Electron Capture in Spin-Trap Capped Peptides. An Experimental Example of Ergodic Dissociation in Peptide Cation-Radicals

Electron capture dissociation was studied with tetradecapeptides and pentadecapeptides that were capped at N-termini with a 2-(4′-carboxypyrid-2′-yl)-4-carboxamide group (pepy), e.g., pepy-AEQLLQEEQLLQEL-NH2, pepy-AQEFGEQGQKALKQL-NH2, and pepy-AQEGSEQAQKFFKQL-NH2. Doubly and triply protonated peptide cations underwent efficient electron capture in the ion-cyclotron resonance cell to yield charge-reduced species. However, the electron capture was not accompanied by backbone dissociations. When the peptide ions were preheated by absorption of infrared photons close to the dissociation threshold, subsequent electron capture triggered ion dissociations near the remote C-terminus forming mainly (b11-14 + 1)+· fragment ions that were analogous to those produced by infrared multiphoton dissociation alone. Ab initio calculations indicated that the N-1 and N-1′ positions in the pepy moiety had topical gas-phase basicities (GB = 923 kJ mol−1) that were greater than those of backbone amide groups. Hence, pepy was a likely protonation site in the doubly and triply charged ions. Electron capture in the protonated pepy moiety produced the ground electronic state of the charge-reduced cation-radical with a topical recombination energy, RE = 5.43-5.46 eV, which was greater than that of protonated peptide residues. The hydrogen atom in the charge-reduced pepy moiety was bound by >160 kJ mol−1, which exceeded the hydrogen atom affinity of the backbone amide groups (21–41 kJ mol−1). Thus, the pepy moiety functioned as a stable electron and hydrogen atom trap that did not trigger radical-type dissociations in the peptide backbone that are typical of ECD. Instead, the internal energy gained by electron capture was redistributed over the peptide moiety, and when combined with additional IR excitation, induced proton-driven ion dissociations which occurred at sites that were remote from the site of electron capture. This example of a spin-remote fragmentation provided the first clear-cut experimental example of an ergodic dissociation upon ECD

Acquiring, archiving, analyzing and exchanging seismic data in real time at the Seismological Research Center of the OGS in Italy

The Centro di Ricerche Sismologiche (CRS, Seismological Research Center) of the Istituto Nazionale di Oceanografia e di Geofisica Sperimentale (OGS, Italian National Institute for Oceanography and Experimental Geophysics) in Udine (Italy) after the strong earthquake (magnitude M=6.4) occurred in 1976 in the Italian Friuli-Venezia Giulia region, started to operate the North-east Italy (NI) seismic network: it currently consists of 11 very sensitive broad band and 23 more simple short period seismic stations, all telemetered to and acquired in real time at the OGS-CRS data center in Udine. Real time data exchange agreements in place with other Italian, Slovenian, Austrian and Swiss seismological institutes lead to a total number of 89 seismic stations acquired in real time, which makes the OGS the reference institute for seismic monitoring of Northeastern Italy. Since 2002 OGS-CRS is using the Antelope software suite as the main tool for collecting, analyzing, archiving and exchanging seismic data in the framework of the EU Interreg IIIA project “Trans-national seismological networks in the South-Eastern Alps”. SeisComP is also used as a real time data exchange server tool. At OGS-CRS we then adapted existing programs and created new ones like: a customized web-accessible server to manually relocate earthquakes, a script for automatic moment tensor determination, scripts for web publishing of earthquake parametric data, waveforms, state of health parameters and shaking maps, noise characterization by means of automatic spectra analysis, plus scripts for email/SMS/fax alerting. A new OGS-CRS real time web site has also been recently designed and made operative in the framework of the DPC-INGV S3 Project

Unique Structural Modifications Are Present in the Lipopolysaccharide from Colistin-Resistant Strains of \u3ci\u3eAcinetobacter baumannii\u3c/i\u3e

Acinetobacter baumannii is a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treat A. baumannii infections. Colistin-resistant strains of A. baumannii with alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistant A. baumannii MAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adapted A. baumannii strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from A. baumannii, which are important for resistance to colistin

The ancestral retinoic acid receptor was a low-affinity sensor triggering neuronal differentiation.

