Arsenic Exposure Increases Monocyte Adhesion to the Vascular Endothelium,a Pro-Atherogenic Mechanism

Epidemiological studies have shown that arsenic exposure increases atherosclerosis, but the mechanisms underlying this relationship are unknown. Monocytes, macrophages and platelets play an important role in the initiation of atherosclerosis. Circulating monocytes and macrophages bind to the activated vascular endothelium and migrate into the sub-endothelium, where they become lipid-laden foam cells. This process can be facilitated by platelets, which favour monocyte recruitment to the lesion. Thus, we assessed the effects of low-to-moderate arsenic exposure on monocyte adhesion to endothelial cells, platelet activation and platelet-monocyte interactions. We observed that arsenic induces human monocyte adhesion to endothelial cells in vitro. These findings were confirmed ex vivo using a murine organ culture system at concentrations as low as 10 ppb. We found that both cell types need to be exposed to arsenic to maximize monocyte adhesion to the endothelium. This adhesion process is specific to monocyte/endothelium interactions. Hence, no effect of arsenic on platelet activation or platelet/leukocyte interaction was observed. We found that arsenic increases adhesion of mononuclear cells via increased CD29 binding to VCAM-1, an adhesion molecule found on activated endothelial cells. Similar results were observed in vivo, where arsenic-exposed mice exhibit increased VCAM-1 expression on endothelial cells and increased CD29 on circulating monocytes. Interestingly, expression of adhesion molecules and increased binding can be inhibited by antioxidants in vitro and in vivo. Together, these data suggest that arsenic might enhance atherosclerosis by increasing monocyte adhesion to endothelial cells, a process that is inhibited by antioxidants.     

Cloning and Functional Characterization of the Maize (Zea mays L.) Carotenoid Epsilon Hydroxylase Gene

The assignment of functions to genes in the carotenoid biosynthesis pathway is necessary to understand how the pathway is regulated and to obtain the basic information required for metabolic engineering. Few carotenoid ε-hydroxylases have been functionally characterized in plants although this would provide insight into the hydroxylation steps in the pathway. We therefore isolated mRNA from the endosperm of maize (Zea mays L., inbred line B73) and cloned a full-length cDNA encoding CYP97C19, a putative heme-containing carotenoid ε hydroxylase and member of the cytochrome P450 family. The corresponding CYP97C19 genomic locus on chromosome 1 was found to comprise a single-copy gene with nine introns. We expressed CYP97C19 cDNA under the control of the constitutive CaMV 35S promoter in the Arabidopsis thaliana lut1 knockout mutant, which lacks a functional CYP97C1 (LUT1) gene. The analysis of carotenoid levels and composition showed that lutein accumulated to high levels in the rosette leaves of the transgenic lines but not in the untransformed lut1 mutants. These results allowed the unambiguous functional annotation of maize CYP97C19 as an enzyme with strong zeinoxanthin ε-ring hydroxylation activity.     

Synthesis and in vitro antitumor effect of vinblastine derivative-oligoarginine conjugates

Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the N-terminal amino function of L-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia (HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg? > Arg? ? Arg?. The conjugate with Arg? exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp. The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvinblastineTrp-Arg?-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR data suggest that 17-desacetylvinblastineTrp-Arg?-1 and 17-desacetylvinblastineTrp-Arg?-2 are epimers at the tryptophan α carbon atom.     

The ��TuRC Revisited: A Comparative Analysis of Interphase and Mitotic Human ��TuRC Redefines the Set of Core Components and Identifies the Novel Subunit GCP8

The γ-tubulin complex is a multi-subunit protein complex that nucleates microtubule polymerization. γ-Tubulin complexes are present in all eukaryotes, but size and subunit composition vary. In Drosophila, Xenopus, and humans large γ-tubulin ring complexes (γTuRCs) have been described, which have a characteristic open ring-shaped structure and are composed of a similar set of subunits, named γ-tubulin, GCPs 2-6, and GCP-WD in humans. Despite the identification of these proteins, γTuRC function and regulation remain poorly understood. Here we establish a new method for the purification of native human γTuRC. Using mass spectrometry of whole protein mixtures we compared the composition of γTuRCs from nonsynchronized and mitotic human cells. Based on our analysis we can define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic γTuRCs. We also identified GCP8/MOZART2 as a novel core subunit that is present in both interphase and mitotic γTuRCs. GCP8 depletion does not affect γTuRC assembly but interferes with γTuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects, suggesting that GCP8 specifically affects the organization of the interphase microtubule network.     

