Guillaume Morissette

Herpesviruses and chromosomal integration.

ABSTRACT Herpesviruses are members of a diverse family of viruses that colonize all vertebrates from fish to mammals. Although more than one hundred herpesviruses exist, all are nearly identical architecturally, with a genome consisting of a linear double-stranded DNA molecule (100 to 225 kbp) protected by an icosahedral capsid made up of 162 hollow-centered capsomeres, a tegument surrounding the nucleocapsid, and a viral envelope derived from host membranes. Upon infection, the linear viral DNA is delivered to the nucleus, where it circularizes to form the viral episome. Depending on several factors, the viral cycle can proceed either to a productive infection or to a state of latency. In either case, the viral genetic information is maintained as extrachromosomal circular DNA. Interestingly, however, certain oncogenic herpesviruses such as Mareks disease virus and Epstein-Barr virus can be found integrated at low frequencies in the hosts chromosomes. These findings have mostly been viewed as anecdotal and considered exceptions rather than properties of herpesviruses. In recent years, the consistent and rather frequent detection (in approximately 1% of the human population) of human herpesvirus 6 (HHV-6) viral DNA integrated into human chromosomes has spurred renewed interest in our understanding of how these viruses infect, replicate, and propagate themselves. In this review, we provide a historical perspective on chromosomal integration by herpesviruses and present the current state of knowledge on integration by HHV-6 with the possible clinical implications associated with viral integration.

Intense pseudotransport of a cationic drug mediated by vacuolar ATPase: procainamide-induced autophagic cell vacuolization.

Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of cellular sequestration. The contributions of organelles and metabolic processes that may mimic drug transport were defined in human vascular smooth muscle cells. We hypothesized that procainamide-induced vacuolar cytopathology is driven by intense pseudotransport mediated by the vacuolar (V)-ATPase and pursued the characterization of vesicular trafficking alterations in this model. Large amounts of procainamide were taken up by intact cells (maximal in 2 h, reversible upon washout, apparent KM 4.69 mM; fluorometric determination of cell-associated drug). Procainamide uptake was extensively prevented or reversed by pharmacological inhibition of the V-ATPase with bafilomycin A1 or FR 167356, decreased at low extracellular pH and preceded vacuolar cell morphology. However, the uptake of procainamide was unaffected by mitochondrial poisons that reduced the uptake of rhodamine 6G. Large vacuoles induced by millimolar procainamide were labeled with the late endosome/lysosome markers Rab7 and CD63 and the autophagy effector LC3; their osmotic formation (but not procainamide uptake) was reduced by extracellular mannitol and parallel to LC3 II formation. Procainamide-induced vacuolization is associated with defective endocytosis of fluorophore-labeled bovine serum albumin, but not with induction of the unfolded protein response. The contents of a vacuole subset slowly (> or =24 h) become positive for Nile red staining (phospholipidosis-like response). V-ATPase-driven ion trapping is a form of intense cation pseudotransport that concerns the uncharged form of the drugs, and is associated with a vacuolar, autophagic and evolutive cytopathology and profound effects on vesicular trafficking.

Inherited chromosomally integrated human herpesvirus 6 as a predisposing risk factor for the development of angina pectoris

