Inhibition of mural thrombus formation by novel nipecotoylpiperazine antiplatelet agents

The effectiveness of two closely related nipecotoylpiperazine derivatives, BPAT-143 and BPAT-117, as antiplatelet agents was measured by their ability to inhibit the accumulation of human blood platelets on collagen-coated (type 1) glass in a parallel plate flow chamber. Whole human blood, with fluorescently labeled platelets, was perfused through the flow chamber, and epi-fluorescent video microscopy was used to visualize the dynamics of individual platelet adhesion and thrombus formation on the collagen-coated surface. Digital image processing was used to analyze the dynamics of thrombus growth on the surfaces. The collagen-coated surface serves as a model for the damaged blood vessel wall, as collagen is a primary component of the matrix beneath endothelial cells. At a concentration of 50 microM, BPAT-117 (the considerably more hydrophobic molecule) inhibited platelet accumulation by striking 90 +/- 2% (+/- S.E.), while it took 2- to 4-fold higher concentrations of BPAT-143 to register meaningful to comparable effects (52 +/- 6% and 80 +/- 4%, respectively). This further corroborates the substantial impact of hydrophobic features within the matrix of appropriately structured molecules on their ability to alter platelet function.     

Resolution of the flavocytochrome p-cresol methylhydroxylase into subunits and reconstitution of the enzyme

An improved procedure is described for the isolation of the flavocytochrome p-cresol methylhydroxylase (PCMH) from Pseudomonas putida as well as methods for the separation of its subunits in native form and their recombination to reconstitute the original flavocytochrome. Under appropriate conditions, the reconstitution is stoichiometric and results in complete recovery of the catalytic activity of the flavocytochrome. The separated flavoprotein subunit shows only 2% of the catalytic activity of the original enzyme on p-cresol and is characterized by converging lines in bisubstrate kinetic analysis, while the intact and reconstituted enzymes show parallel line kinetics in steady-state experiments. van't Hoff plots of the dependence of the dissociation constant of the subunits of PCMH on temperature show a break near 15 degrees C. Above this temperature, KD is characterized by a positive delta H value of 12.6 kcal mol-1; below 15 degrees C, the dissociation is essentially temperature independent. The subunit dissociation is strongly dependent on ionic strength in the oxidized form of PCMH but not in the reduced form of the enzyme. Reduction also lowers the KD significantly, while substrates and nonoxidizable competitive inhibitors lower the dissociation constant even further, suggesting a conformation change. Combination of the subunits to form PCMH entails a small but measurable change in the absorption spectra of the component proteins.     

CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis

Background & Aims

CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.

Methods

Acute inflammation and recovery in wild-type (WT) and CCR9^−/− mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.

Results

CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9^−/− mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9^−/− colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9^−/− animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production.

Conclusions

Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.     

Facilitation within species: a possible origin of group-selected superorganisms

Facilitation (positive interactions) has emerged as a dominant ecological mechanism in many ecosystems. Its importance has recently been expanded to include intraspecific interactions, creating the potential for higher-level natural selection within species. Using multiple lines of evidence, we show that conspecific facilitation within the southern beech tree, Nothofagus pumilio, appears to overcome competition in two life phases. In a seedling experiment addressing stress and planting-density effects, we found that mortality was lowest (～0%) where there was no stress and was indistinguishable across densities. Furthermore, in mature forests (45 years old), genetically variable, merged individuals had lower mortality (-50%) than unmerged individuals in locations without identifiable stress. Thus, a full understanding of the occurrence of facilitation may require a more general model of resource improvements than the commonly cited stress gradient hypothesis. Additionally, the merged trees showed a density-dependent mortality pattern at the level of the group. These data demonstrate a potential mechanism (facilitation) driving natural selection at this higher level, via stem merging. These merged "superorganisms" would confirm theoretical predictions whereby facilitation acts as an ecological mechanism driving group selection.     

The genetic basis of alcoholism: multiple phenotypes,many genes,complex networks

Alcoholism is a significant public health problem. A picture of the genetic architecture underlying alcohol-related phenotypes is emerging from genome-wide association studies and work on genetically tractable model organisms.     

Automated selection of DAB-labeled tissue for immunohistochemical quantification.

The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each one's agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.