scholarly journals Characterization of Bacillus anthracis Germinant Receptors In Vitro

2005 ◽  
Vol 187 (23) ◽  
pp. 8055-8062 ◽  
Author(s):  
Nathan Fisher ◽  
Philip Hanna
Keyword(s):  
Bacillus Anthracis ◽  
Current Model ◽  
Insertion Mutagenesis ◽  
Cell Type ◽  
Dormant Cell ◽  
Temperature Dependent ◽  
Infectious Cycle ◽  
A Current

ABSTRACT Bacillus anthracis begins its infectious cycle as a metabolically dormant cell type, the endospore. Upon entry into a host, endospores rapidly differentiate into vegetative bacilli through the process of germination, thus initiating anthrax. Elucidation of the signals that trigger germination and the receptors that recognize them is critical to understanding the pathogenesis of B. anthracis. Individual mutants deficient in each of the seven putative germinant receptor-encoding loci were constructed via temperature-dependent, plasmid insertion mutagenesis and used to correlate these receptors with known germinant molecules. These analyses showed that the GerK and GerL receptors are jointly required for the alanine germination pathway and also are individually required for recognition of either proline and methionine (GerK) or serine and valine (GerL) as cogerminants in combination with inosine. The germinant specificity of GerS was refined from a previous study in a nonisogenic background since it was required only for germination in response to aromatic amino acid cogerminants. The gerA and gerY loci were found to be dispensable for recognition of all known germinant molecules. In addition, we show that the promoter of each putative germinant receptor operon, except that of the gerA locus, is active during sporulation. A current model of B. anthracis endospore germination is presented.

A temporally regulated, diffusible activity is required for rod photoreceptor development in vitro

Development ◽  
1992 ◽  
Vol 114 (4) ◽  
pp. 947-957 ◽  
Author(s):  
D. Altshuler ◽  
C. Cepko
Keyword(s):  
Cell Fate ◽  
Rod Photoreceptor ◽  
Cell Type ◽  
Retinal Neuron ◽  
Development In Vitro ◽  
Temporally Correlated ◽  
Photoreceptor Development

The retina is a relatively simple and well-characterized CNS structure in which cell-cell interactions have been hypothesized to influence cell type determination. By manipulating cell density in serum-free cultures we show that rat rod photoreceptor development requires a diffusible activity produced by neonatal retinal cells. This effect is not mediated by changes in cell survival or mitosis. Production of the rod promoting activity varies with developmental stage and is temporally correlated with the timing of rod generation in vivo. In low density cultures, which do not support rod development, an increased fraction of cells stain with an antibody specific for another retinal neuron, the bipolar cell. Thus, the diffusible rod promoting activity may influence cell fate determination, and not only terminal differentiation. These results provide an approach for the molecular characterization of developmentally important signals in the vertebrate retina.

scholarly journals Characterization of a Novel Lytic Protein Encoded by the Bacillus cereus E33L GeneampDas a Bacillus anthracis Antimicrobial Protein

2012 ◽  
Vol 78 (8) ◽  
pp. 3025-3027 ◽  
Author(s):  
Feliza A. Bourguet ◽  
Brian E. Souza ◽  
Angela K. Hinz ◽  
Matthew A. Coleman ◽  
Paul J. Jackson
Keyword(s):  
Bacillus Cereus ◽  
Bacillus Anthracis ◽  
Antimicrobial Protein ◽  
Cell Wall Biosynthesis ◽  
Bacterial Genomes ◽  
Content Type ◽  
Highly Active ◽  
Low Concentrations

ABSTRACTLytic proteins encoded by bacterial genomes have been implicated in cell wall biosynthesis and recycling. TheBacillus cereusE33LampDgene encodes a putativeN-acetylmuramoyl-l-alanine amidase. This gene, expressedin vitro, produced a very stable, highly active lytic protein. Very low concentrations rapidly and efficiently lyse vegetativeBacillus anthraciscells.

scholarly journals In Vitro Selection and Characterization of Bacillus anthracis Mutants with High-Level Resistance to Ciprofloxacin

2003 ◽  
Vol 47 (7) ◽  
pp. 2362-2365 ◽  
Author(s):  
Lance B. Price ◽  
Amy Vogler ◽  
Talima Pearson ◽  
Joseph D. Busch ◽  
James M. Schupp ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
In Vitro Selection ◽  
Quinolone Resistance ◽  
Second Step ◽  
Ciprofloxacin Resistance ◽  
High Level ◽  
Level Resistance

