Sequence and Organization of pXO1, the Large Bacillus anthracis Plasmid Harboring the Anthrax Toxin Genes

The Bacillus anthracis Sterne plasmid pXO1 was sequenced by random, "shotgun" cloning. A circular sequence of 181,654 bp was generated. One hundred forty-three open reading frames (ORFs) were predicted using GeneMark and GeneMark.hmm, comprising only 61% (110,817 bp) of the pXO1 DNA sequence. The overall guanine-plus-cytosine content of the plasmid is 32.5%. The most recognizable feature of the plasmid is a "pathogenicity island," defined by a 44.8-kb region that is bordered by inverted IS1627 elements at each end. This region contains the three toxin genes (cya, lef, and pagA), regulatory elements controlling the toxin genes, three germination response genes, and 19 additional ORFs. Nearly 70% of the ORFs on pXO1 do not have significant similarity to sequences available in open databases. Absent from the pXO1 sequence are homologs to genes that are typically required to drive theta replication and to maintain stability of large plasmids in Bacillus spp. Among the ORFs with a high degree of similarity to known sequences are a collection of putative transposases, resolvases, and integrases, suggesting an evolution involving lateral movement of DNA among species. Among the remaining ORFs, there are three sequences that may encode enzymes responsible for the synthesis of a polysaccharide capsule usually associated with serotype-specific virulent streptococci.     

Inhibition of EGF-mediated receptor activity and cell proliferation by HK1-ceramide,a stable analog of the ganglioside GM3-lactone

Gangliosides have been described as modulators of growth factor receptor activity and subsequent cellular function. Due to the lower-pH environment found in tumor cells, ganglosides are thought to be formed (at least to some extent) into their lactone forms. The aim of the study was to analyze the mode of action of the lactone of the ganglioside GM3 on epidermal growth factor (EGF) signaling in human ovarial epidermoid carcinoma A431 cells and cell growth in human oral epidermoid carcinoma KB cells by applying the GM3 lactone analog HK1-ceramide 2, which is stable under hydrolytic conditions. Specific inhibition of EGF-dependent receptor tyrosine phosphorylation was observed by HK1-ceramide 2 at 25 microM, whereas GM3 showed a comparable inhibition at eightfold higher concentrations. In cells exposed to low pH, where GM3 is thought to form its lactone to a higher extent, addition of GM3 showed no further inhibitory effect on EGF-dependent receptor phosphorylation. Similarly to GM3, HK1-ceramide 2 does not affect binding of (125)I-EGF to the cell surface receptor. EGF-dependent growth of KB cells was also found to be inhibited by HK1-ceramide 2 at much lower concentrations compared to GM3. In conclusion, our results indicate that the GM3 lactone analog HK1-ceramide 2 is a specific inhibitor of EGF receptor function and is more potent in reducing EGF-dependent tyrosine phosphorylation of the receptor in A431 cells and in inhibiting EGF-dependent growth of KB cells compared to GM3.     

Optimal packing characteristics of rolled, continuous stationary-phase columns

Rolled, continuous stationary phases were constructed by tightly rolling and inserting a whole textile fabric into a chromatography column. This work reports the column performance, in terms of plate height, void fraction, and resolution, of 10 cellulose-based fabrics. The relation between fabric structural properties of yarn diameter, fabric count, fabric compressibility, and column performance are quantitated. General requirements, including reproducibility of packing, for choosing fabrics to make a good SEC column are identified. This research showed that the packed columns have an optimal mass of fabric that minimizes plate height and maximizes resolution, in a manner that is consistent with chromatography theory. Mass of material packed is then an important column parameter to consider when optimizing columns for the rapid desalting of proteins. Proteins were completely separated from salt and glucose in less than 8 min at a pressure drop less than 500 psi on the rolled, continuous stationary-phase columns. These results, together with stability and reproducibility, suggest potential industrial applications for cellulose-based rolled, continuous stationary-phase columns where speed is a key parameter in the production process.     

Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis

Bacillus anthracis is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the B. anthracis genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (vrrC(1), vrrC(2), vrrB(1), vrrB(2), and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (vrrA). MLVA characterization of 426 B. anthracis isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.     

