Synthesis of Indole Derived Protease-Activated Receptor 4 Antagonists and Characterization in Human Platelets

Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. We sought to develop potent, selective, and novel PAR4 antagonists to test the role of PAR4 in thrombosis and hemostasis. Development of an expedient three-step synthetic route to access a novel series of indole-based PAR4 antagonists also necessitated the development of a platelet based high-throughput screening assay. Screening and subsequent structure activity relationship analysis yielded several selective PAR4 antagonists as well as possible new scaffolds for future antagonist development.     

Parietal endoderm secreted SPARC promotes early cardiomyogenesis in vitro

Cardiomyogenesis proceeds in the presence of signals emanating from extra-embryonic lineages emerging before and during early eutherian gastrulation. In embryonic stem cell derived embryoid bodies, primitive endoderm gives rise to visceral and parietal endoderm. Parietal endoderm undergoes an epithelial to mesenchymal transition shortly before first cardiomyocytes start to contract rhythmically. Here, we demonstrate that Secreted Protein, Acidic, Rich in Cysteine, SPARC, predominantly secreted by mesenchymal parietal endoderm specifically promotes early myocardial cell differentiation in embryoid bodies. SPARC enhanced the expression of bmp2 and nkx2.5 in embryoid bodies and fetal cardiomyocytes. Inhibition of either SPARC or Bmp2 attenuated in both cases cardiomyogenesis and downregulated nkx2.5 expression. Thus, SPARC directly affects cardiomyogenesis, modulates Bmp2 signaling, and contributes to a positive autoregulatory loop of Bmp2 and Nkx2.5 in cardiomyocytes.     

The effects of granulocyte colony-stimulating factor and neutrophil recruitment on the pulmonary chemokine response to intratracheal endotoxin

Although G-CSF has been shown to increase neutrophil (polymorphonuclear leukocyte, PMN) recruitment into the lung during pulmonary infection, relatively little is known about the local chemokine profiles associated with this enhanced PMN delivery. We investigated the effects of G-CSF and PMN recruitment on the pulmonary chemokine response to intratracheal LPS. Rats pretreated twice daily for 2 days with an s.c. injection of G-CSF (50 microg/kg) were sacrificed at either 90 min or 4 h after intratracheal LPS (100 microg) challenge. Pulmonary recruitment of PMNs was not observed at 90 min post LPS challenge. Macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC) concentrations in bronchoalveolar lavage (BAL) fluid were similar in animals pretreated with or without G-CSF at this time. G-CSF pretreatment enhanced pulmonary recruitment of PMNs (5-fold) and greatly reduced MIP-2 and CINC levels in BAL fluid at 4 h after LPS challenge. In vitro, the presence of MIP-2 and CINC after LPS stimulation of alveolar macrophages was decreased by coculturing with circulating PMNs but not G-CSF. G-CSF had no direct effect on LPS-induced MIP-2 and CINC mRNA expression by alveolar macrophages. Pulmonary recruited PMNs showed a significant increase in cell-associated MIP-2 and CINC. Cell-associated MIP-2 and CINC of circulating PMNs were markedly increased after exposure of these cells to the BAL fluid of LPS-challenged lungs. These data suggest that recruited PMNs are important cells in modulating the local chemokine response. G-CSF augments PMN recruitment and, thereby, lowers local chemokine levels, which may be one mechanism resulting in the subsidence of the host proinflammatory response.     

