Privileged structure-based ligands for melanocortin receptors-tetrahydroquinolines, indoles, and aminotetralines

Substitution of the aryl sulfonamide moiety contained in MC4 agonist 1 with bicyclic heterocycles and aminotetralines produced compounds with MC4 activity. The heterocycles represent alternative privileged structures to that contained in 1. Compounds in which the polar group of the privileged structure was displayed in an endocyclic fashion were not as active as the parent agonist 1, while those with an exocyclic polar group afforded activity competitive with 1.     

Regulated targeting of protein phosphatase 1 to the outer kinetochore by KNL1 opposes Aurora B kinase

Regulated interactions between kinetochores and spindle microtubules are essential to maintain genomic stability during chromosome segregation. The Aurora B kinase phosphorylates kinetochore substrates to destabilize kinetochore–microtubule interactions and eliminate incorrect attachments. These substrates must be dephosphorylated to stabilize correct attachments, but how opposing kinase and phosphatase activities are coordinated at the kinetochore is unknown. Here, we demonstrate that a conserved motif in the kinetochore protein KNL1 directly interacts with and targets protein phosphatase 1 (PP1) to the outer kinetochore. PP1 recruitment by KNL1 is required to dephosphorylate Aurora B substrates at kinetochores and stabilize microtubule attachments. PP1 levels at kinetochores are regulated and inversely proportional to local Aurora B activity. Indeed, we demonstrate that phosphorylation of KNL1 by Aurora B disrupts the KNL1–PP1 interaction. In total, our results support a positive feedback mechanism by which Aurora B activity at kinetochores not only targets substrates directly, but also prevents localization of the opposing phosphatase.     

Interacting effects of warming and drought on regeneration and early growth of Acer pseudoplatanus and A. platanoides

Climate change is acting on several aspects of plant life cycles, including the sexual reproductive stage, which is considered amongst the most sensitive life‐cycle phases. In temperate forests, it is expected that climate change will lead to a compositional change in community structure due to changes in the dominance of currently more abundant forest tree species. Increasing our understanding of the effects of climate change on currently secondary tree species recruitment is therefore important to better understand and forecast population and community dynamics in forests. Here, we analyse the interactive effects of rising temperatures and soil moisture reduction on germination, seedling survival and early growth of two important secondary European tree species, Acer pseudoplatanus and A. platanoides. Additionally, we analyse the effect of the temperature experienced by the mother tree during seed production by collecting seeds of both species along a 2200‐km long latitudinal gradient. For most of the responses, A. platanoides showed higher sensitivity to the treatments applied, and especially to its joint manipulation, which for some variables resulted in additive effects while for others only partial compensation. In both species, germination and survival decreased with rising temperatures and/or soil moisture reduction while early growth decreased with declining soil moisture content. We conclude that although A. platanoides germination and survival were more affected after the applied treatments, its initial higher germination and larger seedlings might allow this species to be relatively more successful than A. pseudoplatanus in the face of climate change.     

Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor beta-subunit

The juxtamembrane region of the insulin receptor (IR) beta-subunit contains an unphosphorylated tyrosyl residue (Tyr960) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M.F., Livingston, J.N., Backer, J.M., Lauris, V., Dull, T.J., Ullrich, A., and Kahn, C.R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXY [corrected] is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IRF960, in which Tyr960 has been substituted with phenylalanine, and IR delta 960, in which 12 amino acids (Ala954-Asp965), including the putative consensus sequence NPXY [corrected], were deleted. Although the in vivo autophosphorylation of IRF960 and IR delta 960 was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IRF960 cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IR delta 960 cells was 20-30% of wild-type. However, insulin internalization in the CHO/IR delta 960 cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IRA1018). The degradation of insulin was equally impaired in CHO/IR delta 960 and CHO/IRA1018 cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXY [corrected] may play a general role in directing the internalization of cell surface receptors.     

Multi-residue analysis of eight anticoagulant rodenticides in animal plasma and liver using liquid chromatography combined with heated electrospray ionization tandem mass spectrometry

The determination of C-terminal peptide sequence is critical since the C-terminal peptide contains biologically relevant information and often undergoes post-translational processing. Another important application is in estimating purity of the biopharmaceuticals, especially for determining the presence of ragged processed ends and for N-terminally blocked polypeptides and proteins. In this paper, different isotope coding strategies in combination with reversed phase chromatography (RPC) coupled with electrospray ionization-mass spectrometry (ESI-MS) were evaluated to detect the C-terminal peptide from proteolytic digests. These were (i) O18 (ii) acylation and (iii) esterification based isotope coding strategies. Using reversed phase chromatography, the C-terminal peptide was resolved from other internal peptides. The isotope coding approaches specifically rendered a characteristic MS signature to the C-terminal peptide, thereby facilitating its detection. The unique MS signature, along with accurate mass data for the C-terminal peptide was found to be sufficient for its detection and identification. The advantages and limitations of the three approaches will be discussed.     