Recoverin depletion accelerates cone photoresponse recovery

The neuronal Ca²⁺-binding protein Recoverin has been shown to regulate phototransduction termination in mammalian rods. Here we identify four recoverin genes in the zebrafish genome, rcv1a, rcv1b, rcv2a and rcv2b, and investigate their role in modulating the cone phototransduction cascade. While Recoverin-1b is only found in the adult retina, the other Recoverins are expressed throughout development in all four cone types, except Recoverin-1a, which is expressed only in rods and UV cones. Applying a double flash electroretinogram (ERG) paradigm, downregulation of Recoverin-2a or 2b accelerates cone photoresponse recovery, albeit at different light intensities. Exclusive recording from UV cones via spectral ERG reveals that knockdown of Recoverin-1a alone has no effect, but Recoverin-1a/2a double-knockdowns showed an even shorter recovery time than Recoverin-2a-deficient larvae. We also showed that UV cone photoresponse kinetics depend on Recoverin-2a function via cone-specific kinase Grk7a. This is the first in vivo study demonstrating that cone opsin deactivation kinetics determine overall photoresponse shut off kinetics.     

Comparative Phylogenomics and Evolution of the Brucellae Reveal a Path to Virulence

Brucella species include important zoonotic pathogens that have a substantial impact on both agriculture and human health throughout the world. Brucellae are thought of as “stealth pathogens” that escape recognition by the host innate immune response, modulate the acquired immune response, and evade intracellular destruction. We analyzed the genome sequences of members of the family Brucellaceae to assess its evolutionary history from likely free-living soil-based progenitors into highly successful intracellular pathogens. Phylogenetic analysis split the genus into two groups: recently identified and early-dividing “atypical” strains and a highly conserved “classical” core clade containing the major pathogenic species. Lateral gene transfer events brought unique genomic regions into Brucella that differentiated them from Ochrobactrum and allowed the stepwise acquisition of virulence factors that include a type IV secretion system, a perosamine-based O antigen, and systems for sequestering metal ions that are absent in progenitors. Subsequent radiation within the core Brucella resulted in lineages that appear to have evolved within their preferred mammalian hosts, restricting their virulence to become stealth pathogens capable of causing long-term chronic infections.     

Tricyclic dihydroquinazolinones as novel 5-HT2C selective and orally efficacious anti-obesity agents

Agonists of the 5-HT_2C receptor have been shown to suppress appetite and reduce body weight in animal models as well as in humans. However, agonism of the related 5-HT_2B receptor has been associated with valvular heart disease. Synthesis and biological evaluation of a series of novel and highly selective dihydroquinazolinone-derived 5-HT_2C agonists with no detectable agonism of the 5-HT_2B receptor is described. Among these, compounds (+)-2a and (+)-3c were identified as potent and highly selective agonists which exhibited weight loss in a rat model upon oral dosing.     

Improving the clinical assessment of consciousness with advances in electrophysiological and neuroimaging techniques

In clinical neurology, a comprehensive understanding of consciousness has been regarded as an abstract concept - best left to philosophers. However, times are changing and the need to clinically assess consciousness is increasingly becoming a real-world, practical challenge. Current methods for evaluating altered levels of consciousness are highly reliant on either behavioural measures or anatomical imaging. While these methods have some utility, estimates of misdiagnosis are worrisome (as high as 43%) - clearly this is a major clinical problem. The solution must involve objective, physiologically based measures that do not rely on behaviour. This paper reviews recent advances in physiologically based measures that enable better evaluation of consciousness states (coma, vegetative state, minimally conscious state, and locked in syndrome). Based on the evidence to-date, electroencephalographic and neuroimaging based assessments of consciousness provide valuable information for evaluation of residual function, formation of differential diagnoses, and estimation of prognosis.     

Deep-Etching Electron Microscopy of Cells of <Emphasis Type="Italic">Magnetospirillum magnetotacticum</Emphasis>: Evidence for Filamentous Structures Connecting the Magnetosome Chain to the Cell Surface

Magnetospirillum magnetotacticum are magnetotactic bacteria that form a single chain of magnetite magnetosomes within its cytoplasm. Here, we studied the ultrastructure of M. magnetotacticum by freeze-fracture and deep-etching to understand the spatial correlation between the magnetosome chain and the cell envelope and its possible implications for magnetotaxis. Magnetosomes were found mainly near the cell envelope, forming chains that were closely associated with the granular cytoplasmic material. The membrane surrounding the magnetosomes could be visualized in deep-etching preparations. Thin connections between magnetosome chains and the cell envelope were observed in deep-etching images. These results strengthen the hypothesis for the existence of structures that transfer the torque from the magnetosome chains to the whole cell during the orientation of magnetotactic bacteria to a magnetic field lines.