First Experimental In Vivo Model of Enhanced Dengue Disease Severity through Maternally Acquired Heterotypic Dengue Antibodies

Dengue (DEN) represents the most serious arthropod-borne viral disease. DEN clinical manifestations range from mild febrile illness to life-threatening hemorrhage and vascular leakage. Early epidemiological observations reported that infants born to DEN-immune mothers were at greater risk to develop the severe forms of the disease upon infection with any serotype of dengue virus (DENV). From these observations emerged the hypothesis of antibody-dependent enhancement (ADE) of disease severity, whereby maternally acquired anti-DENV antibodies cross-react but fail to neutralize DENV particles, resulting in higher viremia that correlates with increased disease severity. Although in vitro and in vivo experimental set ups have indirectly supported the ADE hypothesis, direct experimental evidence has been missing. Furthermore, a recent epidemiological study has challenged the influence of maternal antibodies in disease outcome. Here we have developed a mouse model of ADE where DENV2 infection of young mice born to DENV1-immune mothers led to earlier death which correlated with higher viremia and increased vascular leakage compared to DENV2-infected mice born to dengue naïve mothers. In this ADE model we demonstrated the role of TNF-α in DEN-induced vascular leakage. Furthermore, upon infection with an attenuated DENV2 mutant strain, mice born to DENV1-immune mothers developed lethal disease accompanied by vascular leakage whereas infected mice born to dengue naïve mothers did no display any clinical manifestation. In vitro ELISA and ADE assays confirmed the cross-reactive and enhancing properties towards DENV2 of the serum from mice born to DENV1-immune mothers. Lastly, age-dependent susceptibility to disease enhancement was observed in mice born to DENV1-immune mothers, thus reproducing epidemiological observations.Overall, this work provides direct in vivo demonstration of the role of maternally acquired heterotypic dengue antibodies in the enhancement of dengue disease severity and offers a unique opportunity to further decipher the mechanisms involved.     

Quantitation of Cu-containing metallothionein by a Cd-saturation method

A rapid and sensitive method for determining Cu-containing metallothionein (MT) is described. The main features of this Cd-saturation assay are: high-molecular-weight Cd-binding compounds are denatured with acetonitrile (50% final concentration), Cu bound to MT is removed with ammonium tetrathiomolybdate, excessive tetrathiomolybdate and its Cu complexes are removed with DEAE-Sephacel, apothionein is saturated with Cd, and excessive Cd is bound to Chelex 100. The thiomolybdate assay is capable of reliably detecting 14 ng MT and thus is particularly suitable for measuring MT in small tissue samples (e.g., biopsies), in extrahepatic tissues, and in cultured cells. Moreover, the combination of the thiomolybdate assay with the recently developed Cd-Chelex assay also makes it possible to determine the portion of MT which binds Cu (Cu load of MT), provided that the amount of non-Cu-thionein exceeds 100 ng, the detection limit of the Cd-Chelex assay.     

Effects of marginal iron overload on iron homeostasis and immune function in alveolar macrophages isolated from pregnant and normal rats

The effects of changes in macrophage iron status, induced by single or multiple iron injections, iron depletion or pregnancy, on both immune function and mRNA expression of genes involved in iron influx and egress have been evaluated. Macrophages isolated from iron deficient rats, or pregnant rats at day 21 of gestation, either supplemented with a single dose of iron dextran, 10 mg, at the commencement of pregnancy, or not, showed significant increases of macrophage ferroportin mRNA expression, which was paralleled by significant decreases in hepatic Hamp mRNA expression. IRP activity in macrophages was not significantly altered by iron status or the inducement of pregnancy ± a single iron supplement. Macrophage immune function was significantly altered by iron supplementation and pregnancy. Iron supplementation, alone or combined with pregnancy, increased the activities of both NADPH oxidase and nuclear factor kappa B (NFκB). In contrast, the imposition of pregnancy reduced the ability of these parameters to respond to an inflammatory stimuli. Increasing iron status, if only marginally, will reduce the ability of macrophages to mount a sustained response to inflammation as well as altering iron homeostatic mechanisms.     

