Synthesis of all-trans anandamide: a substrate for fatty acid amide hydrolase with dual effects on rabbit platelet activation

Anandamide (AEA) presents the four double bonds in the cis configuration, deriving from the arachidonic acid moiety. In the context of an antisense strategy based on the double bond configuration, all-trans AEA (t-AEA) was synthesized in high yield starting from all-trans methyl arachidonate and ethanolamine in the presence of KCN. t-AEA was assayed on rabbit platelet aggregation, obtaining effect only at high concentrations (>10(-4) M) after an also concentration-dependent lag phase. At lower concentrations it inhibited PAF-induced rabbit platelet aggregation with an IC(50)=4.6 x 10(-6) M. In contrast to anandamide, the activation of platelets was not due to the conversion of t-AEA to trans arachidonic acid, as ascertained by negative results with FAAH inhibitors. However, t-AEA was found to be a substrate for fatty acid amide hydrolase (FAAH), the enzyme that cleaves anandamide and regulates in vivo the magnitude and duration of the signaling induced by this lipid messenger.     

Intracellular mechanisms regulating corticotropin-releasing hormone receptor-2beta endocytosis and interaction with extracellularly regulated kinase 1/2 and p38 mitogen-activated protein kinase signaling cascades

Many important physiological roles of the urocortin (UCN) family of peptides as well as CRH involve the type 2 CRH receptor (CRH-R2) and downstream activation of multiple pathways. To characterize molecular determinants of CRH-R2 functional activity, we used HEK293 cells overexpressing recombinant CRH-R2beta and investigated mechanisms involved in attenuation of CRH-R2 signaling activity and uncoupling from intracellular effectors. CRH-R2beta-mediated adenylyl cyclase activation was sensitive to homologous desensitization induced by pretreatment with either UCN-II or the weaker agonist CRH. CRH-R2beta activation induced transient beta-arrestin1 and beta-arrestin2, as well as clathrin, recruitment to the plasma membrane. Beta-arrestin2 appeared to be the main beta-arrestin subtype associated with the receptor. This was followed by CRH-R2beta endocytosis in a mechanism that exhibited distinct agonist-dependent temporal characteristics. CRH-R2beta also induced transient activation of the ERK1/2 and p38MAPK signaling cascades that peaked at 5 min and returned to basal within 20-30 min. Unlike p38MAPK, activated ERK1/2 was localized both in the cytoplasm and nucleus. Experiments employing inhibitors of receptor endocytosis showed that CRH-R2beta-MAPK interaction does not require beta-arrestin, clathrin, or receptor endocytosis. Site-directed mutagenesis studies on CRH-R2beta C terminus showed that the amino acid cassette TAAV at the end of the C terminus is important for CRH-R2beta signaling because loss of a potential phospho-acceptor site in mutant receptors containing deletion or Ala substitution of the cassette TAAV resulted in reduced ERK1/2 activation and accelerated receptor internalization. These findings provide new insights about the signaling mechanisms regulating CRH-R2beta functional activity and determining its biological responses.     

Detoxification of 2,4-dichlorophenol by the marine microalga Tetraselmis marina

Xenobiotic chlorinated phenols have been found in fresh and marine waters and are toxic to many aquatic organisms. Metabolism of 2,4-dichlorophenol (2,4-DCP) in the marine microalga Tetraselmis marina was studied. The microalga removed more than 1mM of 2,4-DCP in a 2l photobioreactor over a 6 day period. Two metabolites, more polar than 2,4-DCP, were detected in the growth medium by reverse phase HPLC and their concentrations increased at the expense of 2,4-DCP. The metabolites were isolated by a C8 HPLC column and identified as 2,4-dichlorophenyl-beta-d-glucopyranoside (DCPG) and 2,4-dichlorophenyl-beta-d-(6-O-malonyl)-glucopyranoside (DCPGM) by electrospray ionization-mass spectrometric analysis in a negative ion mode. The molecular structures of 2,4-DCPG and 2,4-CPGM were further confirmed by enzymatic and alkaline hydrolyses. Thus, it was concluded that the major pathway of 2,4-DCP metabolism in T. marina involves an initial conjugation of 2,4-DCP to glucose to form 2,4-dichlorophenyl-beta-d-glucopyranoside, followed by acylation of the glucoconjugate to form 2,4-dichlorophenyl-beta-d-(6-O-malonyl)-glucopyranoside. The microalga ability to detoxify dichlorophenol congeners other than 2,4-DCP was also investigated. This work provides the first evidence that microalgae can use a combined glucosyl and malonyl transfer to detoxify xenobiotics such as dichlorophenols.     