Retinoic acid (RA) is an important intercellular signaling molecule in vertebrate development, with a well-established role in the regulation of hox genes during hindbrain patterning and in neurogenesis. However, the evolutionary origin of the RA signaling pathway remains elusive. To elucidate the evolution of the RA signaling system, we characterized RA metabolism and signaling in the marine annelid Platynereis dumerilii, a powerful model for evolution, development, and neurobiology. Binding assays and crystal structure analyses show that the annelid retinoic acid receptor (RAR) binds RA and activates transcription just as vertebrate RARs, yet with a different ligand-binding pocket and lower binding affinity, suggesting a permissive rather than instructive role of RA signaling. RAR knockdown and RA treatment of swimming annelid larvae further reveal that the RA signal is locally received in the medial neuroectoderm, where it controls neurogenesis and axon outgrowth, whereas the spatial colinear hox gene expression in the neuroectoderm remains unaffected. These findings suggest that one early role of the new RAR in bilaterian evolution was to control the spatially restricted onset of motor and interneuron differentiation in the developing ventral nerve cord and to indicate that the regulation of hox-controlled anterior-posterior patterning arose only at the base of the chordates, concomitant with a high-affinity RAR needed for the interpretation of a complex RA gradient

Development of linear free energy relationships for aqueous phase radical-involved chemical reactions

Aqueous phase advanced oxidation processes (AOPs) produce hydroxyl radicals (HO•) which can completely oxidize electron rich organic compounds. The proper design and operation of AOPs require that we predict the formation and fate of the byproducts and their associated toxicity. Accordingly, there is a need to develop a first-principles kinetic model that can predict the dominant reaction pathways that potentially produce toxic byproducts. We have published some of our efforts on predicting the elementary reaction pathways and the HO• rate constants. Here we develop linear free energy relationships (LFERs) that predict the rate constants for aqueous phase radical reactions. The LFERs relate experimentally obtained kinetic rate constants to quantum mechanically calculated aqueous phase free energies of activation. The LFERs have been applied to 101 reactions, including (1) HO• addition to 15 aromatic compounds; (2) addition of molecular oxygen to 65 carbon-centered aliphatic and cyclohexadienyl radicals; (3) disproportionation of 10 peroxyl radicals, and (4) unimolecular decay of nine peroxyl radicals. The LFERs correlations predict the rate constants within a factor of 2 from the experimental values for HO• reactions and molecular oxygen addition, and a factor of 5 for peroxyl radical reactions. The LFERs and the elementary reaction pathways will enable us to predict the formation and initial fate of the byproducts in AOPs. Furthermore, our methodology can be applied to other environmental processes in which aqueous phase radical-involved reactions occur

Use of Fast HPLC Multiple Reaction Monitoring Cubed for Endogenous Retinoic Acid Quantification in Complex Matrices

Retinoic acid (RA), an essential active metabolite of vitamin A, controls numerous physiological processes. In addition to the analytical challenges owing to its geometric isomers, low endogenous abundance, and often localized occurrence, nonspecific interferences observed during liquid chromatography (LC) multiple reaction monitoring (MRM) quantification methods have necessitated lengthy chromatography to obtain accurate quantification free of interferences. We report the development and validation of a fast high performance liquid chromatography (HPLC) multiplexing multiple reaction monitoring cubed (MRM³) assay for selective and sensitive quantification of endogenous RA from complex matrices. The fast HPLC separation was achieved using an embedded amide C18 column packed with 2.7 μm fused-core particles which provided baseline resolution of endogenous RA isomers (all-trans-RA, 9-<i>cis</i>-RA, 13-<i>cis</i>-RA, and 9,13-di-<i>cis</i>-RA) and demonstrated significant improvements in chromatographic efficiency compared to porous particle stationary phases. Multiplexing technology further enhanced sample throughput by a factor of 2 by synchronizing parallel HPLC systems to a single mass spectrometer. The fast HPLC multiplexing MRM³ assay demonstrated enhanced selectivity for endogenous RA quantification in complex matrices and had comparable analytical performance to robust, validated LC-MRM methodology for RA quantification. The quantification of endogenous RA using the described assay was validated on a number of mouse tissues, nonhuman primate tissues, and human plasma samples. The combined integration of fast HPLC, MRM³, and multiplexing yields an analysis workflow for essential low-abundance endogenous metabolites that has enhanced selectivity in complex matrices and increased throughput that will be useful in efficiently interrogating the biological role of RA in larger study populations

Altered RBP1 Gene Expression Impacts Epithelial Cell Retinoic Acid, Proliferation, and Microenvironment

