Ranavirus could facilitate local extinction of rare amphibian species

Oecologia - There is growing evidence that pathogens play a role in population declines and species extinctions. For small populations, disease-induced extinction may be especially probable. We...     

Aeromonas neutral protease: Specificity toward extended substrates

Kinetic measurements on the action of Aeromonas neutral protease toward blocked peptide substrates were made in order to determine the most favorable fit on the enzyme subsites that bind the residues flanking the scissile bond and to define the number of secondary sites involved in catalysis. Variations in the identity of P₁′,³ the residue furnishing the amino group to the scissile bond, produced significant changes in k_rmcat, whereas the identity of P₁′, the residue donating the carboxyl group, was of much less catalytic importance. Comparison of these results with those of previous investigators of other bacterial neutral proteases indicated distinct differences in specificity of the Aeromonas enzyme and revealed that phenylalanyl residues, rather than leucyl, were preferred in the P₁′ position. Additional binding sites on the carboxyl side of the scissile bond were shown to be important to catalytic efficiency and it is evident that at least three residues (P₁t$\text{,}\text{?}$ P₂′, P₃′) are involved while only two residues (P₂, P₁) on the amino terminal side of the sensitive bond are implicated.     

Preparative labeling of proteins with [35S]methionine.

The most common technique for preparative labeling of proteins with radioisotopes for experimental purposes utilizes 125I. This isotope has certain limitations, including the emission of gamma- and X-irradiation, the release of gaseous 125I2 from solutions of Na 125I, and the potential for concentration of 125I in thyroid glands. We have discovered a means for labeling proteins rapidly and simply with [35S]methionine. The technique is applicable to a wide variety of proteins. Antibodies labeled by our technique remain functional.     

Highly efficient photoaffinity labeling of the hormone binding domain of atrial natriuretic factor receptor.

A high-efficiency photoaffinity derivative of atrial natriuretic factor (ANF) was developed for studying the peptide binding domain of the receptor protein and for better characterization of this receptor in tissues with a low density of binding sites. The position of the photosensitive residue was chosen on the basis of a molecular conformational model and on structure-activity relationship studies which both indicate that the carboxy-terminal end of the peptide is part of a hydrophobic pole likely to interact deeply within the ANF binding pocket of the receptor. Selection of the photoreactive residue p-benzoylphenylalanine (BPA) as a substitute for arginine in position 125 of the peptide sequence led to a photoaffinity derivative with a high (63%) efficiency of covalent incorporation to the receptor protein. This derivative (BPA-ANF) has a 10-fold lower affinity when compared with ANF, but it is a full agonist in stimulating cGMP production and inhibiting aldosterone secretion in bovine adrenal zona glomerulosa. Photoaffinity labeling with BPA-ANF specifically identifies ANF-R1 and ANF-R2 receptor proteins with a 10-fold higher efficiency than with azido derivatives of ANF or with cross-linking agents. This new ANF derivative therefore appears to be useful for studying ANF receptors in tissues with low levels of expression, for locating receptor following cellular internalization, and for tagging proteolytic fragments of the receptor amenable to amino acid microsequencing.     

Synthesis and biological actions of melanin concentrating hormone

A melanin (melanosome) concentrating hormone, MCH, was synthesized and the methodology for its synthesis is detailed. This heptadecapeptide, H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH , stimulated melanosome concentration (centripetal aggregation) within melanophores of all species of teleost fishes studied. Melanosome aggregation in response to MCH was not blocked by Dibenamine as was the response to norepinephrine (NE), demonstrating that melanosome aggregating responses to MCH and NE are mediated through separate receptors. Melanosome aggregation in response to MCH was reversed by an equimolar concentration of alpha-melanocyte stimulating hormone (alpha-MSH). In contrast, MCH stimulated melanosome dispersion (centrifugal movement) within melanophores of a frog (Rana pipiens) and a lizard (Anolis carolinensis). Therefore, MCH exhibits both melanosome concentrating and dispersing actions depending upon the species studied.     

Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.     

Transforming growth factor beta activation of c-Abl is independent of receptor internalization and regulated by phosphatidylinositol 3-kinase and PAK2 in mesenchymal cultures

Transforming growth factor beta (TGF-beta) modulates a number of cellular phenotypes as divergent as growth stimulation and growth inhibition. Although the Smad pathway is critical for many of these responses, recent evidence indicates that Smad-independent pathways may also have a critical role. One such protein previously shown to regulate TGF-beta action independent of the Smad proteins is the c-Abl nonreceptor tyrosine kinase. In the current study we determined that TGF-beta receptor signaling activates c-Abl kinase activity in a subset of fibroblast but not epithelial cultures. This cell type-specific response occurs in a membrane-proximal locale independent of receptor internalization and upstream of dynamin action. Although c-Abl activation by TGF-beta is independent of Smad2 or Smad3, it is prevented by inhibitors of phosphatidylinositol 3-kinase or PAK2. Thus, c-Abl represents a target downstream of phosphatidylinositol 3-kinase-activated PAK2, which differentiates TGF-beta signaling in fibroblasts and epithelial cell lines and integrates serine/threonine receptor kinases with tyrosine kinase pathways.     

