Characterization of heparin affin regulatory peptide signaling in human endothelial cells

Heparin affin regulatory peptide (HARP) is an 18-kDa secreted growth factor that has a high affinity for heparin and a potent role on tumor growth and angiogenesis. We have previously reported that HARP is mitogenic for different types of endothelial cells and also affects cell migration and differentiation (12). In this study we examined the signaling pathways involved in the migration and tube formation on matrigel of human umbilical vein endothelial cells (HUVEC) induced by HARP. We report for the first time that receptor-type protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), which is a receptor for HARP in neuronal cell types, is also expressed in HUVEC. We also document that HARP signaling through RPTPbeta/zeta leads to activation of Src kinase, focal adhesion kinase, phosphatidylinositol 3-kinase, and Erk1/2. Sodium orthovanadate, chondroitin sulfate-C, PP1, wortmannin, LY294002, and U0126 inhibit HARP-mediated signaling and HUVEC migration and tube formation. In addition, RPTPbeta/zeta suppression using small interfering RNA technology interrupts intracellular signals and HUVEC migration and tube formation induced by HARP. These results establish the role of RPTPbeta/zeta as a receptor of HARP in HUVEC and elucidate the HARP signaling pathway in endothelial cells.     

Minos as a genetic and genomic tool in Drosophila melanogaster

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5′-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient “jump-starting” scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and ~55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila.     

X-ray and thermodynamic studies of staphylococcal nuclease variants I92E and I92K: insights into polarity of the protein interior

We have used crystallography and thermodynamic analysis to study nuclease variants I92E and I92K, in which an ionizable side-chain is placed in the hydrophobic core of nuclease. We find that the energetic cost of burying ionizable groups is rather modest. The X-ray determinations show water molecules solvating the buried glutamic acid under cryo conditions, but not at room temperature. The lysine side-chain does not appear solvated in either case. Guanidine hydrochloride (GnHCl) denaturation of I92E and I92K, done as a function of pH and monitored by tryptophan fluorescence, showed that I92E and I92K are folded in the pH range pH 3.5-9.0 and pH 5.5-9.5, respectively. The stability of the parental protein is independent of pH over a broad range. In contrast, the stabilities of I92E and I92K exhibit a pH dependence, which is quantitatively explained by thermodynamic analysis: the PK(a) value of the buried K92 is 5.6, while that of the buried E92 is 8.65. The free energy difference between burying the uncharged and charged forms of the groups is modest, about 6 kcal/mol. We also found that epsilon(app) for I92K and I92E is in the range approximately 10-12, instead of 2-4 commonly used to represent the protein interior. Side-chains 92E and 92K were uncharged under the conditions of the X-ray experiment. Both are buried completely inside the well-defined hydrophobic core of the variant proteins without forming salt-bridges or hydrogen bonds to other functional groups of the proteins. Under cryo conditions 92E shows a chain of four water molecules, which hydrate one oxygen atom of the carboxyl group of the glutamic acid. Two other water molecules, which are present in the wild-type at all temperatures, are also connected to the water ring observed inside the hydrophobic core. The ready burial of water with an uncharged E92 raises the possibility that solvent excursions into the interior also take place in the wild-type protein, but in a random, dynamic way not detectable by crystallography. Such transient excursions could increase the average polarity, and thus epsilon(app), of the protein interior.     

Neutrophil CD64 expression and serum IL-8: sensitive early markers of severity and outcome in sepsis

The aim of the present study was to investigate which biomarker/s reliably assess severity and mortality early in the sepsis process. In 47 critically-ill patients within the 24h of septic onset, Interleukins (IL)-8, -1beta, -6, -10, and -12p70, tumor necrosis factor-alpha (TNF-alpha), procalcitonin (PCT) and C-reactive protein (CRP) were measured in serum. Additionally, CD64 expression was measured in neutrophils. In early sepsis, neutrophil CD64 expression and IL-8 levels are the only biomarkers that increased with sepsis severity, differentiating disease stages: sepsis, severe sepsis and septic shock (p<0.001). The biomarkers that best evaluate the severity of sepsis (via APACHE II) were CD64, IL-8 and IL-6 (p<0.01), and the severity of organ failure (via SOFA) were CD64 and IL-8 (p<0.01). CD64 expression and IL-8 levels were associated with mortality within 28-days (OR=1.3, p=0.01 for CD64 and OR=1.26, p=0.024 for IL-8 by logistic regression analysis) and ROC curve analysis showed high sensitivity and specificity for predicting sepsis stages and the 28 day mortality. We conclude that there is an early increase of neutrophil CD64 expression and IL-8 levels during sepsis. Based on this single measurement it is possible to reliably assess the stage, detect the severity and predict the 28-day mortality of sepsis.     