ChemMatrix® for complex peptides and combinatorial chemistry

CM resin is a totally PEG‐based resin, made exclusively from primary ether bonds and therefore highly chemically stable. Compared to other PEG resins, it exhibits good loading and is user friendly because of its free‐flowing form upon drying. It shows improved performance over PS resins for the preparation of hydrophobic, highly structured poly‐Arg peptides. In combination with ψPros, it allows the synthesis of small proteins such as the chemokine RANTES. Like other PEG‐based resins, CM resin swells well in biocompatible solvents such as water, thereby allowing on‐bead screening. Furthermore, the high loading of this resin permits the use of a tiny quarter of a bead as a microreactor for HPLC and MALDI‐TOF analysis, thus further extending its applications in the field of combinatorial chemistry. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Quantification of the oxidative damage biomarker 2,3-dinor-8-isoprostaglandin-F2α in human urine using liquid chromatography-tandem mass spectrometry

F₂-isoprostanes are useful biomarkers of oxidative status in humans. We developed an ultraperformance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 2,3-dinor-8-iso prostaglandin F_2α, a urinary metabolite of 8-iso-prostaglandin F_2α. Urine was purified by solid-phase extraction and analyzed by UPLC-MS/MS with negative-ion electrospray ionization. The method was robust with a mean inaccuracy of 9%, interday and intraday imprecision of 7.5% or lower, and a lower limit of quantification of 0.5 μg/L, equivalent to 0.04 pmol injected onto the column. An analysis time of 6 min was shorter than previously published methods and amenable to large studies.     

Evaluation of Drug Release From Coated Pellets Based on Isomalt,Sugar, and Microcrystalline Cellulose Inert Cores

The objective of the present study was to investigate the effect of the pellet core materials isomalt, sugar, and microcrystalline cellulose on the in vitro drug release kinetics of coated sustained-release pellets as well as to evaluate the influence of different ratios of polymethacrylate copolymers exhibiting different permeability characteristics on the drug release rate. For characterization of the drug release process of pellets, the effect of osmolality was studied using glucose as an osmotically active agent in the dissolution medium. The pellet cores were layered with diclofenac sodium as model drug and coated with different ratios of Eudragit® RS30D and Eudragit® RL30D (ERS and ERL; 0:1 and 0.5:0.5 and 1:0 ratio) in a fluid bed apparatus. Physical characteristics such as mechanical strength, shape, and size proved that the inert cores were adequate for further processing. The in vitro dissolution tests were performed using a USP Apparatus I (basket method). The results demonstrated that, besides the ratio of the coating polymers (ERS/ERL), the release mechanism was also influenced by the type of starter core used. Sugar- and isomalt-type pellet cores demonstrated similar drug release profiles.     

Real-time fluorescence detection of ERAD substrate retrotranslocation in a mammalian in vitro system

Secretory proteins unable to assemble into their native states in the endoplasmic reticulum (ER) are transported back or "retrotranslocated" into the cytosol for ER-associated degradation (ERAD). To examine the roles of different components in ERAD, one fluorescence-labeled ERAD substrate was encapsulated with selected lumenal factors inside mammalian microsomes. After mixing microsomes with fluorescence-quenching agents and selected cytosolic proteins, the rate of substrate efflux was monitored continuously in real time by the decrease in fluorescence intensity as cytosolic quenchers contacted dye-labeled substrates. The retrotranslocation kinetics of nonglycosylated pro-alpha factor were not significantly altered by replacing all lumenal proteins with only protein disulfide isomerase or all cytosolic proteins with only PA700, the 19S regulatory particle of the 26S proteasome. Retrotranslocation was blocked by antibodies against a putative retrotranslocation channel protein, derlin-1, but not Sec61alpha. In addition, pro-alpha factor photocrosslinked derlin-1, but not Sec61alpha. Thus, derlin-1 appears to be involved in pro-alpha factor retrotranslocation.