Vitamin A is an essential diet-derived nutrient that has biological activity affected through an active metabolite, all-trans retinoic acid (atRA). Retinol-binding protein type 1 (RBP1) is an intracellular chaperone that binds retinol and retinal with high affinity, protects retinoids from non-specific oxidation, and delivers retinoids to specific enzymes to facilitate biosynthesis of RA. RBP1 expression is reduced in many of the most prevalent cancers, including breast cancer. Here, we sought to understand the relationship between RBP1 expression and atRA biosynthesis in mammary epithelial cells, as well as RBP1 expression and atRA levels in human mammary tissue. We additionally aimed to investigate the impact of RBP1 expression and atRA on the microenvironment as well as the potential for therapeutic restoration of RBP1 expression and endogenous atRA production. Using human mammary ductal carcinoma samples and a series of mammary epithelial cell lines representing different stages of tumorigenesis, we investigated the relationship between RBP1 expression as determined by QPCR and atRA via direct liquid chromatography-multistage-tandem mass spectrometry-based quantification. The functional effect of RBP1 expression and atRA in epithelial cells was investigated via the expression of direct atRA targets using QPCR, proliferation using Ki-67 staining, and collagen deposition via picrosirius red staining. We also investigated the atRA content of stromal cells co-cultured with normal and tumorigenic epithelial cells. Results show that RBP1 and atRA are reduced in mammary tumor tissue and tumorigenic epithelial cell lines. Knock down of RBP1 expression using shRNA or overexpression of RBP1 supported a direct relationship between RBP1 expression with atRA. Increases in cellular atRA were able to activate atRA direct targets, inhibit proliferation and inhibit collagen deposition in epithelial cell lines. Conditions encountered in tumor microenvironments, including low glucose and hypoxia, were able to reduce RBP1 expression and atRA. Treatment with either RAR&alpha; agonist AM580 or demethylating agent Decitabine were able to increase RBP1 expression and atRA. Cellular content of neighboring fibroblasts correlated with the RA producing capacity of epithelial cells in co-culture. This work establishes a direct relationship between RBP1 expression and atRA, which is maintained when RBP1 expression is restored therapeutically. The results demonstrate diseases with reduced RBP1 could potentially benefit from therapeutics that restore RBP1 expression and endogenous atRA

All-Trans Retinoic Acid Activity in Acute Myeloid Leukemia: Role of Cytochrome P450 Enzyme Expression by the Microenvironment

<div><p>Differentiation therapy with all-trans retinoic acid (atRA) has markedly improved outcome in acute promyelocytic leukemia (APL) but has had little clinical impact in other AML sub-types. Cell intrinsic mechanisms of resistance have been previously reported, yet the majority of AML blasts are sensitive to atRA <i>in vitro</i>. Even in APL, single agent atRA induces remission without cure. The microenvironment expression of cytochrome P450 (CYP)26, a retinoid-metabolizing enzyme was shown to determine normal hematopoietic stem cell fate. Accordingly, we hypothesized that the bone marrow (BM) microenvironment is responsible for difference between <i>in vitro</i> sensitivity and <i>in vivo</i> resistance of AML to atRA-induced differentiation. We observed that the pro-differentiation effects of atRA on APL and non-APL AML cells as well as on leukemia stem cells from clinical specimens were blocked by BM stroma. In addition, BM stroma produced a precipitous drop in atRA levels. Inhibition of CYP26 rescued atRA levels and AML cell sensitivity in the presence of stroma. Our data suggest that stromal CYP26 activity creates retinoid low sanctuaries in the BM that protect AML cells from systemic atRA therapy. Inhibition of CYP26 provides new opportunities to expand the clinical activity of atRA in both APL and non-APL AML.</p></div

Retinoic acid biosynthesis is impaired in human and murine endometriosis.

Endometriosis is characterized by the presence of endometrial glands and stroma in extrauterine sites. Our objective was to determine whether endometriotic lesions (ELs) from women with endometriosis have altered retinoid levels compared with their eutopic endometrium, and to test the hypothesis that defects in all-trans retinoic acid (ATRA) biosynthesis in EL is related to reduced expression of cellular retinol-binding protein type 1 (RBP1). Retinoids were evaluated by liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography in eutopic endometrial biopsies (EBs) and ELs from 42 patients with pathologically confirmed endometriosis. The ATRA levels were reduced, whereas the retinol and retinyl ester concentrations were elevated in EL compared with EB tissue. Similar results were found in a mouse model of endometriosis that used green fluorescent protein-positive endometrial tissue injected into the peritoneum of syngeneic hosts to mimic retrograde menses. The ATRA biosynthesis in vitro in retinol-treated primary human endometrial stromal cell (ESC) cultures derived from ELs was reduced compared with that of ESCs derived from patient-matched EBs. Correspondingly, RBP1 expression was reduced in tissue and ESCs derived from EL versus EB. Rbp1(-/-) mice showed reduced endometrial ATRA concentrations compared with wild type, associated with loss of tissue organization and hypercellularity. These findings provide the first quantitative measurements of ATRA in human endometrium and endometriosis, demonstrating reduced ATRA in ectopic tissue and corresponding ESC cultures. Quantitation of retinoids in murine endometriosis and in Rbp1(-/-) mice supports the contention that impaired ATRA synthesis caused by reduced RBP1 promotes an "endometriosis phenotype" that enables cells to implant and grow at ectopic sites