Deficits in spontaneous burrowing behavior in the rat bilateral monosodium iodoacetate model of osteoarthritis: an objective measure of pain-related behavior and analgesic efficacy.

OBJECTIVE: To characterize deficits in burrowing behavior - an ethologically-relevant rodent behavior - in the monosodium iodoacetate (MIA) rat model of osteoarthritis (OA), and the sensitivity of these deficits to reversal by analgesic drugs of both prototypical and novel mechanisms of action. A second objective was to compare the burrowing assay to a spontaneous locomotor activity (sLA) assay. METHOD: Male Wistar Han rats (200-220 g) received intrarticular (i.a.) injections of MIA or saline for sham animals. A deficit in the amount of sand burrowed from steel tubes filled with 2.5 kg of sand was used as a measure of pain-related behavior, and sensitivity to reversal of these deficits by analgesic drugs was assessed in bilaterally MIA-injected rats. RESULTS: Bilateral MIA injections induced a significant impairment of burrowing behavior, which was concentration-dependent. The temporal pattern of the deficits was biphasic: a large deficit at 3 days post-injection, resolving by day 14 and returning at the 21 and 28 day time points. At the 3 day time point ibuprofen, celecoxib and an anti-nerve growth factor (NGF) monoclonal antibody (mAb) were able to significantly reinstate burrowing behavior, whereas the fatty acid amide hydrolase (FAAH) inhibitor PF-04457845 and morphine displayed no reversal effect. Morphine impaired burrowing behavior at 3 mg/kg in sham animals. Deficits in rearing frequency in the locomotor activity assay proved irreversible by analgesics. CONCLUSION: Burrowing behavior provides an objective, non-reflexive read-out for pain-related behavior in the MIA model that has predictive validity in detecting analgesic efficacy of nonsteroidal anti-inflammatory drugs (NSAIDs) and an anti-NGF mAb.

Tularemia in deer mice (peromyscus maniculatus) during a population irruption in Saskatchewan, Canada.

Type B tularemia caused by Francisella tularensis subsp. holarctica was diagnosed in deer mice (Peromyscus maniculatus) found dead at four sites in west-central Saskatchewan during April and May 2005. The occurrence of tularemia coincided with a decline in the number of deer mice in part of a large area (>22000 km(2) ) in which deer mice had been extremely abundant during the autumn of 2004 and spring of 2005, and in which mice caused damage to crops in the autumn of 2004. This is apparently the first report of tularemia as a cause of death of wild deer mice. The bacterium isolated from deer mice was atypical in that cysteine was not required in the media used for isolation. Three isolates tested were genotypes not previously identified in Canada. There were no reports of human disease in the area.

Flagella as a potential marker for Campylobacter jejuni strains associated with Guillain-Barré syndrome.

Campylobacter jejuni recovered from patients with Guillain-Barré syndrome (GBS) in different geographical locations and bearing different heat-labile and heat-stable antigens were found to have identical amino acid sequences in their flagellar flaA short variable region, suggesting that it may be a potentially useful marker for GBS association.

Metronidazole activation is mutagenic and causes DNA fragmentation in Helicobacter pylori and in Escherichia coli containing a cloned H. pylori RdxA(+) (Nitroreductase) gene.

Much of the normal high sensitivity of wild-type Helicobacter pylori to metronidazole (Mtz) depends on rdxA (HP0954), a gene encoding a novel nitroreductase that catalyzes the conversion of Mtz from a harmless prodrug to a bactericidal agent. Here we report that levels of Mtz that partially inhibit growth stimulate forward mutation to rifampin resistance in rdxA(+) (Mtz(s)) and also in rdxA (Mtz(r)) H. pylori strains, and that expression of rdxA in Escherichia coli results in equivalent Mtz-induced mutation. A reversion test using defined lac tester strains of E. coli carrying rdxA(+) indicated that CG-to-GC transversions and AT-to-GC transitions are induced more frequently than other base substitutions. Alkaline gel electrophoretic tests showed that Mtz concentrations near or higher than the MIC for growth also caused DNA breakage in H. pylori and in E. coli carrying rdxA(+), suggesting that this damage may account for most of the bactericidal action of Mtz. Coculture of Mtz(s) H. pylori with E. coli (highly resistant to Mtz) in the presence of Mtz did not stimulate forward mutation in E. coli, indicating that the mutagenic and bactericidal products of Mtz metabolism do not diffuse significantly to neighboring (bystander) cells. Our results suggest that the widespread use of Mtz against other pathogens in people chronically infected with H. pylori may stimulate mutation and recombination in H. pylori, thereby speeding host-specific adaptation, the evolution of virulence, and the emergence of resistance against Mtz and other clinically useful antimicrobials.