Significance Based on several studies, including ours, we estimate that 40–70 million individuals carry a chromosomally integrated copy of the human herpesvirus 6 (HHV-6) genome in every cell of their body. This condition is referred to as inherited chromosomally integrated HHV-6 (iciHHV-6). The regions targeted for integration are telomeres, which play important roles in the self-renewal capacity of cells. Whether iciHHV-6 is associated with disease remains unknown. After conducting a population screen (n = 20,000), our results indicate that the prevalence of angina is three times greater in iciHHV-6+ subjects relative to iciHHV-6− ones. Furthermore, iciHHV-6+ subjects have shorter telomeres, a result that may explain, at least in part, how iciHHV-6 may contribute to the development of angina. Inherited chromosomally integrated human herpesvirus-6 (iciHHV-6) results in the germ-line transmission of the HHV-6 genome. Every somatic cell of iciHHV-6+ individuals contains the HHV-6 genome integrated in the telomere of chromosomes. Whether having iciHHV-6 predisposes humans to diseases remains undefined. DNA from 19,597 participants between 40 and 69 years of age were analyzed by quantitative PCR (qPCR) for the presence of iciHHV-6. Telomere lengths were determined by qPCR. Medical records, hematological, biochemical, and anthropometric measurements and telomere lengths were compared between iciHHV-6+ and iciHHV-6− subjects. The prevalence of iciHHV-6 was 0.58%. Two-way ANOVA with a Holm–Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on continuous outcomes. Two-way logistic regression with a Holm–Bonferroni correction was used to determine the effects of iciHHV6, sex, and their interaction on disease prevalence. Of 50 diseases monitored, a single one, angina pectoris, is significantly elevated (3.3×) in iciHHV-6+ individuals relative to iciHHV-6− subjects (P = 0.017; 95% CI, 1.73–6.35). When adjusted for potential confounding factors (age, body mass index, percent body fat, and systolic blood pressure), the prevalence of angina remained three times greater in iciHHV-6+ subjects (P = 0.015; 95%CI, 1.23–7.15). Analyses of telomere lengths between iciHHV-6− without angina, iciHHV-6− with angina, and iciHHV-6+ with angina indicate that iciHHV-6+ with angina have shorter telomeres than age-matched iciHHV-6− subjects (P = 0.006). Our study represents, to our knowledge, the first large-scale analysis of disease association with iciHHV-6. Our results are consistent with iciHHV-6 representing a risk factor for the development of angina.

B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-bradykinin) Is an Inactivation-Resistant Agonist of the Bradykinin B2 Receptor Derived from the Peptide Antagonist B-9430 (D-Arg-[Hyp3,Igl5,D-Igl7,Oic8]-bradykinin): Pharmacologic Profile and Effective Induction of Receptor Degradation

The bradykinin B2 receptor is a heptahelical receptor regulated by a cycle of phosphorylation, endocytosis, and extensive recycling at the cell surface following agonist stimulation. B-9430 (d-Arg-[Hyp3,Igl5,d-Igl7,Oic8]-bradykinin) is a second generation peptide antagonist found to be competitive at the human B2 receptor and insurmountable at the rabbit B2 receptor (contractility assays, isolated human umbilical and rabbit jugular veins). Two isomers of this peptide were prepared: B-10344 (d-Arg-[Hyp3,Igl5,Oic7,d-Igl8]-bradykinin; inverted sequence Oic7, d-Igl8) and B-9972 (d-Arg-[Hyp3,Igl5,Oic7,Igl8]-bradykinin); they are low- and high-potency agonists, respectively, in vascular preparations. The potency gap between bradykinin and B-9972 is narrow in contractility assays, despite the fact that B-9972 affinity is 7-fold inferior at the rabbit B2 receptor (radioligand binding competition assay). The effects of agonists on receptors were compared using two chimerical constructions based on rabbit B2 receptors: conjugate of the B2 receptor with green fluorescent protein (B2R-GFP) and the N-terminally tagged conjugate of the myc epitope with the B2 receptor. Imaging and immunoblotting showed that B-9972 induced a persistent endocytosis of cell surface B2 receptors in human embryonic kidney 293 cells with slow receptor degradation (weak after 3 h of treatment, important at 12 h) and B2R-GFP desensitization ([3H]bradykinin endocytosis and extracellular signal-regulated kinase 1/2 phosphorylation assays). Bradykinin was not active in this respect but when combined with captopril, induced some degradation. B-9430 reduced the endocytosis and degradation of B2 receptors by the agonists. The results illustrate the agonist-antagonist transition in B2 receptor peptide ligands with a constrained C-terminal structure, the importance of species in their pharmacological profile, and the possibility of selectively degrading receptors using a peptidase-resistant agonist.

Role of Nuclear Factor-κB and Protein Kinase C Signaling in the Expression of the Kinin B1 Receptor in Human Vascular Smooth Muscle Cells

Kinin B1 receptor expression was characterized in human umbilical artery smooth muscle cells to further elucidate the function and specificity of three previously proposed pathways [nuclear factor-κB (NF-κB), protein kinase C, and agonist autoregulation] that regulate this inducible G protein-coupled receptor. Radioligand binding assays, real-time reverser transcription/polymerase chain reaction with an optional actinomycin D treatment period, and NF-κB immunofluorescence were primarily employed in these primary cell cultures. Various stimulatory compounds that increase receptor mRNA stability only (human and bovine sera, cycloheximide) or that stimulate NF-κB nuclear translocation and both mRNA concentration and stability [interleukin (IL)-1β, phorbol 12-myristate 13-acetate (PMA)] all increased the density of binding sites for the tritiated B1 receptor agonist [3H]Lys-des-Arg9-bradykinin (without change in receptor affinity) in cell-based assays. Small interfering RNA assays indicated that NF-κB p65 is necessary for the effective expression of the cell surface B1 receptor under basal or IL-1β, fetal bovine serum (FBS), or PMA stimulation conditions. Dexamethasone cotreatment reproduced these effects. IL-1β-, FBS-, or PMA-induced stabilization of B1 receptor mRNA was inhibited by the addition of the protein kinase C inhibitor 3-[1-[3-(dimethylamino)propyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride (GF-109203x), which also diminished the Bmax under FBS or PMA treatment. Lys-des-Arg9-bradykinin had little effect on NF-κB activation, the Bmax, or receptor mRNA abundance or stability. Both NF-κB and protein kinase C signaling are required for the effective expression of the kinin B1 receptor in human umbilical artery smooth muscle cells.

Vacuolar ATPase-Mediated Cellular Concentration and Retention of Quinacrine: A Model for the Distribution of Lipophilic Cationic Drugs to Autophagic Vacuoles

The antiprotozoal agent quinacrine is a lipophilic cationic drug highly distributed to tissues. It has been used in the present experiments to examine whether the vacuolar and autophagic cytopathology induced by organic amines is independent from the therapeutic class. Furthermore, we tested the presence of the concentrated cationic drug itself in the enlarged vacuoles by exploiting the intense green fluorescence of quinacrine. Finally, the influence of lipophilicity on the apparent affinity of amine pseudotransport has been addressed by comparing quinacrine to another substituted triethylamine, procainamide. Quinacrine was concentration-dependently taken up by human smooth muscle cells (cytosolic granular-vacuolar morphology at and above 25 nM; in cell extracts, uptake nearly maximal in 2 h, apparent Km of 8.7 μM). The vacuolar (V)-ATPase inhibitor bafilomycin A1 prevented quinacrine uptake by cells or released the cell-associated drug in preloaded cells. The lipidated (II) form of microtubule-associated protein light chain 3 accumulated at and above a quinacrine concentration of 2.5 μM (4 h), indicating the conserved macroautophagic nature of the vacuolar cytopathology, although vacuole size was modest. The enlarged vacuoles containing quinacrine excluded cherry fluorescent protein; many vacuoles were lined with cherry fluorescent protein-conjugated Rab7, a GTPase associated with late endosomes/lysosomes. Taken together, these results are compatible with the transition of quinacrine-concentrating vacuoles toward an autophagolysosome identity. Quinacrine is concentrated in cells via V-ATPase-mediated ion trapping with an apparent affinity ∼500-fold higher than that of the less lipophilic drug procainamide, and, despite the small size of ensuing vacuoles, the macroautophagic signature of this cytopathology was observed.

Structural modification of the highly potent peptide bradykinin B1 receptor antagonist B9958

Bradykinin (BK)-related peptides stimulate two major classes of receptors, B1 and B2. The B1 receptor (B1R) plays an important role in various pathophysiological states including chronic inflammation, pain, hypotension, trauma and proliferation of cancer. Therefore, there is interest in the development of highly potent peptide BK B1R antagonists. We previously developed a highly potent and selective BK B1R receptor antagonist, B9958 (Lys-Lys-[Hyp3, CpG5, d-Tic7, CpG8]des-Arg9-BK) (Hyp, trans-4-hydroxyproline; CpG, alpha-cyclopentylglycine; Tic, tetrahydroisoquinoline-3-carboxylic acid). We now report on new BK B1R antagonist analogs of B9958 with N-terminal basic residues in the d-configuration, or Lys-, Orn- derivatives (NiK, epsilon-nicotinoyllysine; PzO, 3-pyrazinoylornithine) and/or having hindered unusual amino acids at position 5 (Igl, alpha-(2-indanyl)glycine). These changes were designed to prevent enzyme degradation while keeping an acceptable affinity. However, these new analogs do not show higher B1R antagonist activity than B9958, but its N-terminal acylated derivative with a bulky and hydrophobic 2,3,4,5,6-pentafluorocinnamic acid (F5c), B10324, retains a B1R antagonist activity close to that of B9958 and, in addition, has high inhibition in vivo against lung cancer (SCLC, 86 %) and moderate inhibition against prostate cancer (PC3, 43%) xenografts. This class of compounds offers hope for the development of new BK antagonist peptide drugs for lung or prostate cancer.

Lack of direct interaction between enalaprilat and the kinin B1 receptors

It has been recently proposed that the second extracellular loop of the human bradykinin (BK) B1 receptor (B1R) contains a conserved HExxH motif also present in peptidases possessing a Zn2+ prosthetic group, such as angiotensin converting enzyme (ACE), and that ACE inhibitors directly activate B1R signaling in endothelial cells. However, the binding of ACE inhibitors to the B1Rs has never been directly evaluated. Information about binding of a radiolabeled inhibitor to natural or recombinant ACE in intact cells (physiologic ionic composition) was also collected. We used the tritiated form of an ACE inhibitor previously proposed to activate the B1R, enalaprilat, to address these questions using recombinant human B1Rs and naturally expressed or recombinant ACE. [3H]Lys-des-Arg9-BK bound to the human recombinant B1Rs with high affinity (KD 0.35 nM) in HEK 293a cells. [3H]Enalaprilat (0.25-10 nM) did not bind to cells expressing recombinant human B1R, but bound with a subnanomolar affinity to recombinant ACE or to naturally expressed ACE in human umbilical vein endothelial cells. The radioligand was further validated using a binding competition assay that involved unlabeled ACE inhibitors or their prodrug forms in endothelial cells. Membranes of HEK 293a cells that expressed B1Rs did not hydrolyze hippuryl-glycylglycine (an ACE substrate). Enalaprilat did not stimulate calcium signaling in HEK 293a cells that expressed B1Rs. A typical ACE inhibitor did not bind to nor stimulate the human B1Rs; nevertheless, several other indirect mechanisms could connect ACE inhibition to B1R stimulation in vivo.

The antiwrinkle effect of topical concentrated 2-dimethylaminoethanol involves a vacuolar cytopathology.

Background  The ‘cosmeceutical’ agent 2‐dimethylaminoethanol (DMAE) is a tertiary amine found in high concentration in numerous topical antiwrinkle preparations.

Fluorescent Ligands of the Bradykinin B1 Receptors: Pharmacologic Characterization and Application to the Study of Agonist-Induced Receptor Translocation and Cell Surface Receptor Expression

Unlike the widely distributed and preformed B2 receptors, the bradykinin B1 receptors exhibit a highly regulated expression and minimal agonist-induced endocytosis. To evaluate the potential usefulness of fluorescent B1 receptor probes applicable to live cell microscopy and cytofluorometry, combined chemical synthesis and pharmacologic evaluation have been conducted on novel 5(6)-carboxyfluorescein [5(6)CF]-containing peptides. Representative agents are the antagonist B-10376 [5(6)CF-ϵ-aminocaproyl-Lys-Lys-[Hyp3, CpG5, d-Tic7, CpG8]des-Arg9-bradykinin] and the agonist B-10378 [5(6)CF-ϵ-aminocaproyl-Lys-des-Arg9-bradykinin]. B-10376 has a Ki of 10 to 20 nM to displace [3H]Lys-des-Arg9-bradykinin from rabbit or human recombinant B1 receptors expressed in human embryonic kidney (HEK) 293 cells and is a surmountable antagonist in the rabbit aorta contractility assay (pA2, 7.49). B-10378 was a full agonist at the naturally expressed B1 receptor (rabbit aorta contraction, calcium transients in human smooth muscle cells) and had a binding competition Ki of 19 or 89 nM at the recombinant rabbit or human receptor, respectively. Both fluorescent probes can label with specificity human or rabbit B1 receptors expressed in HEK 293 cells (epifluorescence or confocal microscopy), but the agonist was associated with discontinuous plasma membrane labeling, which coincided with that of a red-emitting caveolin-1 conjugate. Cytofluorometry with B-10376 was applied to recombinant and, in human vascular smooth muscle cells, to naturally expressed B1 receptors. In all fluorescent applications, the specific labeling was reduced by an excess of a B1 receptor nonpeptide antagonist. Despite the loss of affinity determined by the introduction of a fluorophore in B1 receptor agonist or antagonist peptides, the resulting agents allow original applications (imaging in live cells, cytofluorometry).