20-Aminosteroids as a novel class of selective and complete androgen receptor antagonists and inhibitors of prostate cancer cell growth

Here, the synthesis and the evaluation of novel 20-aminosteroids on androgen receptor (AR) activity is reported. Compounds 11 and 18 of the series inhibit both the wild type and the T877A mutant AR-mediated transactivation indicating AR antagonistic function. Interestingly, minor structural changes such as stereoisomers of the amino lactame moiety exhibit preferences for antagonism among wild type and mutant AR. Other tested nuclear receptors are only weakly or not affected. In line with this, the prostate cancer cell growth of androgen-dependent but not of cancer cells lacking expression of the AR is inhibited. Further, the expression of the prostate specific antigen used as a diagnostic marker is also repressed. Finally steroid 18 enhances cellular senescence that might explain in part the growth inhibition mediated by this derivative. Steroids 11 and 18 are the first steroids that act as complete AR antagonists and exhibit AR specificity.     

Peptidic models for the binding of Pb(II), Bi(III) and Cd(II) to mononuclear thiolate binding sites

Herein, we evaluate the binding of Pb(II) and Bi(III) to cysteine-substituted versions of the TRI peptides [AcG-(LKALEEK)₄G-NH₂] which have previously been shown to bind Hg(II) and Cd(II) in unusual geometries as compared with small-molecule thiol ligands in aqueous solutions. Studies of Pb(II) and Bi(III) with the peptides give rise to complexes consistent with the metal ions bound to three sulfur atoms with M–S distances of 2.63 and 2.54 Å, respectively. Competition experiments between the metal ions Pb(II), Cd(II), Hg(II) and Bi(III) for the peptides show that Hg(II) has the highest affinity, owing to the initial formation of the extremely strong HgS₂ bond. Cd(II) and Pb(II) have comparable binding affinities at pH > 8, while Bi(III) displays the weakest affinity, following the model, M(II) + (TRI LXC)₃ ^3? → M(II)(TRI LXC)₃ ^?. While the relevant equilibria for Hg(II) binding to the TRI peptides corresponds to a strong first step forming Hg(TRI LXC)₂(HTRI LXC), followed by a single deprotonation to give Hg(TRI LXC)₃ ^?, the binding of Cd(II) and Pb(II) is consistent with initial formation of M(II)(TRI LXC)(HTRI LXC)₂ ⁺ at pH < 5 followed by a two-proton dissociation step (pK _a2) yielding M(II)(TRI LXC)₃ ^?. Pb(II)(TRI LXC)(HTRI LXC)₂ ⁺ converts to Pb(II)(TRI LXC)₃ ^? at slightly lower pH values than the corresponding Cd(II)–peptide complexes. In addition, Pb(II) displays a lower pK _a of binding to the “d”-substituted peptide, (TRI L12C, pK _a2 = 12.0) compared with the “a”-substituted peptide, (TRI L16C, pK _a2 = 12.6), the reverse of the order seen for Hg(II) and Cd(II). Pb(II) also showed a stronger binding affinity for TRI L12C (K _bind = 3.2 × 10⁷ M^?1) compared with that with TRI L16C (K _bind = 1.2 × 10⁷ M^?1) at pH > 8.     

Evolution at the subgene level: domain rearrangements in the Drosophila phylogeny

Although the possibility of gene evolution by domain rearrangements has long been appreciated, current methods for reconstructing and systematically analyzing gene family evolution are limited to events such as duplication, loss, and sometimes, horizontal transfer. However, within the Drosophila clade, we find domain rearrangements occur in 35.9% of gene families, and thus, any comprehensive study of gene evolution in these species will need to account for such events. Here, we present a new computational model and algorithm for reconstructing gene evolution at the domain level. We develop a method for detecting homologous domains between genes and present a phylogenetic algorithm for reconstructing maximum parsimony evolutionary histories that include domain generation, duplication, loss, merge (fusion), and split (fission) events. Using this method, we find that genes involved in fusion and fission are enriched in signaling and development, suggesting that domain rearrangements and reuse may be crucial in these processes. We also find that fusion is more abundant than fission, and that fusion and fission events occur predominantly alongside duplication, with 92.5% and 34.3% of fusion and fission events retaining ancestral architectures in the duplicated copies. We provide a catalog of ～9,000 genes that undergo domain rearrangement across nine sequenced species, along with possible mechanisms for their formation. These results dramatically expand on evolution at the subgene level and offer several insights into how new genes and functions arise between species.