Expression stability of commonly used reference genes in canine articular connective tissues

Background

Pain of mild to moderate grade is difficult to detect in laboratory mice because mice are prey animals that attempt to elude predators or man by hiding signs of weakness, injury or pain. In this study, we investigated the use of telemetry to identify indicators of mild-to-moderate post-laparotomy pain.

Results

Adult mice were subjected to laparotomy, either combined with pain treatment (carprofen or flunixin, 5 mg/kg s/c bid, for 1 day) or without pain relief. Controls received anesthesia and analgesics or vehicle only. Telemetrically measured locomotor activity was undisturbed in all animals, thus confirming that any pain experienced was of the intended mild level. No symptoms of pain were registered in any of the groups by scoring the animals' outer appearance or spontaneous and provoked behavior. In contrast, the group receiving no analgesic treatment after laparotomy demonstrated significant changes in telemetry electrocardiogram recordings: increased heart rate and decreased heart rate variability parameters pointed to sympathetic activation and pain lasting for 24 hours. In addition, core body temperature was elevated. Body weight and food intake were reduced for 3 and 2 days, respectively. Moreover, unstructured cage territory and destroyed nests appeared for 1–2 days in an increased number of animals in this group only. In controls these parameters were not affected.

Conclusion

In conclusion, real-time telemetric recordings of heart rate and heart rate variability were indicative of mild-to-moderate post-laparotomy pain and could define its duration in our mouse model. This level of pain cannot easily be detected by direct observation.     

GQ-16, a Novel Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand, Promotes Insulin Sensitization without Weight Gain

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.     

Stereoselective synthesis of β-arabino glycosyl sulfones as potential inhibitors of mycobacterial cell wall biosynthesis

A series of β-arabino glycosyl sulfones with varying alkyl chain lengths were synthesised in a stereoselective fashion as putative mimics of decaprenolphosphoarabinose (DPA), and as potential inhibitors of mycobacterial cell wall biosynthesis. Biological testing against Mycobacterium bovis BCG revealed low to moderate anti-mycobacterial activity with marked dependence on alkyl chain length, which was maximal for a C-12 chain.     

Periplasmic Domains of Pseudomonas aeruginosa PilN and PilO Form a Stable Heterodimeric Complex

Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 Å resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.     

Effects of aerosolized dust in goats on lung clearance of Pasteurella and Mannheimia species

The objective was to determine whether the inhalation of large quantities of feedyard dust predisposed the animals to pulmonary bacterial proliferation. Two control groups, C1 and C2, did not receive dust treatments, and two principal groups (P1 and P2) received a total of 14 dust treatments each. The C1 and P1 groups of goats each received a transthoracic challenge of live Mannheimia haemolytica (4 x 10(6) colony forming units, CFU) The C2 and P2 groups of goats each received a transthoracic challenge of live Pasteurella multocida (1.0 x 10(6) CFU/goat). The results showed that dusted animals had fever when compared with non-dusted controls. In addition, dusted animals demonstrated a leukocytosis with neutrophilia after the first dust treatment that was not sustainable. Finally, dusted animals demonstrated pulmonary clearance of two potential bacterial pathogens that was not significantly different from that shown by control (not dusted) animals.     

Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation

Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of β-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis.     

Host-Pathogen Interactions: XII. Response of Suspension-cultured Soybean Cells to the Elicitor Isolated from Phytophthora megasperma var. sojae, a Fungal Pathogen of Soybeans

The glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae, the fungus which causes stem and root rot in soybeans, stimulates the activity of phenylalanine ammonia-lyase and the accumulation of glyceollin in suspension-cultured soybean cells. Nigeran, a commercially available fungal wall glucan, was the only other compound tested which has any activity in this system. Glyceollin is a phenylpropanoid-derived phytoalexin which is toxic to P. megasperma var. sojae. Evidence is presented to support the hypothesis that the action of elicitors in stimulating phytoalexin synthesis is not species or variety specific but, rather, is part of a general defensive response of plants.