Distribution and Diversity of Escherichia coli Populations in the South Nation River Drainage Basin,Eastern Ontario,Canada

We investigated the prevalence and diversity of Escherichia coli strains isolated from surface waters from multiple watersheds within the South Nation River basin in eastern Ontario, Canada. The basin is composed of mixed but primarily agricultural land uses. From March 2004 to November 2007, a total of 2,004 surface water samples were collected from 24 sampling sites. E. coli densities ranged from undetectable to 1.64 × 10⁵ CFU 100 ml⁻¹ and were correlated with stream order and proximity to livestock production systems. The diversity of 21,307 E. coli isolates was characterized using repetitive extragenic palindromic PCR (rep-PCR), allowing for the identification of as many as 7,325 distinct genotypes, without capturing all of the diversity. The community was temporally and spatially dominated by a few dominant genotypes (clusters of more than 500 isolates) and several genotypes of intermediary abundance (clustering between 10 and 499 isolates). Simpson diversity indices, assessed on a normalized number of isolates per sample, ranged from 0.050 to 0.668. Simpson indices could be statistically discriminated on the basis of year and stream order, but land use, discharge, weather, and water physical-chemical properties were not statistically important discriminators. The detection of Campylobacter species was associated with statistically lower Simpson indices (greater diversity; P < 0.05). Waterborne E. coli isolates from genotypes of dominant and intermediary abundance were clustered with isolates obtained from fecal samples collected in the study area over the same period, and 90% of the isolates tested proved to share genotypes with fecal isolates. Overall, our data indicated that the densities and distribution of E. coli in these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne E. coli populations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized E. coli strains.Escherichia coli is ubiquitously distributed in fecal material from humans and warm-blooded animals (38). The detection of E. coli in water is an implicit indicator of recent fecal contamination and therefore of the risk of cooccurrence of enteric pathogens that can cause illness in susceptible populations (62). Many jurisdictions evaluate and mandate compliance with drinking and recreational water quality standards on the basis of the presence and abundance of E. coli (14, 44). For example, Canadian recreational water quality standards stipulate that E. coli densities in excess of a geometric mean of 200 CFU per 100 ml indicate that the water is unsuitable for swimming and bathing (23).In a background of increasing occurrence of microbial contamination of surface water, a variety of methods for elucidating the sources of fecal contamination have been developed, and these microbial source tracking (MST) methods are recommended components of fecal pollution abatement strategies (16, 57). So-called library-dependent MST methods compare environmental isolates to collections of isolates obtained from likely sources of fecal pollution in the area of investigation. The host source is distinguished on the basis of the similarity of environmental isolates to reference fecal isolates. Comparison can be undertaken on the basis of genomic fingerprinting methods, including repetitive extragenic palindromic PCR (rep-PCR), ribotyping, or pulsed-field gel electrophoresis (PFGE) (13, 17, 31, 54, 57). A variety of studies using these methods have revealed enormous diversity in the fecal and environmental E. coli populations. For example, 461 distinct PFGE genotypes and 175 distinct enterobacterial repetitive intergenic consensus (ERIC)-PCR genotypes were detected in a collection of 555 E. coli strains isolated from river water in Texas (10). As many as 291 and 94 rep-PCR genotypes were distinguished in collections of 643 river isolates and 353 beach water E. coli isolates, respectively (43). Significant diversity was also revealed using multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) on 185 E. coli isolates from freshwater beaches, where an average of 40 alleles per locus were detected (59). Almost 60% of 657 E. coli isolates in a fecal reference collection had unique (i.e., detected in only one individual) fingerprints determined by rep-PCR (32). Extensive diversity of E. coli was also observed in soils in temperate climates, where the growth and persistence of “naturalized” populations without any known fecal input have been found (7, 28, 30). Naturalized populations have been dominated by the B1 phylogroup and may have adapted in ways that enhance their survival in temperate secondary habitats (59). The temporal and spatial diversity of E. coli may not be a significant factor in coarse-source (e.g., human versus animal) classification of E. coli by means of ribotyping procedures (48). Ultimately, the characterization and understanding of the diversity of populations of selected microorganisms in surface watercourses affected by multiple sources of fecal pollution (as in agricultural watershed settings, for example) may be more critical for assessing the specific impacts of contamination-mitigating measures than previously thought. For instance, restricting the access of cattle on pasture to adjacent water by implementing vegetative buffering along watercourses creates habitat for varied wildlife, which then contribute to fecal pollution. In this context, the diversity in populations of indicator bacteria could be useful for better understanding how changes in landscape use influence fecal source inputs.As part of a research program evaluating the impact of agriculture on water quality and the efficacy of better agricultural management practices to mitigate agricultural pollution, we have conducted a multiyear study of the microbiological water quality for a suite of different-sized watersheds in the South Nation River basin in eastern Ontario, Canada (41, 46, 61). Land use in this river basin is mixed, consisting primarily of agricultural activities, light urban development, and interspersed wildlife habitat. Surface water systems in the study region differ widely in their contributing areas and therefore in their discharges (61).In the work undertaken here, we sought to determine the spatial and seasonal variability in the density and the structure of populations of E. coli in surface waters within the South Nation River basin. The specific objectives of the study were (i) to characterize the seasonal distribution and abundance of E. coli in different watershed settings within the river basin, (ii) to evaluate the spatial distribution of E. coli densities and diversity with respect to upstream land use activities, (iii) to use rep-PCR to elucidate the dominant E. coli genotypes and the diversity of E. coli populations and to explore linkages to pathogen presence, season, and environmental and land use variables, and (iv) using rep-PCR, to evaluate the concordance between waterborne isolates and fecal isolates obtained from within the study area. The study is distinguished by an intensive 4-year sampling of numerous (n = 24) sites that differed in their stream order and proximal land use activity; the number of E. coli isolates (≈21,000) included in the analysis; and the use of two distinct rep-PCR fingerprinting methods (ERIC and BOXA1R) to characterize the isolates. Furthermore, we used classification and Regression Tree (CART) analysis to evaluate relationships between the abundance and diversity of E. coli in water samples and environmental and land use variables.