Helicobacter pylori with separate beta- and beta'-subunits of RNA polymerase is viable and can colonize conventional mice.

The genes encoding the beta- and beta'-subunits of RNA polymerase (rpoB and rpoC respectively) are fused as one continuous open reading frame in Helicobacter pylori and in other members of this genus, but are separate in other bacterial taxonomic groups, including the closely related genus Campylobacter. To test whether this beta-beta' tethering is essential, we used polymerase chain reaction-based cloning to separate the rpoB and rpoC moieties of the H. pylori rpoB-rpoC fusion gene with a non-polar chloramphenicol resistance cassette containing a new translational start, and introduced this construct into H. pylori by electro-transformation. H. pylori containing these separated rpoB and rpoC genes in place of the native fusion gene produced non-tethered beta and beta' RNAP subunits, grew well in culture and colonized and proliferated well in conventional C57BL/6 mice. Thus, the extraordinary beta-beta' tethering is not essential for H. pylori viability and gastric colonization.

Antimicrobial activities of synthetic bismuth compounds against Clostridium difficile.

Clostridium difficile is a major nosocomial pathogen responsible for pseudomembranous colitis and many cases of antibiotic-associated diarrhea. Because of potential relapse of disease with current antimicrobial therapy protocols, there is a need for additional and/or alternative antimicrobial agents for the treatment of disease caused by C. difficile. We have synthesized a systematic series of 14 structurally simple bismuth compounds and assessed their biological activities against C. difficile and four other gastrointestinal species, including Helicobacter pylori. Here, we report on the activities of six compounds that exhibit antibacterial activities against C. difficile, and some of the compounds have MICs of less than 1 microgram/ml. Also tested, for comparison, were the activities of bismuth subcitrate and ranitidine bismuth citrate obtained from commercial sources. C. difficile and H. pylori were more sensitive both to the synthetic bismuth compounds and to the commercial products than were Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis, and the last three species were markedly resistant to the commercial bismuth salts. Testing with human foreskin fibroblast cells revealed that some of the synthetic compounds were more cytotoxic than others. Killing curves for C. difficile treated with the more active compounds revealed rapid death, and electron microscopy showed that the bismuth of these compounds was rapidly incorporated by C. difficile. Energy dispersive spectroscopy X-ray microanalysis of C. difficile cells containing electron-dense material confirmed the presence of internalized bismuth. Internalized bismuth was not observed in C. difficile treated with synthetic bismuth compounds that lacked antimicrobial activity, which suggests that the uptake of the metal is required for killing activity. The nature of the carrier would seem to determine whether bismuth is transported into susceptible bacteria like C. difficile.

ROn-1 SINEs: a tRNA-derived, short interspersed repetitive DNA element from Oreochromis niloticus and its species-specific distribution in Old World cichlid fishes.

A SINE-like repetitive element (ROn-1) has been cloned from the tilapiine cichlid fish Oreochromis niloticus. The element is 345 base pairs (bp) in length and consists of a transfer-RNA-like domain with putative RNA polymerase III recognition sequences, a tRNA-unrelated region, and a poly(A) tail. Approximately 6000 copies of ROn-1 occur in the haploid genome of O. niloticus. Southern blot analysis revealed that ROn-1 is an abundant element in the genomes of many African cichlid fishes, but absent from the genome of the Indian cichlid Etroplus.

Tandem repeat polymorphism and heteroplasmy in the mitochondrial control region of redfishes (Sebastes: Scorpaenidae).

Three species of redfish (Sebastes) share a common pattern of mitochondrial DNA tandem repeat polymorphism and heteroplasmy in the northwest Atlantic Ocean. All three species exhibit 9-17 copies of an approximately 275 base pair (bp) tandem repeat situated within the 3' domain of the control region. Sequence analysis of cloned mtDNA from S. mentella revealed that the tandem array is adjacent to the tRNA(phe) gene, and that the repeat shares 53% identity with the tRNA(phe) gene and part of the 12S rRNA gene. These features, as well as potential secondary structure assumed by the repeat, are consistent with previously proposed models explaining tandem duplications in the 3' end of the control region. In a sample comprising 36 S. fasciatus, 52 S. mentella, and 13 S. marinus taken near Newfoundland, neither the mean number of repeats per fish (12.2-12.7) nor the frequency of heteroplasmy varied significantly among species. A total of 42% of the redfishes were heteroplasmic, bearing either two or three repeat variants (33% and 9%, respectively). The similarity of the frequency distributions of tandem repeat variants in the three species suggests either a common balance between mutation and selection in the three species, or mitochondrial gene flow between them.