Virulence and antibiotic susceptibility of Aeromonas spp. isolated from drinking water

Aeromonas isolates from tap water, mineral water, and artesian well water were investigated for their ability to produce different potential virulence factors or markers such as hemolysins, cytotoxins, phospholipase, DNase, hydrophobicity and their ability to adhere to epithelial cells and to abiotic surfaces. The susceptibility to antibiotics of Aeromonas isolates was also examined. Majority of the isolates displayed hemolytic activity against sheep erythrocytes, while only 7 of the 23 Aeromonas strains displayed DNase activity and 4 of the 23 Aeromonas strains tested were regarded as positive for phospholipase production. Most of the isolates showed cytotoxic activities in culture filtrate dilutions at titer of 1/8 or lower. No general relation between the strain isolated and the ability to interact with epithelial cells could be established. Using the bacterial adherence to hydrocarbons method, most of the strains were classified as highly hydrophilic. All five Aeromonas jandaei strains isolates, 9 of the 12 Aeromonas sp strains and four of the five Aeromonas hydrophila were multidrug resistant. The most active antimicrobial was ciprofloxacin (susceptible in 100% of the isolates), and the least active antibiotic was ampicillin (resistance in 92% of the isolates). The majority of the isolates tested were not killed by chlorine at 1.2 mg/l. Whether the high tolerance to chlorine of Aeromonas isolates can be linked to greater virulence is not know.     

Solution structure of C-terminal Escherichia coli translation initiation factor IF2 by small-angle X-ray scattering

Initiation of protein synthesis in bacteria involves the combined action of three translation initiation factors, including translation initiation factor IF2. Structural knowledge of this bacterial protein is scarce. A fragment consisting of the four C-terminal domains of IF2 from Escherichia coli was expressed, purified, and characterized by small-angle X-ray scattering (SAXS), and from the SAXS data, a radius of gyration of 43 +/- 1 A and a maximum dimension of approximately 145 A were obtained for the molecule. Furthermore, the SAXS data revealed that E. coli IF2 in solution adopts a structure that is significantly different from the crystal structure of orthologous aIF5B from Methanobacterium thermoautotrophicum. This crystal structure constitutes the only atomic resolution structural knowledge of the full-length factor. Computer programs were applied to the SAXS data to provide an initial structural model for IF2 in solution. The low-resolution nature of SAXS prevents the elucidation of a complete and detailed structure, but the resulting model for C-terminal E. coli IF2 indicates important structural differences between the aIF5B crystal structure and IF2 in solution. The chalice-like structure with a highly exposed alpha-helical stretch observed for the aIF5B crystal structure was not found in the structural model of IF2 in solution, in which domain VI-2 is moved closer to the rest of the protein.     

The proapoptotic protein Smac/DIABLO dimer has the highest stability as measured by pressure and urea denaturation

Apoptosis is an essential mechanism of cell death required for normal development and homeostasis of all multicellular organisms. Smac/DIABLO is a dimeric protein important in the control of apoptosis by removing the inhibitory activity of IAPs (inhibitor of apoptosis proteins). In vitro studies reveal that dimerization is required for its function. Here we investigate the structural and thermodynamic features of folding and dimerization of Smac/DIABLO. To disturb the folded, dimeric structure, we used high hydrostatic pressure, low and high temperatures, and chemical denaturing agents. Conformational changes were monitored using spectroscopic techniques such as fluorescence and circular dichroism (CD) as well as gel filtration chromatography. Our data show that Smac/DIABLO is very stable under pressures up to 3.1 kbar, even at subzero temperatures. A complete denaturation/dissociation process is obtained when we use high concentrations of urea, which affect its secondary structure as assessed by CD. The association of pressure and subdenaturing urea concentrations also results in complete denaturation/dissociation of the protein. Under these conditions, unfolding of the protein shows concentration dependence that is in accordance with the dimer-monomer dissociation equilibrium, confirming Smac/DIABLO dissociation. These results suggest that most of the treatments lead to a reversible disruption of the dimeric structure with a dissociation constant ( K d) of 34 x 10 (-21) M (34 zM). This tight dimer is biologically relevant, considering that monomeric mutants bind IAP with low affinity. The extremely high stability of the dimeric form of Smac/DIABLO also implies that once expressed in the cell the protein has a low probability of dissociation and, consequently, loss of function. In addition, the stability in the zeptomolar range is the highest so far measured for a dimeric protein. It also indicates that under most circumstances Smac/DIABLO does not exist as a monomer in the cell and suggests that the dimer-to-monomer equilibrium does not play a regulatory role in the Smac/DIABLO-IAP interaction.     