Embryoid bodies from mouse stem cells express oxytocin receptor, Oct-4 and DAZL

Oxytocin is a nine amino acid peptide involved in a wide spectrum of physiological functions; predominantly those concerning reproduction and differentiation are of interest. Oxytocin receptors are expressed at early developmental stages of mammals, suggesting that oxytocin might be involved in the determination of the germ stem cell line, at the very early stages of mammalian development. In this respect, the proximate aim of the present study was to confirm and further analyze the existence of oxytocin receptors at a very early level of cell commitment, that is, the determination of germ cells derived from embryoid bodies. To achieve our purpose we have cultured mouse embryonic stem cells under conditions inducing formation of embryoid bodies. In this work, ES cells were allowed to aggregate in a novel medium, “Stefanidis medium” from day 0 to day 14 until formed EBs. RNA was isolated from EBs and using RT-PCR we showed that EBs expressed Oct-4, OTR, OT, and DAZL. To demonstrate simultaneous expression immunocytochemistry was preformed, in which EBs showed strong immunoreactivity for both, OTR and DAZL molecular markers. We found that 35% of the cells displayed OTR, using flow cytometry. In addition, this novel medium showed to increase OTR mRNA. We propose, that at least in murine induced embryoid bodies there is simultaneous expression of oxytocin receptors and germ cell markers (DAZL) in many cells (expressing Oct-4). We thus conclude that, the oxytocin might indeed be a molecule playing a leading role in germ cell determination.     

Organotin meclofenamic complexes: Synthesis, crystal structures and antiproliferative activity of the first complexes of meclofenamic acid - Novel anti-tuberculosis agents

The complexes [Me₂(Meclo)SnOSn(Meclo)Me₂]₂ (2) and [Ph₃Sn(Meclo)] (3) where HMeclo is meclofenamic acid, N-(2,6-dichloro-m-tolylanthranilic acid)], have been prepared and structurally characterized by means of vibrational, ¹H and ¹³C NMR spectroscopies. The crystal structure of complexes (2) and (3) have been determined by X-ray crystallography. Three distannoxane rings are present to the dimeric tetraorganodistannoxane of planar ladder arrangement of (2). The structure is centro symmetric and features a central rhombus Sn₂O₂ unit two additional tin atoms linked at the oxygen atoms. Five- and six-coordinated tin centers are present in the dimer distannoxane. X-ray analysis of (3) revealed a penta-coordinated structure containing Ph₃Sn coordinated to the chelated carboxylato group. The polar imino hydrogen atom participates in intra-molecular hydrogen bonds. Complexes (2) and (3) are self-assembled via π → π, C-H-π, stacking interactions and intra-molecular hydrogen bonds. Meclofenamic acid and [Ph₃Sn(Meclo)] have been evaluated for antiproliferative activity in vitro against three human cancer cell lines: MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line), A-549 (non-small cell lung carcinoma) and a mouse L-929 (a fibroblast-like cell line cloned from strain L). The [Ph₃Sn(Meclo)] complex exhibited high cytotoxic activity against all the cancer cell lines. Meclofenamic and [Ph₃Sn(Meclo)] were tested for anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv. The [Ph₃Sn(Meclo)] complex was found to be a promising anti-mycobacterial lead compound, displaying high activity against M. tuberculosis H37Rv.     

Complex dynamics of microbial competition in the gradostat

We examine the conditions necessary for the emergence of complex dynamic behavior in systems of microbial competition. In particular, we study the effect of spatial heterogeneity and substrate-inhibition on the dynamics of such a system. This is accomplished through the study of a mathematical model of two microbial populations competing for a single nutrient in a configuration of two interconnected chemostats. Microbial growth is assumed to follow substrate-inhibited kinetics for both species. Such a system with sterile feed has been shown in a previous work to exhibit stable periodic states. In the present work we study the system for the case of non-sterile feed, i.e., when the two species are present in the feed of the chemostats. The analysis is done by numerical bifurcation theory methods. We demonstrate that, in addition to periodic states, the system possesses stable quasi-periodic states resulting from Neimark-Sacker bifurcations of limit cycles. Also, periodic states may undergo successive period doublings leading to periodic states of increasing period and indicating that chaotic states might be possible. Multistability is also observed, consisting in the coexistence of several stable steady states and possibly stable periodic or quasi-periodic states for given operating conditions. It appears that substrate-inhibition, spatial heterogeneity and presence of microorganisms in the inflow are all necessary conditions for complex dynamics to arise in a microbial system of pure and simple competition.     

Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Monoclonal anti‐HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L ‐Phe/β‐Ala bi‐substituted 1,3,5‐triazine (Trz) scaffold (β‐Ala‐Trz‐L ‐Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10‐binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K_D) of 0.41 ± 0.05 µM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60–80%). Analysis of the antibody preparation by SDS‐PAGE, enzyme‐linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extract

Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract.