Cadaver-detection dogs (also known as human remains detection dogs) are used worldwide to locate deceased victims and human remains. Ethical restrictions often prevent the dog handlers from using cadavers as training aids, resulting in a reliance on pseudoscents or human tissues, such as blood, bone, and decomposition f luid. Often these aids must be re-used many times because of the diff iculty in obtaining new materials. The aim of this study was to investigate the dogs’ sensitivity to aged human decomposition f luid samples that are used as a training aid. Human decomposition f luid was collected and serially diluted to 1 part-per-trillion (10-12) and aged up to two years. The samples were presented throughout the aging process to three police accredited cadaver-detection dog teams under standard indoor training conditions. The dogs were capable of detecting the oldest and lowest dilution levels of decomposition f luid samples. Ongoing training to retain this level of sensitivity is recommended. The results of these trials indicate human decomposition f luid is a valid training aid for cadaver-detection dogs.

Pseudomonas aeruginosa LasB elastase gene (lasB) transcription depends on cell density-dependent quorum-sensing mechanisms of gene activation. Previously, we collected several non-mucoid P. aeruginosa veterinary isolates and showed that the total matrix protease phenotype was similar for isolates regardless of host and site of isolation. In contrast, isolates from chronic canine ear infections (otitis externa) were significantly more likely to exhibit less elastase activity as measured by elastin Congo red than from any other site [Clin. Diag. Lab. Immun. 8 (2001) 632]. In this study, we found that the elastase deficiency phenotype is stable upon passage in broth culture. Transcript amplification analyses indicated that the elastase deficiency appears to be strain-specific, with each isolate exhibiting a unique expression profile relative to strain PAO1. Although a number of strain-specific transcriptional differences were observed, the overall pattern that emerges is a quorum sensing deficiency among canine ear P. aeruginosa isolates.

ABSTRACTPseudomonas aeruginosaveterinary isolates were assayed for elastase and total matrix protease activity. The elastase activity of canine ear isolates was much less than that of strain PAO1 and that of all other veterinary isolates (P< 0.0001). The results indicate that canine ear isolates have a distinct elastase phenotype.

Chromobacterium violaceum was recovered at necropsy from the lungs, liver, spleen, and an interscapular abscess of a Chinese red panda (strain 98-9187) [J. Vet. Diagn. Invest. 12 (2000) 177]. As the lungs exhibited extensive, necrotizing lesions harboring bacterial aggregates, we sought to determine whether C. violaceum produced an elastase that might in part account for these lesions. The C. violaceum type strain (ATCC 12472(T)) and strain 98-9187 were shown to exhibit elastolytic activity by elastin Congo red and elastin nutrient agar assays. The activity was isolated from the periplasmic fraction and was present throughout the growth cycle. Activity increased markedly in late logarithmic phase growth. In elastin-limiting medium, activity rapidly decreased in early stationary phase indicating a tight regulation of yield. The activity was optimal at neutral pH and was sensitive to the metalloproteinase inhibitors EDTA and 1,10-phenanthroline. Activity was restored upon addition of zinc indicating the enzyme is a zinc metalloproteinase. A band corresponding to purified elastase activity was present at approximately 30kDa in a denaturing polyacrylamide gel.

ABSTRACT Pseudomonas aeruginosa LasB elastase gene ( lasB ) transcription is controlled by the two-component quorum-sensing system of LasR, and the autoinducer, 3OC 12 -HSL ( N -3-[oxododecanoyl]homoserine lactone). LasR and 3OC 12 -HSL-mediated lasB activation requires a functional operator sequence (OP1) in the lasB promoter region. Optimal activation of lasB , however, requires a second sequence of 70% identity to OP1, named OP2, located 43 bp upstream of OP1. In this study, we used sequence substitutions and insertion mutations in lasB p- lacZ fusion plasmids to explore the role of OP2 in lasB activation. Our results demonstrate that (i) OP1 and OP2 synergistically mediate lasB activation; (ii) OP2, like OP1, responds to LasR and 3OC 12 -HSL; and (iii) the putative autoinducer-binding domain of LasR is not required for synergistic activation from OP1 and OP2.

The enzyme elastase is an important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa. Previous studies have shown that expression of the P. aeruginosa elastase gene (lasB) requires both an activator protein, LasR, and an N-acylhomoserine lactone compound termed Pseudomonas autoinducer (PAI). In this study, we analyzed the lasB promoter region to learn more about lasB activation by LasR and PAI. We report that the lasB transcriptional start is located 141 nucleotides upstream from the lasB translational start. It was also discovered that the lasB promoter region contains two putative operator sequences (OP1 and OP2) that are similar to each other and the Vibrio fischeri lux operator. OP1 is located directly upstream from, and may overlap with, the lasB promoter region, and OP2 is centered 102 nucleotides upstream from the lasB transcriptional start site. To study the effects of these putative operators and other sequences upstream from the lasB transcriptional start site on lasB activation, a series of transcriptional lasBp-lacZ gene fusions was constructed. Data from these fusions indicate that both putative operators are involved in LasR- and PAI-mediated lasB activation, with OP1 being more important than OP2.