ABSTRACT Mutants of attenuated Bacillus anthracis with high-level ciprofloxacin resistance were isolated using a three-step in vitro selection. Ciprofloxacin MICs were 0.5 μg/ml for first-step mutants, which had one of two gyrA quinolone resistance-determining region (QRDR) mutations. Ciprofloxacin MICs were 8 and 16 μg/ml for second-step mutants, which had one of three parC QRDR mutations. Ciprofloxacin MICs for third-step mutants were 32 and 64 μg/ml. Mutants for which MICs were 64 μg/ml had one of two additional mutations within the gyrA QRDR or one of two mutations within the gyrB QRDR. Mutants for which MICs were 32 μg/ml had no additional target modifications but showed evidence of enhanced ciprofloxacin efflux.

scholarly journals Isolation and characterization of a transfectant influenza B virus altered in RNA segment 6

1999 ◽  
Vol 80 (9) ◽  
pp. 2353-2359 ◽  
Author(s):  
Kate V. Rowley ◽  
Ruth Harvey ◽  
Wendy S. Barclay
Keyword(s):  
Helper Virus ◽  
Influenza B Virus ◽  
Influenza B ◽  
System A ◽  
Isolation And Characterization ◽  
B Virus ◽  
Infectious Cycle ◽  
Selection Of

This report describes the successful generation of an influenza B transfectant virus altered in RNA segment 6, which encodes the neuraminidase (NA) protein. The procedure for selection of the transfectant virus relies on the use of strain-specific anti-NA monoclonal antibodies to inhibit growth of the helper virus within the system. A transfectant virus has been engineered which has a coding change in the NA protein. This change resulted in attenuated growth in vitro that could be rescued by addition of exogenous bacterial NA. The mutant virus-associated NA activity was unstable as a result of the engineered changes. The ability to genetically manipulate influenza B virus segment 6 will allow us to assess the function of both NA and the small protein NB, also coded from this RNA, within the context of the virus infectious cycle.

scholarly journals Genome Characterization of Lipid-Containing Marine Bacteriophage PM2 by Transposon Insertion Mutagenesis

2006 ◽  
Vol 80 (18) ◽  
pp. 9270-9278 ◽  
Author(s):  
Mart Krupovič ◽  
Heikki Vilen ◽  
Jaana K. H. Bamford ◽  
Hanna M. Kivelä ◽  
Juha-Matti Aalto ◽  
...  
Keyword(s):  
Lipid Vesicle ◽  
Insertion Mutagenesis ◽  
Transposon Insertion ◽  
New Genes ◽  
Double Stranded Dna ◽  
Marine Phage ◽  
Protein Rich ◽  
Insertion Mutants

ABSTRACT Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed- and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.

scholarly journals Characterization of VAMP isoforms in 3T3-L1 adipocytes: implications for GLUT4 trafficking

2015 ◽  
Vol 26 (3) ◽  
pp. 530-536 ◽  
Author(s):  
Jessica B. A. Sadler ◽  
Nia J. Bryant ◽  
Gwyn W. Gould
Keyword(s):  
Plasma Membrane ◽  
Snare Complex ◽  
Cell Type ◽  
Attachment Protein ◽  
Systematic Analysis ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Factor Attachment Protein Receptor

The fusion of GLUT4-containing vesicles with the plasma membrane of adipocytes is a key facet of insulin action. This process is mediated by the formation of functional soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complexes between the plasma membrane t-SNARE complex and the vesicle v-SNARE or VAMP. The t-SNARE complex consists of Syntaxin4 and SNAP23, and whereas many studies identify VAMP2 as the v-SNARE, others suggest that either VAMP3 or VAMP8 may also fulfil this role. Here we characterized the levels of expression, distribution, and association of all the VAMPs expressed in 3T3-L1 adipocytes to provide the first systematic analysis of all members of this protein family for any cell type. Despite our finding that all VAMP isoforms form SDS-resistant SNARE complexes with Syntaxin4/SNAP23 in vitro, a combination of levels of expression (which vary by >30-fold), subcellular distribution, and coimmunoprecipitation analyses lead us to propose that VAMP2 is the major v-SNARE involved in GLUT4 trafficking to the surface of 3T3-L1 adipocytes.

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