Biomedical subjects

M Mock

Publications and source records attributed to M Mock.

At least 37 records · Page 2Linked to original sources

PubMed1999

Co-existence of clpB and clpC in the Bacillaceae.

The gene encoding ClpC in Bacillus anthracis was amplified from the chromosome by polymerase chain reaction using degenerate oligonucleotide primers. These primers also amplified a second DNA fragment identified as a clpB homolog. Both genes were suggested to be functional. Contrary to Bacillus subtilis which possesses clpC but not clpB, many Bacillus species were found to harbor both clpC and clpB. We also found that Clostridium strains could possess clpB, clpC, or both. All the Gram-negative strains tested had clpB only.

Bacillaceae↗

PubMed1999

The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community.

Plasmid genes that are responsible for virulence of Bacillus anthracis are important targets for the DNA-based detection of anthrax. We evaluated the distribution of the Ba813 chromosomal DNA sequence (Ba813) within closely related Bacillus species. Ba813 was systematically identified from 47 strains or isolates of B. anthracis tested, thus indicating its reliability as a tracer for that species. From the 60 strains of closely related Bacillus spp. examined, three bona fide B. cereus and one bona fide B. thuringiensis were found to harbour Ba813. This marker was also detected in Bacillus sp. isolates that were present at high levels in soil samples collected in a place where an anthrax outbreak had occurred. The significance and the possible function of the Ba813 locus is discussed.

Animals↗

PubMed1999

The S-layer homology domain as a means for anchoring heterologous proteins on the cell surface of Bacillus anthracis.

Bacillus anthracis synthesizes two S-layer proteins, each containing three S-layer homology (SLH) motifs towards their amino-terminus. In vitro experiments suggested that the three motifs of each protein were organized as a structural domain sufficient to bind purified cell walls. Chimeric genes encoding the SLH domains fused to the levansucrase of Bacillus subtilis were constructed and integrated on the chromosome. Cell fractionation and electron microscopy studies showed that both heterologous polypeptides were targeted to the cell surface. In addition, surface-exposed levansucrase retained its enzymatic and antigenic properties. Preliminary results concerning applications of this work are presented.

Animals↗

PubMed1999

Bacillus anthracis surface: capsule and S-layer.

Two abundant surface proteins, EA1 and Sap, are components of the Bacillus anthracis surface layer (S-layer). Their corresponding genes have been cloned, shown to be clustered on the chromosome and sequenced. EA1 and Sap each possess three 'S-layer homology' motifs. Single and double disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of both the non-capsulated and capsulated bacilli. When present, the capsule is exterior to, and completely covers, the S-layer proteins, which form an array beneath it. Nevertheless, the presence of these proteins is not required for normal capsulation of the bacilli. Thus both structures are compatible, and yet neither is required for the correct formation of the other. Bacillus anthracis has, therefore, a very complex cell wall organization for a gram-positive bacterium.

Bacillus anthracis↗

PubMed1999

Anthrax lethal factor cleaves the N-terminus of MAPKKS and induces tyrosine/threonine phosphorylation of MAPKS in cultured macrophages

The lethal toxin (LeTx) of Bacillus anthracis is the major virulence factor responsible for the death of infected animals and for cytolysis of cultured macrophages. Its catalytic component, LF, contains the characteristic zinc-binding motif of metalloproteases, it binds zinc and indirect evidence suggests that this hydrolytic activity is essential for LeTx cytotoxicity (Limpel et al. 1994; Kochi et al. 1994). To identify substrates of LF, we have used the yeast two-hybrid system, employing an LF inactive mutant as bait. This approach has led to the identification of the MAP kinase kinases (MAPKKs) Mek1 and Mek2 as proteins capable of specific interaction with LF. LF cleaves Mek1 and Mek2 within their N-terminus in vitro and in vivo, hydrolysing a Pro8-Ile9 and a Pro10-Arg11 peptide bond in Mek1 and Mek2, respectively (Vitale et al. 1998), similarly to that found with a different approach by Duesbery et al. (1998). The removal of the amino terminus of MAPKKs eliminates the 'docking site' involved in the specific interaction with MAPKs and interferes with the phospho-activation of the MAPKs ERK1 and ERK2, which become phosphorylated in cultured macrophages following toxin challenge. We are currently investigating the relevance of MAPKKs cleavage for LeTx cytotoxicity and the consequences for the activity of the MAP pathway.

Journal Article↗

PubMed1999

Antigen delivery by attenuated Bacillus anthracis: new prospects in veterinary vaccines.

This report summarizes the recent investigations on the use of Bacillus anthracis as a live vector for delivery of antigens. Recombinant strains were constructed by engineering the current live Sterne vaccine. This vaccine, used to prevent anthrax in cattle, causes side-effects due to anthrax toxin activities. Bacteria producing a genetically detoxified toxin factor were devoid of lethal effects and were as protective as the Sterne strain against experimental anthrax. Moreover, B. anthracis expressing a foreign antigen controlled by an in vivo inducible promoter were able to generate either antibody or cellular protective responses against heterologous diseases.

Animals↗

PubMed1999

Germination of Bacillus anthracis spores within alveolar macrophages.

The fatal character of the infection caused by inhalation of Bacillus anthracis spores results from a complex pathogenic cycle involving the synthesis of toxins by the bacterium. We have shown using immunofluorescent staining, confocal scanning laser microscopy and image cytometry analysis that the alveolar macrophage was the primary site of B. anthracis germination in a murine inhalation infection model. Bacillus anthracis germinated inside murine macrophage-like RAW264.7 cells and murine alveolar macrophages. Germination occurred in vesicles derived from the phagosomal compartment. We have also demonstrated that the toxin genes and their trans-activator, AtxA, were expressed within the macrophages after germination.

Animals↗

PubMed1999

Identification and characterization of a germination operon on the virulence plasmid pXO1 of Bacillus anthracis.

The spores of Bacillus anthracis, the agent of anthrax disease, germinate within professional phagocytes, such as murine macrophage-like RAW264.7 cells and alveolar macrophages. We identified a cluster of germination genes extending for 3608 nucleotides between the pag and atxA genes on the B. anthracis virulence plasmid pXO1. The three predicted proteins (40, 55 and 37 kDa in size) have significant sequence similarities to B. subtilis, B. cereus and B. megaterium germination proteins. Northern blot analysis of total RNA from sporulating cells indicated that the gerX locus was organized as a tricistronic operon (gerXB, gerXA and gerXC). Primer extension analysis identified a major potential transcriptional start site 31 bp upstream from the translation initiation codon of gerXB. Expression of the gerX operon was studied using a gerXB-lacZ transcriptional fusion. Expression began 2.5-3 h after the initiation of sporulation and was detected exclusively in the forespore compartment. A gerX null mutant was constructed. It was less virulent than the parental strain and did not germinate efficiently in vivo or in vitro within phagocytic cells. These data strongly suggest that gerX-encoded proteins are involved in the virulence of B. anthracis.

Animals↗

PubMed1999

Functional analysis of the carboxy-terminal domain of Bacillus anthracis protective antigen.

Protective antigen (PA) is the common receptor-binding component of the two anthrax toxins. We investigated the involvement of the PA carboxy-terminal domain in the interaction of the protein with cells. A deletion resulting in removal of the entire carboxy-terminal domain of PA (PA608) or part of an exposed loop of 19 amino acids (703 to 722) present within this domain was introduced into the pag gene. PA608 did not induce the lethal-factor (LF)-mediated cytotoxic effect on macrophages because it did not bind to the receptor. In contrast, PA711- and PA705-harboring lethal toxins (9- and 16-amino-acid deletions in the loop, starting after positions 711 and 705, respectively) were 10 times less cytotoxic than wild-type PA. After cleavage by trypsin, the mutant PA proteins formed heptamers and bound LF. The capacity of PA711 and PA705 to interact with cells was 1/10 that of wild-type PA. In conclusion, truncation of the carboxy-terminal domain or deletions in the exposed loop resulted in PA that was less cytotoxic or nontoxic because the mutated proteins did not efficiently bind to the receptor.

Animals↗

PubMed1999

Cell surface-exposed tetanus toxin fragment C produced by recombinant Bacillus anthracis protects against tetanus toxin.

Bacillus anthracis, the causal agent of anthrax, synthesizes two surface layer (S-layer) proteins, EA1 and Sap, which account for 5 to 10% of total protein and are expressed in vivo. A recombinant B. anthracis strain was constructed by integrating into the chromosome a translational fusion harboring the DNA fragments encoding the cell wall-targeting domain of the S-layer protein EA1 and tetanus toxin fragment C (ToxC). This construct was expressed under the control of the promoter of the S-layer component gene. The hybrid protein was stably expressed on the cell surface of the bacterium. Mice were immunized with bacilli of the corresponding strain, and the hybrid protein elicited a humoral response to ToxC. This immune response was sufficient to protect mice against tetanus toxin challenge. Thus, the strategy developed in this study may make it possible to generate multivalent live veterinary vaccines, using the S-layer protein genes as a cell surface display system.

Amino Acid Sequence↗

PubMed1998

Use of a photoactivatable lipid to probe the topology of PA63 of Bacillus anthracis in lipid membranes.

The protective antigen of Bacillus anthracis is a key protein that promotes the translocation of the enzymatic moieties of the two toxins of B. anthracis into the cell cytoplasm. The membrane topology of the active form of the protective antigen (PA63) was investigated by proteolysis of PA63 inserted into liposomes containing a photoactivatable, radioactive lipid, and characterization of the N-terminal moiety of the deeply-inserted (and therefore radiolabeled) peptides. A single sequence starting at residue Ala258 was identified. Fourier-transform infrared spectroscopy showed that the protected peptide was mainly adopting a beta-sheet structure whose orientation was compatible with a transmembrane organization.

Antigens, Bacterial↗

PubMed1998

Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages.

Lethal factor (LF) is the major virulence factor produced by Bacillus anthracis. LF is sufficient to cause death in laboratory animals and cytolysis of peritoneal macrophages and macrophage cell lines. LF contains the characteristic zinc binding motif of metalloproteases and indirect evidence suggest that this hydrolytic activity is essential for its cytotoxicity. To identify the substrate(s) of LF, we have used the yeast two-hybrid system, employing a LF inactive mutant as bait. This approach has led to the identification of the MAP kinase kinases (MAPKKs) Mek1 and Mek2 as proteins capable of specific interaction with LF. LF cleaves Mek1 and Mek2 within their N-terminus in vitro and in vivo, hydrolyzing a Pro8-Ile9 and a Pro10-Arg11 peptide bond in Mek1 and Mek2 respectively. The removal of the amino terminus of MAPKKs eliminates the "docking site" for the MAPKs ERK1 and ERK2, which become phosphorylated in cultured macrophages following toxin challenge. The possible implications of these findings for the cytolysis of macrophage cells induced by LF are discussed. These results open the way to the design and screening of specific inhibitors of LF.

Amino Acid Sequence↗

PubMed1998

[Analysis of different application systems and CT-controlled planning variants in treatment of primary endometrial carcinomas. Is brachytherapy treatment of the entire uterus technically possible?].

BACKGROUND: Intracorporal brachytherapy is regarded as the definitive component of treatment for inoperable patients with endometrial carcinoma. Until now the whole uterus has been claimed to represent the target volume independent of individual tumor spread. The purpose of this work is to analyse the correlation between target volume and treated volume using different application- and planning procedures. PATIENTS AND METHODS: In a consecutive series of 10 patients with primary irradiated endometrial carcinoma we analyzed the correlation between target volume and treated volume using either standard 1-channel applicators or individual Heyman-applicators. Application of the ovoids was followed by a planning CT scan for all patients. Based on this, target volume (uterus volume) was estimated on a 3D-planning system. According to the measurable length of the uterus cavity we determined the corresponding standard 1-channel applicator and calculated the respectively treated volume. Estimating the advantages of an optimized treatment planning strategy for individual Heyman-applications were compared the treated volumes, which result from a standardized and optimized treatment planning procedure. RESULTS: The mean uterus volume was 180 cm3 (range 57 to 316 cm3). Asymmetric uterus configurations with longitudinal or sagittal side differences exceeding 1 cm were found in 40% of the cases. Using standard 1-channel applicators on average 47% (range 25 to 89%) of the uterus volume were enclosed by the treated volume compared to 70% for Heyman-applications. Differentiating these individual applications according to the variable treatment modality values of mean 66% (range 36 to 110%) for the standardized and 73% (range 48 to 95%) for the optimized treatment planning strategy were found. Moreover optimized planning modalities led to an improved coverage of the target volume in 5 out of 10 cases with an increase in volume of 20% on average (range 11 to 32%). In 3 cases changes of less than 5% were noticed (no improvement). In order to protect organs at risk treated volume had to be decreased in 2 cases for 19% and 40% respectively. CONCLUSIONS: Intracavitary brachytherapy of primary endometrial carcinoma was improved by individualized application- and planning procedures, which led to better adaptations of the treated volumes to the target volumes. Nevertheless a complete coverage--corresponding to the primary intent--was not possible. Individualized and optimized brachy-therapy must be performed according to the individual tumor spread and uterus configuration. Therefore, different applicators are required.

