The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL

For more than two decades, there has been continuing evidence of lipid oxidation playing a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways. The failure of antioxidant vitamins to influence clinical outcomes may have many explanations, including the inability of vitamin E to prevent the formation of these oxidized phospholipids and other lipid oxidation products of the myeloperoxidase pathway. Preliminary data suggest that the oxidation hypothesis of atherogenesis and the reverse cholesterol transport hypothesis of atherogenesis may have a common biological basis. The levels of specific oxidized lipids in plasma and lipoproteins, the levels of antibodies to these lipids, and the inflammatory/anti-inflammatory properties of HDL may be useful markers of susceptibility to atherogenesis. Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides may both promote a reduction in oxidized lipids and enhance reverse cholesterol transport and therefore may have therapeutic potential.     

Two novel hexadepsipeptides with several modified amino acid residues isolated from the fungus Isaria

Two new cyclohexadepsipeptides have been isolated from the fungus Isaria. Fungal growth in solid media yielded hyphal strands from which peptide fractions were readily isolable by organic-solvent extraction. Two novel cyclodepsipeptides, isaridin A and isaridin B, have been isolated by reverse-phase HPLC, and characterized by ESI-MS and 1H-NMR. Single crystals of both peptides have been obtained, and their 3D structures were elucidated by X-ray diffraction. The isaridins contain several unusual amino acid residues. The sequences are cyclo(beta-Gly-HyLeu-Pro-Phe-NMeVal-NMePhe) and cyclo(beta-Gly-HyLeu-beta-MePro-Phe-NMeVal-NMePhe), where NMeVal is N-methylvaline, NMePhe N-methylphenylalanine, and HyLeu hydroxyleucine (= 2-hydroxy-4-methylpentanoic acid). The two peptides differ from one another at residue 3, isaridin A having an (S)-proline at this position, while beta-methyl-(S)-proline (= (2S,3S)-2,3,4,5-tetrahydro-3-methyl-1H-pyrrole-2-carboxylic acid) is found in isaridin B. The solid-state conformations of both cyclic depsipeptides are characterized by the presence of two cis peptide bonds at HyLeu(2)-Pro(3)/HyLeu(2)-beta-MePro(3) and NMeVal(5)-NMePhe(6), respectively. In isaridin A, a strong intramolecular H-bond is observed between Phe(4)CO...HNbeta-Gly(1), and a similar, but weaker, interaction is observed between beta-Gly(1)CO...HNPhe(4). In contrast, in isaridin B, only a single intramolecular H-bond is observed between beta-Gly(1)CO...HNPhe(4).     

Polar substitutions in the benzenesulfonamide ring of celecoxib afford a potent 1,5-diarylpyrazole class of COX-2 inhibitors

Several chemical modifications in the N(1)-benzenesulfonamide ring of celecoxib are presented. The series with a hydroxymethyl group adjacent to the sulfonamide was found to be the most potent modification that yielded many compounds selectively active against COX-2 enzyme in vitro.     

Pharmaceutical development and manufacturing of a parenteral formulation of a novel antitumor agent, VNP40101M

The objective of this study was to develop and manufacture a stable parenteral formulation for Phase I clinical trials of VNP40101M (1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(2-methylamino)carbonyl] hydrazine), a novel antitumor agent. The solubility and stability of the drug was determined. Solubility studies suggested that VNP40101M exhibited poor aqueous solubility but showed appreciable solubility in nonaqueous solvents. The aqueous solubility of the drug could not be increased by adjusting the pH. At a pH above 7, basecatalyzed decomposition of VNP40101M occurred. The low octanol-water partition coefficient of 0.75 suggested poor solubility in lipophilic solvents. Based on these preformation observations, a parenteral formulation containing 10 mg/mL of VNP40101M was prepared in a solvent system consisting of 30% ethyl alcohol and 70% polyethylene glycol-300 (PEG-300). To minimize base-catalyzed hydrolytic degradation. citric acid at 0.6% concentration was included to acidify the formulation. Rubber closures, filter membranes, and liquid transfer tubing were selected on the basis of compatibility studies and absence of loss of drug the of adsorption of these components. The formulation was subjected to accelerated stability studies and dilution studies with large volume parenteral (LVP) solutions, normal saline, and 5% dextrose injection (D5W). The results of the dilution study indicated that the formulation could be diluted in these solutions up to 2 mg/mL for 8 hours without drug precipitation and degradation. Accelerated stability studies suggested that the product should be kept at 2°C to 8°C for long-term storage. The developed formulation was successfully scaled up and manufactured for use in clinical trials. Published: August 26, 2001.     

