection was equivalent to that obtained by vaccinating with live BCG, whereas immunizing with the protein was ineffective . Protection was also obtained with DNA encoding another mycobacterial antigen (36-kDa proline-rich antigen) . These results suggest that DNA vaccination might yield improved vaccines to replace BCG. Appl Environ Microbiol, 1996 Aug, 62(8), 3061 - 5 Production of kanosamine by Bacillus cereus UW85; Milner JL et al.; Bacillus cereus UW85 produces two antibiotics that contribute to its ability to suppress certain plant diseases (L . Silo-Suh, B . Lethbridge, S . J . Raffel, H . He, J . Clardy, and J . Handelsman, Appl . Environ . Microbiol . 60:2023-2030, 1994) . To enhance the understanding of disease suppression by UW85, we determined the chemical structure, regulation, and the target range of one of the antibiotics . The antibiotic was identified as 3-amino-3-deoxy-D-glucose, also known as kanosamine . Kanosamine was highly inhibitory to growth of plant-pathogenic oomycetes and moderately inhibitory to certain fungi and inhibited few bacterial species tested . Maximum accumulation of kanosamine in B . cereus UW85 culture supernatants coincided with sporulation . Kanosamine accumulation was enhanced by the addition of ferric iron and suppressed by addition of phosphate to rich medium . Kanosamine accumulation was also enhanced more than 300% by the addition of alfalfa seedling exudate to minimal medium. Appl Environ Microbiol, 1996 Aug, 62(8), 2932 - 9 The Bacillus thuringiensis insecticidal toxin binds biotin-containing proteins; Du C et al.; Brush border membrane vesicles from larvae of the tobacco hornworm, Manduca sexta, contain protein bands of 85 and 120 kDa which react directly with streptavidin conjugated to alkaline phosphatase . The binding could be prevented either by including 10 microM biotin in the reaction mixture or by prior incubation of the brush border membrane vesicles with an activated 60- to 65-kDa toxin from Bacillus thuringiensis HD-73 . The ability of B . thuringiensis toxins to recognize biotin-containing proteins was confirmed by their binding to pyruvate carboxylase, a biotin-containing enzyme, as well as to biotinylated ovalbumin and biotinylated bovine serum albumin but not to their nonbiotinylated counterparts . Activated HD-73 toxin also inhibited the enzymatic activity of pyruvate carboxylase . The biotin binding site is likely contained in domain III of the toxin . Two highly conserved regions within domain III are similar in sequence to the biotin binding sites of avidin, streptavidin, and a biotin-specific monoclonal antibody . In particular, block 4 of the B . thuringiensis toxin contains the YAS biotin-specific motif . On the basis of its N-terminal amino acid sequence, the 120-kDa biotin-containing protein is totally distinct from the 120-kDa aminopeptidase N reported to be a receptor for Cry1Ac toxin. Appl Environ Microbiol, 1996 Aug, 62(8), 2845 - 9 Aminopeptidase N purified from gypsy moth brush border membrane vesicles is a specific receptor for Bacillus thuringiensis CryIAc toxin; Lee MK et al.; We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV) . CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN . An in vitro competition binding study demonstrated that the binding of CryIAc to L . dispar BBMV was inhibited by APN . Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L . dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane . In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa . These data suggest that APN is the major functional receptor for CryIAc in L . dispar BBMV . A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L . dispar BBMV . In contrast, CryIAa and CryIAb bound to both the 120- and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide . The 120-kDa peptide (APN) in L . dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein. Appl Environ Microbiol, 1996 Aug, 62(8), 2839 - 44 Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins; Tabashnik BE et al.; We compared responses to six insecticidal crystal proteins from Bacillus thuringiensis by a Cry1A-resistant strain (NO-QA) and a susceptible strain (LAB-P) of the diamondback moth, Plutella xylostella . The resistant strain showed > 100-fold cross-resistance to Cry1J and to H04, a hybrid with domains I and II of Cry1Ab and domain III or Cry1C . Cross-resistance was sixfold to Cry1Bb and threefold to Cry1D . The potency of Cry1I did not differ significantly between the resistant and susceptible strains . Cry2B did not kill resistant or susceptible larvae . By combining these new data with previously published results, we classified responses to 14 insecticidal crystal proteins by strains NO-QA and LAB-P . NO-QA showed high levels of resistance to Cry1Aa, Cry1Ab, and Cry1Ac and high levels of cross-resistance to Cry1F, Cry1J, and H04 . Cross-resistance was low or nil to Cry1Ba, Cry1Bb, Cry1C, Cry1D, Cry1I, and Cry2A . Cry1E and Cry2B showed little or no toxicity to susceptible or resistant larvae . In dendrograms based on levels of amino acid sequence similarity among proteins, Cry1F and Cry1J clustered together with Cry1A proteins for domain II, but not for domain I or III . High levels of cross-resistance to Cry1Ab-Cry1C hybrid H04 show that although Cry1C is toxic to NO-QA, domain III or Cry1C is not sufficient to restore toxicity when it is combined with domains I and II of Cry1Ab . Thus, diamondback moth strain NO-QA cross-resistance extends beyond the Cry1A family of proteins to at least two other families that exhibit high levels of amino sequence similarity with Cry1A in domain II (Cry1F and Cry1J) and to a protein that is identical to Cry1Ab in domain II (H04) . The results of this study imply that resistance to Cry1A alters interactions between the insect and domain II. Appl Environ Microbiol, 1996 Aug, 62(8), 2753 - 7 Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots; de Maagd RA et al.; We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) delta-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots . A collection of wild-type and CryIC-CryIA hybrid toxins was used for this purpose . As demonstrated elsewhere (R . A . de Maagd, M . S . G . Kwa, H . van der Klei, T . Yamamoto, B . Schipper, J . M . Vlak, W . J . Stiekema, and D . Bosch, Appl . Environ . Microbiol . 62:1537-1543, 1996), CryIA(b) domain III recognized a 205-kDa protein on S . exigua blots, while no specific binding by domain I or II could be detected . In contrast, on ligand blots of M . sexta proteins CryIA(b) domain II recognized a 210-kDa protein and CryIA(b) domain III recognized a 250-kDa protein . Domain III is responsible for the interaction of CryIA(c) with 120-kDa major binding proteins of both S . exigua and M . sexta . In addition, in M . sexta CryIA(c) also reacts with a 210-kDa binding protein through its domain I and/or domain II . These results show that besides domain II, domain III of delta-endotoxins plays a major role in binding to putative receptors on ligand blots . However, for S . exigua there was no clear correlation between binding of toxins on ligand blots and the in vivo toxicity of the toxins . These and previous results suggest that interactions of insect membrane proteins with both domain II and domain III can occur and that detection of these interactions depends on the type of binding assay used. J Am Acad Dermatol, 1996 Aug, 35(2 Pt 2), 285 - 7 Widespread cutaneous bacillary angiomatosis and a large fungating mass in an HIV-positive man; Fagan WA et al.; Bacillary angiomatosis (BA), an infection caused by a gram-negative rod, can be a multiorgan disease . The usual causative organism, Bartonella (formerly Rochalimaea) hensalae, has only recently been identified . Bartonella quintana has also been shown to cause some cases of cutaneous BA . We describe a patient with widespread cutaneous BA with probable bone involvement and a large fungating mass. Am J Ophthalmol, 1996 Aug, 122(2), 272 - 3 Ochrobactrum anthropi endophthalmitis after uncomplicated cataract surgery; Braun M et al.; PURPOSE: To treat a case of Ochrobactrum anthropi endophthalmitis after uneventful cataract surgery . METHODS: A 66-year-old patient in good general health underwent uncomplicated cataract surgery in his right eye . Seven weeks later, pars plana vitrectomy with removal of the intraocular lens became necessary because of progressive low-grade endophthalmitis resistant to topical and systemic erythromycin, cephalosporins, aminoglycosides, and colistin . RESULTS: Microbiologic examination of the vitreous biopsy, capsule, and anterior chamber fluid disclosed O . anthropi, a nonfermentative gram-negative bacillus sensitive to imipenem, amikacin, ciprofloxacin, and vancomycin . CONCLUSION: Ochrobactrum anthropi and its natural resistance against many antibiotics should be considered in the treatment of low-grade endophthalmitis after uneventful cataract surgery. J Urol, 1996 Aug, 156(2 Pt 1), 372 - 6 A randomized prospective study comparing long-term intravesical instillations of mitomycin C and bacillus Calmette-Guerin in patients with superficial bladder carcinoma; Lundholm C et al.; PURPOSE: We compared the efficacy and toxicity of long-term mitomycin C versus bacillus Calmette-Guerin (BCG) instillation in patients at high risk for recurrence and progression of superficial bladder carcinoma . MATERIALS AND METHODS: Our randomized comparison study included 261 patients with primary dysplasia, or stage Tis, stage T1, grade 3 and multiple recurrent stage Ta/T1, grade 1 or 2 disease . Mitomycin C (40 mg.) or Pasteur strain BCG (120 mg.) was instilled weekly for 6 weeks, then monthly for up to 1 year and every 3 months during year 2 . RESULTS: After a median followup of 39 months 49% of the patients given BCG and 34% given mitomycin C were disease-free (p < 0.03), compared to 48 and 35%, respectively, of those with stage Ta or T1 disease, and 54 and 33%, respectively, of those with dysplasia or stage Tis tumor . Tumor progressed in 13% of patients, with no statistically significant difference observed regarding progression between the mitomycin C and BCG groups . Side effects were more common after BCG instillation, with 5 cases of severe side effects compared to 1 in the mitomycin C group . Treatment was stopped due to toxicity in 10% of the patients . CONCLUSIONS: The majority of patients tolerated long-term intravesical therapy well . BCG instillation was hampered by more frequent side effects . BCG was superior regarding recurrence prophylaxis, since patients given BCG had fewer recurrences and a significantly longer time to treatment failure compared to those treated with mitomycin C . No statistically significant difference was observed regarding progression. J Biotechnol, 1996 Jul 31, 48(3), 209 - 19 A specific chromophoric substrate for activity assays of 1,3-1,4-beta-D-glucan 4-glucanohydrolases; Malet C et al.; The synthesis of 4-methylumbelliferyl 3-beta-O-cellobiosyl-beta-D-glucopyranoside (3a) and its use as specific substrate to monitor enzyme activity of 1,3-1,4-beta-D-glucan 4-glucanohydrolases are described . The chromophoric substrate 3a is prepared by a chemoenzymatic approach starting from barley grain, whose beta-D-glucan polysaccharide is degraded down to a tri- and tetrasaccharide by an extracellular extract of recombinant E . coli expressing and secreting Bacillus licheniformis 1,3-1,4-beta-glucanase . The trisaccharide 1 is further chemically transformed into the title compound . Its use as substrate for an enzyme activity assay, the specificity of cleavage, and kinetic parameters are reported . As it undergoes a single glycosidic bond hydrolysis with release of 4-methylumbelliferone, direct UV monitoring of the reaction provides a sensitive kinetic assay of the enzyme action. Biochem Biophys Res Commun, 1996 Jul 25, 224(3), 779 - 83 Bacillus thuringiensis crystal proteins CRY1Ab and CRY1Fa share a high affinity binding site in Plutella xylostella (L.); Granero F et al.; The future success of Bacillus thuringiensis based insecticides depends in part on our ability to prevent insects from developing resistance against their insecticidal crystal proteins . Two recent papers indicated cross-resistance between Cry1A proteins and Cry1Fa in two different insect species (Tabashnik et al., 1994, Appl . Environ . Microbiol . 60, 4627-4629; Gould et al., 1995, J . Econ . Entomol . 88, 1545-1559) . Brush border membrane vesicles were prepared from Plutella xylostella and used in binding assays with 125I-labeled trypsin-activated crystal proteins . Competition experiments showed that Cry1Fa competed with Cry1Ab for a same binding site, though the latter still bound to a different minor binding site with apparently the same affinity . Cry1Ca did not compete for Cry1Ab binding sites nor Cry1Fa for Cry1Ca binding sites . Based on these results, a modification of the receptor shared by Cry1Ab and Cry1Fa might confer multiple resistance to Cry1A and Cry1Fa proteins. Biochemistry, 1996 Jul 23, 35(29), 9533 - 8 Identification of significant residues in the substrate binding site of Bacillus stearothermophilus farnesyl diphosphate synthase; Koyama T et al.; Farnesyl diphosphate synthases have been shown to possess seven highly conserved regions (I-VII) in their amino acid sequences {Koyama et al . (1993) J . Biochem . (Tokyo) 113, 355-363} . Site-directed mutants of farnesyl diphosphate synthase from Bacillus stearothermophilus were made to evaluate the roles of the conserved aspartic acids in region VI and lysines in regions I, V, and VI . The aspartate at position 224 was changed to alanine or glutamate (mutants designated as D224A and D224E, respectively); aspartates at positions 225 and 228 were changed to isoleucine and alanine (D225I, D228A); lysine at position 238 was changed to either alanine or arginine (K238A, K238R) . The lysines at positions 47 and 183 were changed to isoleucine and alanine (K471, K183A), respectively . Kinetic analyses of the wild-type and mutant enzymes indicated that the mutagenesis of Asp-224 and Asp-225 resulted in a decrease of Kcat values of approximately 10(4)- to 10(5)-fold compared to the wild type . On the other hand, D228A showed a Kcat value approximately one-tenth of that of the wild type, and the k(m) value for isopentenyl diphosphate increased approximately 10-fold . Both K471 and K183A showed k(m) values for isopentenyl diphosphate 20-fold larger and kcat values 70-fold smaller than the wild type . These results suggest that the two conserved lysines in regions I and V contribute to the binding of isopentenyl diphosphate and that the first and the second aspartates in region VI are involved in catalytic function . Aspartate-228 is also important for the binding of isopentenyl diphosphate rather than for catalytic reaction. Biochemistry, 1996 Jul 23, 35(29), 9496 - 504 Crystal structure of phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with glucosaminyl(alpha 1-->6)-D-myo-inositol, an essential fragment of GPI anchors; Heinz DW et al.; Numerous proteins on the external surface of the plasma membrane are anchored by glycosylated derivatives of phosphatidylinositol (GPI), rather than by hydrophobic amino acids embedded in the phospholipid bilayer . These GPI anchors are cleaved by phosphatidylinositol-specific phospholipases C (PI-PLCs) to release a water-soluble protein with an exposed glycosylinositol moiety and diacylglycerol, which remains in the membrane . We have previously determined the crystal structure of Bacillus cereus PI-PLC, the enzyme which is widely used to release GPI-anchored proteins from membranes, as free enzyme and also in complex with myo-inositol {Heinz, D.W., Ryan, M . Bullock, T.L., & Griffith, O . H . (1995) EMBO J . 14, 3855-3863} . Here we report the refined 2.2 A crystal structure of this enzyme complexed with a segment of the core of all GPI anchors, glucosaminyl(alpha 1-->6)-D-myo-inositol {GlcN-(alpha 1-->6)Ins } . The myo-inositol moiety of GlcN(alpha 1-->6)Ins is well-defined and occupies essentially the same position in the active site as does free myo-inositol, which provides convincing evidence that the enzyme utilizes the same catalytic mechanism for cleavage of PI and GPI anchors . The myo-inositol moiety makes several specific hydrogen bonding interactions with active site residues . In contrast, the glucosamine moiety lies exposed to solvent at the entrance of the active site with minimal specific protein contacts . The glucosamine moiety is also less well-defined, suggesting enhanced conformational flexibility . On the basis of the positioning of GlcN(alpha 1-->6)Ins in the active site, it is predicted that the remainder of the GPI-glycan makes little or no specific interactions with B . cereus PI-PLC . This explains why B . cereus PI-PLC can cleave GPI anchors having variable glycan structures. Mutat Res, 1996 Jul 22, 354(2), 203 - 9 SOS-inducing ability of native and mutant microbial ribonucleases; Ilinskaya ON et al.; The results of genotoxicity testing of microbial ribonucleases from Bacillus species with different catalytic activity obtained by site-directed mutagenesis in SOS chromotest are reported . At the concentrations 0.1-1 mg/ml, the induction factor for wild-type bacillar binase, barnase and mutant Arg58Lys binase with 100% activity was found to be significantly higher than 1.5 (1.8-2.8) . Mutant RNases having decreased catalytic activity (binases with replacements Lys26Ala, Arg61Gln, His101Glu) or through natural inhibitor barstar inactivated wild-type RNase exhibited no SOS-inducing potency . The ability of native bacillar RNases and mutant enzymes possessing high catalytic activity comparable with the activity of wild-type RNase to cause the SOS response indicates that genotoxicity is mediated through the probable cleavage of cellular RNA. J Biotechnol, 1996 Jul 18, 48(1-2), 81 - 96 Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector; Sanchis V et al.; Bacillus thuringiensis (Bt) delta-endotoxins are safe biological insecticidal proteins whose usefulness has long been recognized . The first commercialized Bt insecticidal formulations were composed of spore-crystal preparations derived from wild-type strains . These products generally have a limited insecticidal host range and several genetically modified strains have, therefore, been constructed using transformation procedures . However, addition of a new delta-endotoxin gene to strains already harboring other delta-endotoxin genes often resulted in broader-spectrum but less potent products because they produced significantly less of each of the crystal proteins . We report expression of the coding sequence of the sporulation specific cryIC gene from the non-sporulation-dependent cryIIIA promoter . Large amounts of CryIC accumulated in various Bt strains with different genetic backgrounds . Sporulation deficient Spo0A mutants, acrystalliferous derivatives and wild-type Bt strains expressing the engineered cryIII-cryIC gene were obtained . Introduction of the cryIII-cryIC gene whose product is highly active against Spodoptera littoralis into the Kto strain harboring the cryIA(c) gene active against Ostrinia nubilalis resulted in the construction of a new strain with increased potency and broader activity spectrum than the parent strain . Large amounts of each toxin were produced and the expression of the two genes seemed to be summed, presumably because the expression systems of the two genes are different . The plasmid shuttle vector used to introduce the cryIII-cryIC gene into the different Bt hosts utilizes the specific resolution site of transposon Tn4430 to enable construction of recombinant Bt strains that are free of foreign non-Bt DNA . This should facilitate the approval and acceptance for environmental release of the insecticidal recombinant products. Biochim Biophys Acta, 1996 Jul 18, 1295(2), 195 - 200 Structural consequences of neopullulanase mutations; Lamminmaki U et al.; Bacillus stearothermophilus neopullulanase (NPL) structure was modeled based on Aspergillus oryzae alpha-amylase (TAA) to understand the structure-function relationships of this pullulan hydrolyzing enzyme . The NPL structure seems to consist of a central (alpha/beta)8 barrel to which the other domains are attached . The immediate surroundings of the NPL catalytic site were found to have very similar structure to TAA . The more distant sites are different due to the stereochemical requirements of accommodating in the substrate alpha-1,6-linkages at every third position instead of alpha-1,4-linkages . The substrate binding cleft is wider than in alpha-amylases . The NPL structure, function, substrate binding and the consequences of mutations were discussed based on the modeled structure. Biochim Biophys Acta, 1996 Jul 18, 1295(2), 187 - 94 Phosphoserine aminotransferase from Bacillus circulans subsp . alkalophilus: purification, gene cloning and sequencing; Battchikova N et al.; Two peaks of aspartate aminotransferase (AspAT) catalytic activity were observed during DEAE chromatography of a protein extract from alkalophilic B . circulans . The enzyme purified from the major peak appeared to be not aspartate but phosphoserine aminotransferase (PSAT) with a considerably high AspAT side activity . The sequence of the enzyme N-terminus was determined, and the PSAT gene was cloned as two separate fragments . DNA sequencing revealed the open reading frame for the PSAT starting from TTG, putative ribosomal binding site and terminator of transcription . The PSAT gene encodes a protein of 361 amino acids (M(r) 39793) which shows moderate homology to other known phosphoserine aminotransferases (36-46% of identity, 60-64% of similarity) . The PSAT from the alkalophile shares with all of them the consensus sequence pattern around the pyridoxal 5'-phosphate attachment site. Structure, 1996 Jul 15, 4(7), 823 - 36 Crystal structure of the wide-spectrum binuclear zinc beta-lactamase from Bacteroides fragilis; Concha NO et al.; BACKGROUND: The metallo-beta-lactamase from Bacteroides fragilis hydrolyzes a wide range of beta-lactam antibiotics, and is not clinically susceptible to any known beta-lactamase inhibitors . B . fragilis is associated with post-surgery hospital infections, and there has been a recent report of plasmid-mediated dissemination of the enzyme . Effective inhibitors are therefore urgently needed . Knowledge of the three-dimensional structure will aid in the drug design effort . RESULTS: The crystal structure of the enzyme has been determined by using multiwavelength anomalous diffraction at the zinc absorption edge and refined to 1.85 A resolution . The structure is a four-layer alpha/beta/beta/alpha molecule . The active site, found at the edge of the beta sandwich contains a binuclear zinc center with several novel features . One zinc is tetrahedrally coordinated, the other has a trigonal bipyramidal coordination; a water/hydroxide molecule serves as a ligand for both metals . The residues that coordinate the two zincs are invariant in all metallo-beta-lactamases that have been sequenced, except for two conservative replacements . Despite the existence of the pattern for binuclear zinc binding, the reported structure of the Bacillus cereus enzyme contains only a single zinc . CONCLUSIONS: Structural analysis indicates that affinity for the penta-coordinated zinc can be modulated by neighboring residues, perhaps explaining the absence of the second zinc in the B . cereus structure . Models of bound substrates suggest that the active-site channel can accommodate a wide variety of beta-lactams . We propose that the zinc cluster prepares an hydroxide, probably the hydroxide that ligates both zincs, for nucleophilic attack on the carbonyl carbon atom of the beta-lactam . The resulting negatively charged tetrahedral intermediate implicated in catalysis is stabilized by an oxyanion hole formed by the side chain of the invariant Asn 193 and the tetrahedral zinc. Cancer Res, 1996 Jul 15, 56(14), 3315 - 9 Augmenting the immunogenicity of synthetic MUC1 peptide vaccines in mice; Zhang S et al.; Human mucin MUC1 is abundantly expressed in some cancers of epithelia} origin and is largely restricted to the apical surface of secretory cells in normal tissues . It is, therefore, a potential target for cancer immunotherapy . In preparation for clinical trials, vaccines containing synthetic MUC1 peptides of different lengths and sequences mixed with various adjuvants or covalently attached, using different linker methods, to protein carrier keyhole limpet hemocyanin (KLH) were studied in mice . MUC1 peptides (containing 30 amino acids), plus adjuvants QS-21 or Bacillus Calmette-Guerin, were incapable of inducing antibody . However, MUC1 peptides conjugated to KLH (MUCl-KLH), plus QS-21, induced high titer antibody against the immunizing peptides and against MUC1-expressing tumor cells . Although T-cell responses, including delayed-type hypersensitivity, lymphocyte proliferation, and CTL, were not observed in mice immunized with these vaccines, significant protection from MUC1-expressing tumor cell challenge in mice immunized with MUC1-KLH was observed . Based on these studies, a vaccine containing MUC1-KLH conjugate prepared with m-maleimidobenzoyl-N-hydroxysuccinimide ester linker, plus QS-21, has been constructed for testing in clinical trials. J Immunol, 1996 Jul 15, 157(2), 806 - 14 Calpain is the target antigen of a Th1 clone that transfers protective immunity against Schistosoma mansoni; Jankovic D et al.; A CD4+ clone (clone B), characterized as Th1 based on its selective production of IFN-gamma and IL-2, was established from C57Bl/6 mice protectively immunized against Schistosoma mansoni by intradermal vaccination with soluble worm Ags, plus bacillus Calmette Guerin . In agreement with previous results demonstrating an IFN-gamma-dependent cell-mediated protective mechanism in this vaccination model, Ag-elicited peritoneal macrophages from syngeneic recipients of this clone were activated to kill schistosome larvae (schistosomula) in vitro . Moreover, recipients of clone B displayed significant resistance against cercarial challenge . By screening a battery of lambda(gt11) clones from an adult worm cDNA library, one recombinant (25B) was identified that stimulated clone B specifically . Analysis of the 25B cDNA insert revealed a nucleotide sequence identical with that of the large subunit of schistosome calpain, a Ca2+-activated neutral proteinase . By expressing the products of PCR subcloning, we identified a 146-amino acid region of the 25B gene containing immunologic activity equivalent to the whole polypeptide . Overlapping peptides spanning this region were synthesized, and a core epitope was identified with the sequence EWKGAWCDGS . Since clone B responds to supernatants from cultured schistosomula, we postulate that the recognition of calpain released by invading larvae and resulting induction of Th1 cytokines accounts for the protection mediated by the adoptively transferred clone . Our findings thus implicate calpain as a target of protective immunity in schistosomes and provide the first example of a candidate vaccine Ag for this parasite identified on the basis of T cell reactivity. Eur J Biochem, 1996 Jul 15, 239(2), 403 - 9 Probing electron transfer in flavocytochrome P-450 BM3 and its component domains; Munro AW et al.; Rapid events in the processes of electron transfer and substrate binding to cytochrome P-450 BM3 from Bacillus megaterium and its constituent haem-containing and flavin-containing domains have been investigated using stopped-flow spectrophotometry . The formation of a blue semiquinone flavin form occurs during the NADPH-dependent reduction of the flavin domain and a species with a similar absorption maximum is also seen during reduction of the holoenzyme by NADPH . EPR spectroscopy confirms the formation of the flavin semiquinone . The formation of this semiquinone is transient during fatty acid monooxygenation by the holoenzyme, but in the presence of excess NADPH the species reforms once fatty acid is exhausted . Electron transfers through the reductase domain are too rapid to limit the fatty acid monooxygenation reaction . The substrate-binding-induced haem iron spin-state shift also occurs much faster than the Kcat at 25 degrees C . The rate of first electron transfer to the haem domain is also rapid; but it is of the order of 5-10-times larger than the Kcat for the enzyme (dependent on the fatty acid used) . Given that two successive electron transfers to haem iron are required for the oxygenation reaction, these rates are likely to exert some control over the rate of fatty acid oxygenation reactions . The presence of large amounts of NADPH also results in decreased rates of electron transfer from flavin to haem iron . In the difference spectrum of the active fatty acid hydroxylase, features indicative of a high-spin iron haem accumulate . These are in accordance with the presence of large amounts of an Fe(3+)-product bound enzyme during turnover and indicate that product release may also contribute to rate limitation . Taken together, these data suggest that the catalytic rate is not determined by the accumulation of a single intermediate in the reaction scheme, but rather that it is controlled in a series of steps. Rev Argent Microbiol, 1996 Jul-Sep, 28(3), 118 - 22 {Poly-(3-hydroxybutyrate) granules in Bacillus megaterium . Isolation and analysis of associated proteins}; Vazquez GJ et al.; Bacillus megaterium accumulates poly-(3-hydroxybutyrate) (PHB) as a reserve material in intracellular granules . In this work we described a method for the preparation of PHB granules from B . megaterium PV447 and the analysis by polyacrylamide gel electrophoresis of the associated proteins . By comparison with another species a function is proposed for some of these proteins. J Ind Microbiol, 1996 Jul, 17(1), 56 - 61 Interactions between gramicidin S and its producer, Bacillus brevis; Azuma T et al.; Gramicidin S (GS) inhibition of germination outgrowth of Bacillus brevis spores was reversed completely by a short pretreatment with sodium dodecyl sulfate, moderately by ethanol or by incubation at pH 10 but not by incubation at pH 4 . Of five metal ions tested (Na+, Mg2+, Fe2+, Cu2+, Ca2+), only Ca2+ reversed GS inhibition . When Ca2+ (but not the other four metal ions) was added to the growth medium, there was a considerable portion of the biosynthesized GS found in the extracellular fluid . These findings are interpreted in terms of the binding of GS to the external layers