Adenocarcinoma↗

PubMed1998

The capsule and S-layer: two independent and yet compatible macromolecular structures in Bacillus anthracis.

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive spore-forming bacterium. Fully virulent bacilli are toxinogenic and capsulated. Two abundant surface proteins, including the major antigen, are components of the B. anthracis surface layer (S-layer). The B. anthracis paracrystalline S-layer has previously only been found in noncapsulated vegetative cells. Here we report that the S-layer proteins are also synthesized under conditions where the poly-gamma-D-glutamic acid capsule is present. Structural and immunological analyses show that the capsule is exterior to and completely covers the S-layer proteins. Nevertheless, analysis of single and double S-layer protein mutants shows that the presence of these proteins is not required for normal capsulation of the bacilli. Similarly, the S-layer proteins assemble as a two-dimensional crystal, even in the presence of the capsule. Thus, both structures are compatible, and yet neither is required for the correct formation of the other.

Animals↗

PubMed1998

Molecular characterization of Bacillus strains involved in outbreaks of anthrax in France in 1997.

Outbreaks of anthrax zoonose occurred in two regions of France in 1997. Ninety-four animals died, and there were three nonfatal cases in humans. The diagnosis of anthrax was rapidly confirmed by bacteriological and molecular biological methods. The strains of Bacillus anthracis in animal and soil samples were identified by a multiplex PCR assay. They all belonged to the variable-number tandem repeat (VNTR) group (VNTR)3. A penicillin-resistant strain was detected. Nonvirulent bacilli related to B. anthracis, of all VNTR types, were also found in the soil.

Adult↗

PubMed1998

Tinea capitis dermatophytes: susceptibility to antifungal drugs tested in vitro and in vivo.

BACKGROUND: Tinea capitis is a worldwide-spread infection of the scalp caused by dermatophytes and is predominantly seen in children. The clinical manifestations range from mild scaling lesions to widespread alopecia or highly inflammatory suppurating lesions. Terbinafine and itraconazole seem to be promising therapies with shorter treatment durations than griseofulvin. OBJECTIVE: The objective of the present study was to test the sensitivity of different species of dermatophytes towards terbinafine and itraconazole, and to compare the results with a retrospective study on 35 immunocompetent patients with tinea capitis who were treated with terbinafine (Lamisil(R)). METHODS: Minimal inhibitory concentrations (MIC) were measured with an agar dilution method. RESULTS: Each tested species of dermatophyte was sensitive to terbinafine and itraconazole at different concentration ranges. The MIC for terbinafine ranged from 0.005 to 0.5 microg/ml and for itraconazole from 40 to 80 microg/ml. Microsporum canis was the dermatophyte least sensitive to terbinafine. Our retrospective study showed that the cure rate was excellent for Trichophyton violaceum and T. soudanense, variable for T. mentagrophytes and poor for M. canis and M. langeronii. CONCLUSIONS: (i) Regarding the results of susceptibility tests obtained with species involved in tinea capitis, clinical efficacy is not related to MIC measured in vitro; (ii) identification of the isolated dermatophyte from tinea capitis seems to be important for choosing the appropriate treatment.

Antifungal Agents↗

PubMed1998

[Anthrax toxins].

Bacillus anthracis, a Gram positive bacterium, is the causative agent of anthrax. This organism is capsulogen and toxinogenic. It secretes two toxins which are composed of three proteins: the protective antigen (PA), the lethal factor (LF) and the edema factor (EF). The lethal toxin (PA + LF) provokes a subite death in animals, the edema toxin (PA + EF) induces edema. The edema and the lethal factors are internalised into the target cells via the protective antigen. EF and LF exert an adenylate cyclase and a metalloprotease activity respectively. The structure-function relationship of these three proteins were defined using in vitro and in vivo approaches.

Animals↗

PubMed1997

Anthrax lethal toxin-induced mitogenic response of human T-cells.

Bacillus anthracis lethal toxin (PALF) stimulated the proliferation of human peripheral blood T-cells in vitro. Activation of T-lymphocytes by PALF required the presence of monocytes and did not result from a collaborative effect between T-cells and B-cells. PALF acted directly on monocytes and independently of T-cells. The monocytes contributed to the proliferation of T-cells by secretion of mediator(s). The mitogenic activity of the lethal toxin was dependent on its metalloprotease activity.

Antigens, Bacterial↗