PRR5L degradation promotes mTORC2-mediated PKC-δ phosphorylation and cell migration downstream of Gα12

Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates AGC protein kinases including protein kinase C (PKC) and regulates cellular functions such as cell migration. However, its regulation remains poorly understood. Here we show that lysophosphatidic acid (LPA) induces two phases of PKC-δ hydrophobic motif phosphorylation. The late phase is mediated by Gα(12), which specifically activates ARAF, leading to upregulation of the RFFL E3 ubiquitin ligase and subsequent ubiquitylation and degradation of the PRR5L subunit of mTORC2. Destabilization of PRR5L, a suppressor of mTORC2-mediated hydrophobic motif phosphorylation of PKC-δ, but not AKT, results in PKC-δ hydrophobic motif phosphorylation and activation. This Gα(12)-mediated signalling pathway for mTORC2 regulation is critically important for fibroblast migration and pulmonary fibrosis development.     

Local loperamide injection reduces mechanosensitivity of rat cutaneous, nociceptive C-fibers

Loperamide reverses signs of mechanical hypersensitivity in an animal model of neuropathic pain suggesting that peripheral opioid receptors may be suitable targets for the treatment of neuropathic pain. Since little is known about loperamide effects on the responsiveness of primary afferent nerve fibers, in vivo electrophysiological recordings from unmyelinated afferents innervating the glabrous skin of the hind paw were performed in rats with an L5 spinal nerve lesion or sham surgery. Mechanical threshold and responsiveness to suprathreshold stimulation were tested before and after loperamide (1.25, 2.5 and 5 µg in 10 µl) or vehicle injection into the cutaneous receptive field. Loperamide dose-dependently decreased mechanosensitivity in unmyelinated afferents of nerve-injured and sham animals, and this effect was not blocked by naloxone pretreatment. We then investigated loperamide effects on nerve conduction by recording compound action potentials in vitro during incubation of the sciatic nerve with increasing loperamide concentrations. Loperamide dose-dependently decreased compound action potentials of myelinated and unmyelinated fibers (ED50 = 8 and 4 µg/10 µl, respectively). This blockade was not prevented by pre-incubation with naloxone. These results suggest that loperamide reversal of behavioral signs of neuropathic pain may be mediated, at least in part, by mechanisms independent of opioid receptors, most probably by local anesthetic actions.     

Spectroscopic and theoretical insights into sequence effects of aminofluorene-induced conformational heterogeneity and nucleotide excision repair

A systematic spectroscopic and computational study was conducted in order to probe the influence of base sequences on stacked (S) versus B-type (B) conformational heterogeneity induced by the major dG adduct derived from the model carcinogen 7-fluoro-2-aminofluorene (FAF). We prepared and characterized eight 12-mer DNA duplexes (-AG*N- series, d[CTTCTAG*NCCTC]; -CG*N- series, d[CTTCTCG*NCCTC]), in which the central guanines (G*) were site-specifically modified with FAF with varying flanking bases (N = G, A, C, T). S/B heterogeneity was examined by CD, UV, and dynamic 19F NMR spectroscopy. All the modified duplexes studied followed a typical dynamic exchange between the S and B conformers in a sequence dependent manner. Specifically, purine bases at the 3'-flanking site promoted the S conformation (G > A > C > T). Simulation analysis showed that the S/B energy barriers were in the 14-16 kcal/mol range. The correlation times (tau = 1/kappa) were found to be in the millisecond range at 20 degrees C. The van der Waals energy force field calculations indicated the importance of the stacking interaction between the carcinogen and neighboring base pairs. Quantum mechanics calculations showed the existence of correlations between the total interaction energies (including electrostatic and solvation effects) and the S/B population ratios. The S/B equilibrium seems to modulate the efficiency of Escherichia coli UvrABC-based nucleotide excision repair in a conformation-specific manner: i.e., greater repair susceptibility for the S over B conformation and for the -AG*N- over the -CG*N- series. The results indicate a novel structure-function relationship, which provides insights into how bulky DNA adducts are accommodated by UvrABC proteins.     

Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.