The role of the exonucleolytic activity of the calf 5' to 3' exo/endonuclease, a RAD2 homolog 1 (RTH-1) class nuclease, in lagging-strand DNA replication has been examined using model Okazaki fragment substrates. These substrates exemplify the situation in Okazaki fragment processing which occurs after the initiator RNA primer is cleaved off, and released intact, by calf RNase HI, leaving a single ribonucleotide at the 5' end of the RNA-DNA junction. This final RNA is then removed by the calf RTH-1 nuclease [Turchi et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9803-9807]. The cleavage specificity of calf RTH-1 nuclease for different junction ribonucleotides was compared. These were removed without the usual requirement of calf RTH-1 for an immediately adjacent upstream primer. In most cases, the presence of an upstream DNA or RNA primer, separated from the monoribonucleotide-DNA segment by either a nick or a gap, reduced the efficiency of removal of the monoribonucleotide compared to the removal seen with no upstream primer. Substrates in which the monoribonucleotide-DNA segment had been replaced by an oligomer of the same sequence but consisting entirely of DNA also exhibited upstream primer inhibition. Results with various sequences indicated that the upstream primer is generally inhibitory for ribonucleotide removal but is sometimes neutral. For deoxynucleotide removal it could be stimulatory, neutral, or inhibitory. Possible reasons for the unexpected lack of upstream primer dependence have been explored. The ratio of RNase HI to RTH-1 was also shown to be critical for both enzymes to work together efficiently. These results suggest that regions of upstream primer inhibition within the genome may play a role in determining the mechanism by which mammalian Okazaki fragments are processed.

Calf 5' to 3' exo/endonuclease, the counterpart of the human FEN-1 and yeast RTH-1 nucleases, performs structure-specific cleavage of both RNA and DNA and is implicated in Okazaki fragment processing and DNA repair, The substrate for endonuclease activity is a primer annealed to a template but with a 5' unannealed tail, The results presented here demonstrate that the nuclease must enter the 5' end of the unannealed tail and then slide to the region of hybridization where the cleavage occurs. The presence of bound protein or a primer at any point on the single-stranded tail prevents cleavage, However, biotinylation of a nucleotide at the 5' end or internal to the tail does not prevent cleavage. The sliding process is bidirectional, If the nuclease slides onto the tail, later binding of a primer to the tail traps the nuclease between the primer binding site and the cleavage site, preventing the nuclease from departing from the 5' end, A model for 5' entry, sliding, and cleavage is presented, The possible role of this unusual mechanism in Okazaki fragment processing, DNA repair, and protection of the replication fork from inappropriate endonudeolytic cleavage is presented.

Two methods of P. aeruginosa elastase purification are described: Method 1 involves concentration of sample supernatants, followed by DEAE-Sepharose liquid chromatography, whereas Method 2 involves initial fractionations followed by molecular sieving and hydrophobic interaction high-performance liquid chromatography. The choice of methods depends on the available equipment and supplies. The methods of assaying elastase activity described as useful for a variety of applications. The elastin-nutrient agar plate method is a qualitative assay to determine the presence of elastase activity produced by a given culture or colony. Use of the quantitative elastin-Congo red assay is appropriate for determining elastase activities of mid-to-high elastase-producing cultures. For more sensitive determinations of P. aeruginosa elastase activity, use of the fluorogenic substrate is advisable.

The lasA gene was the first of the Pseudomonas aeruginosa genes involved in proteolysis and elastolysis to be cloned and sequenced. Its function and significance have been studied by genetic approaches (D. S. Toder, M. J. Gambello, and B. H. Iglewski, Mol. Microbiol. 5:2003-2010, 1991) and by attempts to purify an active fragment of the protein (J. E. Peters and D. R. Galloway, J. Bacteriol. 172:2236-2240, 1990). To further study LasA in vivo, we have constructed and characterized an insertional mutant in the lasA gene in strain PAO1 (PAO-A1) and in the lasB insertional mutant, PAO-B1. Analysis of these isogenic strains demonstrates that the lasA lesion diminished elastolysis more than proteolysis and that LasA is required for staphylolytic activity. Despite previous suggestions that lasB elastase cleaves the LasA protein, the size of the LasA protein was the same whether or not lasB elastase was present. Expression of lasA in a lasR-negative mutant, PAO-R1, demonstrated that the LasA protein is produced in an active form in the absence of (lasB) elastase or alkaline protease and is itself a protease with elastolytic activity. We also observed that PAO-A1 was closer to the parental phenotype, with respect to elastolytic and proteolytic activities, than the previously characterized, chemically induced lasA mutant PAO-E64. Quantification of promoter activity with lasA::lacZ and lasB::lacZ fusions suggests that PAO-E64 harbors a mutation in a gene which regulates expression of both lasA and lasB.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes a variety of infections in immunocompromised hosts and individuals with cystic fibrosis. Expression of elastase, one of the virulence factors produced by this organism, requires the transcriptional activator LasR. Experiments with gene fusions show that gene lasl is essential for high expression of elastase. The lasl gene is involved in the synthesis of a diffusible molecule termed Pseudomonas autoinducer (PAI). PAI provides P. aeruginosa with a means of cell-to-cell communication that is required for the expression of virulence genes and may provide a target for therapeutic approaches.

Transcriptional patterns of lasB and algD were compared in isogenic mucoid and non-mucoid Pseudomonas aeruginosa isolates from cystic fibrosis patients. The lasB gene encodes elastase, a major proteolytic enzyme secreted by P. aeruginosa, while algD is required for the synthesis of alginate, an exopolysaccharide frequently overproduced by strains infecting cystic fibrosis patients. A possible coregulation at the transcriptional level of these major virulence determinants was analysed. The lasB and algD genes showed inverse levels of promoter activity. The lasB promoter was active in non-mucoid cells and inactive in mucoid cells (in four out of five tested pairs), while the algD promoter was active in mucoid cells and silent in non-mucoid cells in all cases. When PAO568, a model strain for the analysis of control of the alginate system, was grown under conditions promoting mucoidy, the algD promoter was activated, whereas lasB mRNA could not be detected. This effect was reversed when the cells were grown in a medium suppressing mucoidy. Insertional inactivation of algR, a member of the signal-transduction systems regulating algD transcription, although abolishing algD expression and rendering cells non-mucoid, did not alter the nature of the induction and repression patterns of lasB seen in the parental strain PAO568. These results suggest that the lasB gene and the alginate system are co-ordinately regulated at a level parallel to or above the algR gene.

A murine IgG2a monoclonal antibody, termed 6-19, was characterized in terms of its ability to bind to human cell lines and tissues. The hybridoma was selected for antibody binding to multiple human neuroblastoma cultured cell lines but not to peripheral blood mononuclear cells. 6-19 binds to the cell surface of all cultured human nonhematopoietic tumor cell lines tested, to cultured human fibroblasts and endothelial cells, and to nonhematopoietic tumors of many types. It does not bind detectably to any hematopoietic cells, leukemia cells, or lymphomas. In the presence of complement, 6-19 is very cytotoxic to cultured human neuroblastoma cells but not to bone marrow granulocyte-macrophage colony-forming cells. The 6-19 monoclonal antibody may prove useful in the identification or destruction of tumor and stromal cells in bone marrow.

In order to identify common mechanisms of action by which both the platelet-derived growth factor (PDGF) and the tumor promoter tetradecanoyl phorbol acetate (TPA) initiate cell growth, the effects of PDGF and TPA on phosphorylation of cellular proteins were examined in density-inhibited Balb/c-3T3 cells. Cultures were incubated with 32Pi and growth factor, and 32P-labeled cellular proteins were examined after separation by SDS-polyacrylamide gel electrophoresis and autoradiography. TPA and PDGF each induced phosphorylation of a major cytosol protein of approximately 75,000 molecular weight (pp75). Phosphorylation of this protein was not induced by either epidermal growth factor or insulin, neither of which initiate 3T3 cell growth but enhance growth later in the 3T3 cell cycle. pp75 was a single band under reduced and non-reduced conditions, and a single spot was seen on two-dimensional gels. Phosphorylation did not occur at 4 degrees C. Phosphorylation of the protein was observed within 3 min and reached a maximum in 10-30 min. Submitogenic doses of TPA and PDGF induced submaximal phosphorylation. The phosphoprotein was labeled only on serine. Cell free phosphorylation of pp75 occurred at 4 degrees C in the presence of Mg++ and Ca2+. Homogenates from cultures pretreated with TPA phosphorylated pp75 in the presence or absence of Ca2+. Phosphorylation of this protein may possibly be related to activation of the Ca2+-dependent, phospholipid sensitive protein kinase C.