RhoGAP18B Isoforms Act on Distinct Rho-Family GTPases and Regulate Behavioral Responses to Alcohol via Cofilin

Responses to the effects of ethanol are highly conserved across organisms, with reduced responses to the sedating effects of ethanol being predictive of increased risk for human alcohol dependence. Previously, we described that regulators of actin dynamics, such as the Rho-family GTPases Rac1, Rho1, and Cdc42, alter Drosophila’s sensitivity to ethanol-induced sedation. The GTPase activating protein RhoGAP18B also affects sensitivity to ethanol. To better understand how different RhoGAP18B isoforms affect ethanol sedation, we examined them for their effects on cell shape, GTP-loading of Rho-family GTPase, activation of the actin-severing cofilin, and actin filamentation. Our results suggest that the RhoGAP18B-PA isoform acts on Cdc42, while PC and PD act via Rac1 and Rho1 to activate cofilin. In vivo, a loss-of-function mutation in the cofilin-encoding gene twinstar leads to reduced ethanol-sensitivity and acts in concert with RhoGAP18B. Different RhoGAP18B isoforms, therefore, act on distinct subsets of Rho-family GTPases to modulate cofilin activity, actin dynamics, and ethanol-induced behaviors.     

Monitoring the mission blue butterfly using immature stages

Monitoring of the Mission blue butterfly has been restricted to periodic counts of adults sighted during the flight season during transect walks. However, adult count data is a reliable index of population size only if the probability of detection is high and stable across varying ecological conditions. Determining these probabilities requires marking and capturing of a large number of individuals, which is harmful and difficult to implement for protected species. Therefore, we explored the potential of using immature stages (eggs, late-instar larvae and the feeding damage inflicted by larvae) as potential measures of population status for monitoring purposes. For each of these measures we examined its correlation with adult counts, its detection probability, and its repeatability. We found that immature stages and feeding damage are highly correlated with adult counts, are highly repeatable, and have detection probabilities that exceed estimates of the probability of detecting Mission blue adults. We conclude that late instar larval counts and foliar feeding damage are superior to adults as measures of population status for population monitoring. Furthermore, if sighting-resighting based counts of immatures or feeding damage are made on the same set of host plants on successive days, then estimates of abundance and its standard error adjusted for detection probabilities can be obtained.     

Gly25-Ser26 Amyloid β-Protein Structural Isomorphs Produce Distinct Aβ42 Conformational Dynamics and Assembly Characteristics

One of the earliest events in amyloid β-protein (Aβ) self-association is nucleation of Aβ monomer folding through formation of a turn at Gly25-Lys28. We report here the effects of structural changes at the center of the turn, Gly25-Ser26, on Aβ42 conformational dynamics and assembly. We used “click peptide” chemistry to quasi-synchronously create Aβ42 from 26-O-acyliso-Aβ42 (iAβ42) through a pH jump from 3 to 7.4. We also synthesized N_α-acetyl-Ser26-iAβ42 (Ac-iAβ42), which cannot undergo O → N acyl chemistry, to study the behavior of this ester form of Aβ42 itself at neutral pH. Data from experiments monitoring increases in β-sheet formation (thioflavin T, CD), hydrodynamic radius (R_H), scattering intensity (quasielastic light scattering spectroscopy), and extent of oligomerization (ion mobility spectroscopy–mass spectrometry) were quite consistent. A rank order of Ac-iAβ42 > iAβ42 > Aβ42 was observed. Photochemically cross-linked iAβ42 displayed an oligomer distribution with a prominent dimer band that was not present with Aβ42. These dimers also were observed selectively in iAβ42 in ion mobility spectrometry experiments. The distinct biophysical behaviors of iAβ42 and Aβ42 appear to be due to the conversion of iAβ42 into “pure” Aβ42 monomer, a nascent form of Aβ42 that does not comprise the variety of oligomeric and aggregated states present in pre-existent Aβ42. These results emphasize the importance of the Gly25-Ser26 dipeptide in organizing Aβ42 monomer structure and thus suggest that drugs altering the interactions of this dipeptide with neighboring side-chain atoms or with the peptide backbone could be useful in therapeutic strategies targeting formation of Aβ oligomers and higher-order assemblies.