Review of Florida Red Tide and Human Health Effects

This paper reviews the literature describing research performed over the past decade on the known and possible exposures and human health effects associated with Florida red tides. These harmful algal blooms are caused by the dinoflagellate, Karenia brevis, and similar organisms, all of which produce a suite of natural toxins known as brevetoxins. Florida red tide research has benefited from a consistently funded, long term research program, that has allowed an interdisciplinary team of researchers to focus their attention on this specific environmental issue-one that is critically important to Gulf of Mexico and other coastal communities. This long-term interdisciplinary approach has allowed the team to engage the local community, identify measures to protect public health, take emerging technologies into the field, forge advances in natural products chemistry, and develop a valuable pharmaceutical product. The Review includes a brief discussion of the Florida red tide organisms and their toxins, and then focuses on the effects of these toxins on animals and humans, including how these effects predict what we might expect to see in exposed people.     

RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.     

Local signaling by the EGF receptor

Differing spatial scales of signaling cascades are critical for cell orientation during chemotactic responses. We used biotin EGF bound to streptavidin-coupled magnetic beads to locally stimulate cells overexpressing the EGF receptor. We have found that EGF-induced actin polymerization remains localized even under conditions of receptor overexpression. Conversely, EGF-induced ERK activation spreads throughout the cell body after EGF bead stimulation. The localized actin polymerization is independent of PI3-kinase and rho protein activity and requires Arp2/3 complex and cofilin function. Thus, we find differing spatial scales of signaling from the EGF receptor, supporting models of chemotaxis that integrate short- and long-range signaling.     

Cytokine polymorphisms and histologic expression in autopsy studies: contribution of TNF-alpha and TGF-beta 1 to the pathogenesis of autoimmune-associated congenital heart block

Although Abs to SSA/Ro-SSB/La are necessary for the development of congenital heart block (CHB), the low frequency suggests that fetal factors are contributory. Because CHB involves a cascade from inflammation to scarring, polymorphisms of the TNF-alpha promoter region and codons 10 and 25 of the TGF-beta gene were evaluated in 88 children (40 CHB, 17 rash, 31 unaffected siblings) and 74 mothers from the Research Registry for Neonatal Lupus (NL). Cytokine expression was assessed in autopsy material from two fetuses with CHB. Significantly increased frequency of the -308A (high-producer) allele of TNF-alpha was observed in all NL groups compared with controls. In contrast, the TGF-beta polymorphism Leu(10) (associated with increased fibrosis) was significantly higher in CHB children (genotypic frequency 60%, allelic frequency 78%) than unaffected offspring (genotypic frequency 29%, p = 0.016; allelic frequency 56%, p = 0.011) and controls, while there were no significant differences between controls and other NL groups. For the TGF-beta polymorphism, Arg(25), there were no significant differences between NL groups and controls. In fetal CHB hearts, protein expression of TGF-beta, but not TNF-alpha, was demonstrated in septal regions, extracellularly in the fibrous matrix, and intracellularly in macrophage infiltrates. Age-matched fetal hearts from voluntary terminations expressed neither cytokine. TNF-alpha may be one of several factors that amplify susceptibility; however, the genetic studies, backed by the histological data, more convincingly link TGF-beta to the pathogenesis of CHB. This profibrosing cytokine and its secretion/activation circuitry may provide a novel direction for evaluating fetal factors in the development of a robust animal model of CHB as well as therapeutic strategies in humans.     

Identification of α2 adrenergic receptors in human frontal cortex membranes by binding of [H]RX 821002, the 2-methoxy analog of [H]idazoxan

The antagonist [³H]idazoxan binds with comparable affinity to α₂ adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-³H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300–1200 times lower affinity for the non-stereoselective sites. Their affinity for the α₂ receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [³H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the α₂ receptors (K_D = 2.8 nM than [³H]idazoxan (K_D = 24 nM) and about equal affinity as [³H]rauwolscine (K_D = 3.6 nM).[³H]Rauwolscine binds with comparable affinity to α₂ receptors and to 5-HT_1A receptors, and competition studies indicate that the K_i value of unlabelled RX 821002 for the 5-HT_1A receptors (30 nM) is about one order in magnitude above its K_i value for the α₂ receptors (4.1 nM). Labelling of the 5-HT_1A receptors by [³H]RX 821002 and by [³H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 μM). Under these conditions, specific binding of [³H]RX 821002 to the α₂ receptors represents 84% of total binding (at its K_D), as compared to 77% for [³H]rauwolscine and 20% for [³H]idazoxan.[³H]RX 821002 labels the α₂ receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37°C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [³H]RX821002. About 60% of the α₂ receptors possess high agonist affinity.