Size and lipid content of nonmyrmecochorous diaspores: effects on the interaction with litter-foraging ants in the Atlantic rain forest of Brazil

Ants are often attracted to diaspores not adapted for dispersal by ants. These diaspores may occasionally benefit from this interaction. We selected six nonmyrmecochorous plant species (Virola oleifera, Eugenia stictosepala, Cabralea canjerana, Citharexylum myrianthum, Alchornea glandulosa and Hyeronima alchorneoides) whose diaspores differ in size and lipid content, and investigated how these features affect the outcome of ant-diaspore interactions on the floor of a lowland Atlantic forest of Southeast Brazil. A total of 23 ant species were seen interacting with diaspores on the forest floor. Ants were generally rapid at discovering and cleaning the diaspore pulp or aril. Recruitment rate and ant attendance were higher for lipid-rich diaspores than for lipid-poor ones. Removal rate and displacement distance were higher for small diaspores. The large ponerine ant Pachycondyla striata, one of the most frequent attendants to lipid-rich arillate diaspores, transported the latter into their nests and discarded clean intact seeds on refuse piles outside the nest. Germination tests with cleaned and uncleaned diaspores revealed that the removal of pulp or aril may increase germination success in Virola oleifera, Cabralea canjerana, Citharexylum myrianthum and Alchornea glandulosa. Gas chromatography analyses revealed a close similarity in the fatty acid composition of the arils of the lipid-rich diaspores and the elaiosome of a typical myrmecochorous seed (Ricinus communis), corroborating the suggestion that some arils and elaiosomes are chemically similar. Although ant-derived benefits to diaspores – secondary dispersal and/or increased germination – varied among the six plant species studied, the results enhanced the role of ant-diaspore interactions in the post-dispersal fates of nonmyrmecochorous seeds in tropical forests. The size and the lipid-content of the diaspores were shown to be major determinants of the outcome of such interactions.     

Simultaneous determination of retinol, β-carotene and α-tocopherol in adipose tissue by high-performance liquid chromatography

A method is described for evaluation of fat-soluble vitamin in human adipose tissue with the aim to obtain, accurately and within the shortest analysis time, a time-integrated measure of exposure to vitamins from the diet. Fat tissue was deproteinized with ethanol and extracted with n-hexane. Normal-phase HPLC was performed in a Lichrosorb Si60 column with a gradient of n-hexane–2-propanol at 1 ml/min. Detection was accomplished using a diode-array system (for retinol and β-carotene) in series with a fluorescence detector (α-tocopherol). The method was validated and applied to human adipose tissue in a total of 140 subjects. The mean contents found were 0.43, 0.84, 240.3 μg/g for retinol, β-carotene and α-tocopherol, respectively. The method is sensitive enough for detecting the compounds in 1.6 mg of adipose tissue considering the lowest concentration found.     

Rapid Detection, Identification, and Enumeration of Escherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

An algebraic-combinatorial model for the identification and mapping of biochemical pathways

We develop the mathematical machinery for the construction of an algebraic-combinatorial model using Petri nets to construct an oriented matroid representation of biochemical pathways. For demonstration purposes, we use a model metabolic pathway example from the literature to derive a general biochemical reaction network model. The biomolecular networks define a connectivity matrix that identifies a linear representation of a Petri net. The sub-circuits that span a reaction network are subject to flux conservation laws. The conservation laws correspond to algebraic-combinatorial dual invariants, that are called S-(state) and T-(transition) invariants. Each invariant has an associated minimum support. We show that every minimum support of a Petri net invariant defines a unique signed sub-circuit representation. We prove that the family of signed sub-circuits has an implicit order that defines an oriented matroid. The oriented matroid is then used to identify the feasible sub-circuit pathways that span the biochemical network as the positive cycles in a hyper-digraph.     

Splitting and biopsy for bovine embryo sexing under field conditions.

Improvements on embryo micromanipulation techniques led to the use of embryo bisection technology in commercial embryo transfer programs, and made possible the direct genetic analysis of preimplantation bovine embryos by biopsy. For example, aspiration and microsection, allow bovine embryos sexing by detection of male-specific Y-chromosome in a sample of embryonic cells. We report on the application of the methodologies of splitting and biopsy of bovine embryos in field conditions, and on the results of embryo sex determination by the polymerase chain reaction (PCR). Pregnancy rates achieved with fresh bisected or biopsied embryos (50 to 60%) were similar to the fresh intact embryos (55 to 61%). The PCR protocol used for embryo sexing showed 92% to 94% of efficiency and 90 to 100% of accuracy. These results demonstrate these procedures are suitable for use in field conditions.