Synthetic prostacyclin analogs differentially regulate macrophage function via distinct analog-receptor binding specificities

PGI(2) (prostacyclin) is a lipid mediator with vasodilatory and antithrombotic effects used in the treatment of vasoconstrictive/ischemic diseases including pulmonary artery hypertension. However, emerging research supports a role for PGs, including PGI(2), in the regulation of both innate and acquired immunity. As PGI(2) is unstable, we sought to define the effects of various PGI(2) analogs on resident alveolar macrophage (AM) and peritoneal macrophage (PM) innate immune functions. The effects of iloprost, carbaprostacyclin, and treprostinil on the regulation of phagocytosis, bacterial killing, and inflammatory mediator production were determined in both macrophage populations from rats. Iloprost failed to suppress AM functions to the same degree that it did in PMs, a characteristic shared by carbaprostacyclin. This difference reflected greater expression of the G(alphas) protein-coupled I prostanoid receptor and greater cAMP generation in PMs than AMs. Treprostinil inhibited phagocytosis, bacterial killing, and cytokine generation in AMs to a much greater degree than the other PGI(2) analogs and more closely resembled the effects of PGE(2). Studies with the E prostanoid (EP) 2 receptor antagonist AH-6809 and EP2-null macrophages indicated that this was due in part to the previously unknown ability of treprostinil to stimulate the EP2 receptor. The present investigation for the first time identifies differences in immunoregulatory properties of clinically administered PGI(2) analogs. These studies are the first to explore the capacity of PGI(2) to regulate bacterial killing and phagocytosis in macrophages, and our findings may hold important consequences regarding the risk of infection for patients receiving such agents.     

Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase

Mur ligases play an essential role in the intracellular biosynthesis of bacterial peptidoglycan, the main component of the bacterial cell wall, and represent attractive targets for the design of novel antibacterials. UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) catalyses the addition of D-glutamic acid to the cytoplasmic intermediate UDP-N-acetylmuramoyl-L-alanine (UMA) and is the second in the series of Mur ligases. MurD ligase is highly stereospecific for its substrate, D-glutamic acid (D-Glu). Here, we report the high resolution crystal structures of MurD in complexes with two novel inhibitors designed to mimic the transition state of the reaction, which contain either the D-Glu or the L-Glu moiety. The binding modes of N-sulfonyl-D-Glu and N-sulfonyl-L-Glu derivatives were also characterised kinetically. The results of this study represent an excellent starting point for further development of novel inhibitors of this enzyme.     

A novel phosphatase family, structurally related to dual-specificity phosphatases, that displays unique amino acid sequence and substrate specificity

Members of the superfamily of protein tyrosine phosphatases (PTPs) share the presence of an evolutionarily conserved PTP catalytic domain. Among them, the dual-specificity phosphatases (DSPs) constitute a diverse group of enzymes in terms of substrate specificity, including nonprotein substrates. In recent years, an increasing number of novel DSPs, whose functions and biological substrates are not well defined, have been discovered in a variety of organisms. In this study, we define the structural and functional properties of evolutionarily related atypical DSPs from different phyla. Sets of conserved motifs were defined that (i) uniquely segregated mammalian atypical DSPs from closely related enzymes and (ii) exclusively characterised a novel family of atypical DSPs present in plants, fungi, and kinetoplastids [plant and fungi atypical (PFA)-DSPs]; despite having different sequence “fingerprints,” the PTP tertiary structure of PFA-DSPs is conserved. Analysis of the catalytic properties of PFA-DSPs suggests the existence of a unique substrate specificity for these enzymes. Our findings predict characteristic functional motifs for the diverse members of the DSP families of PTPs and provide insights into the functional properties of DSPs of unknown function.     

Harvesting in a random environment: Itô or Stratonovich calculus?

We extend to harvesting stochastic differential equation (SDE) models in a random environment our previous work on models without harvesting concerning the resolution of the It?-Stratonovich controversy. The resolution is obtained for the very general class of models dN/dt=N (r(N)-h(N)+sigmaepsilon(t)), where N=N(t) is the population size at time t, r(N) is the (density-dependent) "mean" per capita growth rate, h(N) is the (density-dependent) harvesting effort, epsilon(t) is a standard white noise (representing environmental random fluctuations), and sigma is a noise intensity parameter. It? and Stratonovich calculus in the resolution of SDEs apparently give different qualitative and quantitative results, leading to controversy on which calculus is more appropriate and creating an obstacle on the use of this modeling approach. We show that the apparent difference between the two calculi is due to a semantic confusion based on the fallacious assumption that we are working with the same type of mean rates. After clearing the confusion, the two calculi yield exactly the same results and we obtain important common conditions for extinction and for existence of a stationary density. The resolution of the controversy is intertwined with and sheds light on the estimation issues.     

Obestatin-mediated proliferation of human retinal pigment epithelial cells: regulatory mechanisms

In this work, we have evaluated the effect of the new discovered peptide obestatin on cell proliferation in primary cultures of human retinal epithelial cells (hRPE cells). The results showed that this peptide induced, in a dose-dependent manner, cell proliferation by MEK/ERK 1/2 phosphorylation. A sequential analysis of the obestatin transmembrane signaling pathway showed that the ERK 1/2 activity is partially blocked after preincubation of the cells with pertussis toxin (PTX), as well as by wortmannin (an inhibitor of PI3K), claphostin C (an inhibitor of PKC), and PP2 (which inhibits the non receptor tyrosine kinase Src). Upon administration of obestatin, the intracellular levels of phospho-PKCepsilon-, theta-, and micro-isoenzymes rise with different time courses, from which PKCepsilon might be responsible for ERK 1/2 response. Based on the experimental data, a signaling pathway involving the consecutive activation of Gi, PI3K, novel PKC (probably PKCepsilon), and Src for ERK 1/2 activation is proposed. These results incorporate a new mitogenic factor to the group of factors that regulate proliferation of hRPE cells.     

A pegylated derivative of alpha-galactosylceramide exhibits improved biological properties

The glycolipid alpha-galactosylceramide (alphaGalCer) has immunomodulatory properties, which have been exploited to combat cancer, chronic inflammatory diseases, and infections. However, its poor solubility makes alphaGalCer a suboptimal compound for in vivo applications. In this study, a pegylated derivative of alphaGalCer is characterized, which exhibits improved physical and biological properties. The new compound, alphaGalCerMPEG, is water-soluble and retains the specificity for the CD1d receptor of alphaGalCer. The in vitro stimulatory properties on immune cells (e.g., dendritic cells and splenocytes) are maintained intact, even when tested at a 33-fold lower concentration of the active moiety than alphaGalCer. NK cells isolated from mice treated with alphaGalCerMPEG also had stronger cytotoxic activity on YAC-1 cells than those obtained from animals receiving either alphaGalCer or CpG. Intranasal immunization studies performed in mice showed that alphaGalCerMPEG exerts stronger adjuvant activities than the parental compound alphaGalCer when tested at 0.35 vs 11.7 nM/dose. Coadministration of beta-galactosidase with alphaGalCerMPEG resulted not only in high titers of Ag-specific Abs in serum (i.e., 1:512,000), but also in the stimulation of stronger Th2 and secretory IgA responses, both at local and remote mucosal effector sites (i.e., nose, lung, and vagina). The new synthetic derivative alphaGalCerMPEG represents a promising tool for the development of immune interventions against infectious and noninfectious diseases.