An in vitro screening assay based on synthetic prion protein peptides for identification of fibril-interfering compounds

Transmissible spongiform encephalopathies are neurodegenerative diseases and are considered to be caused by malformed prion proteins accumulated into fibrillar structures that can then aggregate to form larger deposits or amyloid plaques. The identification of fibril-interfering compounds is of therapeutic and prophylactic interest. A robust and easy-to-perform, high-throughput, in vitro fluorescence assay was developed for the detection of such compounds. The assay was based on staining with the fluorescent probe thioflavin S in polystyrene microtiter plates to determine the amyloid state of synthetic peptides, representing a putative transmembrane domain of human and mouse prion protein. In determining optimal test conditions, it was found that drying peptides from phosphate buffer prior to staining resulted in good reproducibility with an interassay variation coefficient of 8%. Effects of thioflavin S concentration and staining time were established. At optimal thioflavin S concentration of 0.2mg/ml, the fluorescence signals of thioflavin S with five different prion protein-based fibrillogenic peptides, as well as peptide Abeta((1-42)), were found to show a peptide-dependent linear correlation within a peptide concentration range of 10-400 microM. The ability of the assay to identify compounds that interfere with fibril formation and/or dissociate preformed fibrils was demonstrated for tetracyclic compounds by preceding coincubation with human prion protein peptide huPrP106-126.     

Oral bacteria resistant to mercury and to antibiotics are present in children with no previous exposure to amalgam restorative materials

Dental plaque from 76 children without amalgam restorations was screened for bacteria resistant to mercuric chloride. Seventy-one per cent of the children harboured mercury-resistant oral bacteria and the median percentage of the total oral microflora resistant to mercuric chloride was 0.007% (range 0-5.3%). Eighty-seven mercury-resistant bacteria were isolated and 86% of these were streptococci with Streptococcus mitis predominating. Sixty per cent of the mercury-resistant isolates were also resistant to at least one of the four antibiotics tested (penicillin, ampicillin, erythromycin and tetracycline) with resistance to tetracycline (40% of isolates) most frequently encountered.     

Is there a correlation between taurine levels and xenobioticinduced perturbations in protein synthesis?: A study with tetracycline in rats

Summary Changes in urinary levels of taurine have been reported in rats following treatment with various xenobiotics including those which alter protein synthesis and/or are hepatotoxic. This paper reports on the time course of the urinary elevation of taurine following treatment of rats with tetracycline (50, 150 and 200mg.kg^-1). Maximum taurine excretion occurred 8–12h following dosing. Serum albumin and total protein were significantly lower after 24h (200mg.kg^-1). The increase in urinary taurine was dose-related and reflected in the raised serum levels of taurine 24h after dosing. Serum and urinary protein and [³H]-leucine incorporation into acid precipitable protein in liver and muscle were reduced by tetracycline (100, 150 and 200mg.kg^-1) 10h after dosing. The reduction in protein synthesis was correlated with increased urinary and serum levels of taurine at 10h. The use of taurine as a non-invasive marker of protein synthesis is discussed.     

Detection of high-level tetracycline resistance in clinical isolates of Helicobacter pylori using PCR-RFLP

Tetracycline is one of four antibiotics commonly used for the treatment of Helicobacter pylori infection, but its effectiveness is decreasing as the incidence of tetracycline resistance is increasing. In five Brazilian tetracycline-resistant (Tet(R)) H. pylori isolates, high-level tetracycline resistance is mediated by the triple-base-pair substitution AGA(926-928)-->TTC in both 16S rRNA genes, as was previously observed in two independent high-level Tet(R) H. pylori strains. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay was developed for the detection of the AGA(926-928)-->TTC substitution, and confirmed the presence of the aforementioned triple-base-pair substitution in all five Brazilian Tet(R) isolates. This PCR-RFLP-based approach distinguishes the high-level Tet(R) isolates from low-level Tet(R) and Tet(S) H. pylori strains and thus allows the direct detection of Tet(R) H. pylori isolates.     

Chromosomal integration of the green fluorescent protein gene in lactic acid bacteria and the survival of marked strains in human gut simulations

An integration vector was constructed to allow introduction of the gfp gene into the chromosomes of Gram-positive bacteria. Integration depends on homologous recombination between a short 458-nt sequence of the tet(M) gene in the vector and a copy of Tn916 in the host chromosome. Strains of Lactococcus lactis IL1403, Enterococcus faecalis JH2-SS, and Streptococcus gordonii DL1 stably marked with single chromosomal copies of the gfp were readily visualised by epifluorescence microscopy. The marked L. lactis strain survived poorly in a continuous culture system inoculated with human faecal flora, while the laboratory E. faecalis strain was lost at approximately the dilution rate of the fermenter.     

Characterization of transferable tetracycline resistance genes in Enterococcus faecalis isolated from raw food

The prevalence of tetracycline resistance, and of specific genetic determinants for this resistance was investigated in 1003 strains of Enterococcus faecalis isolated from various raw food products originating from five categories including chicken meat, other poultry meat, beef, pork, and 'other'. For the 238 resistant isolates identified, the ability to transfer the resistant phenotype to a given recipient in vitro was investigated. New and interesting observations were that the tet(L) resistance determinant was more readily transferred than tet(M), and that the presence of Tn916-like elements known to encode tet(M) did not correlate with increased transferability of the resistant phenotype.     

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C. Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria.     

Adaptable doxycycline-regulated gene expression systems for Drosophila

We have engineered two new versions of the doxycycline (dox) inducible system for use in Drosophila. In the first system, we have used the ubiquitously expressed Drosophila actin5C promoter to express the Tet-Off transactivator (tTA) in all tissue. Induction of a luciferase target transgene begins 6 h after placing the flies on dox-free food. Feeding drug-free food to mothers results in universal target gene expression in their embryos. Larvae raised on regular food also show robust expression of a target reporter gene. In the second version, we have used the Gal4-UAS system to spatially limit expression of the transactivator. Dox withdrawal results in temporally- and spatially-restricted, inducible expression of luciferase in the adult head and embryo. Both the actin5C and Gal4-UAS versions produce more than 100-fold induction of luciferase in the adult, with virtually no leaky expression in the presence of drug. Reporter gene expression is also undetectable in larvae or embryos from mothers fed dox-containing food. Such tight control may be due to the incorporation of Drosophila insulator elements (SCS and SCS′) into the transgenic vectors. These systems offer a practical, effective alternative to currently available expression systems in the Drosophila research community.