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1.
J Nat Prod ; 77(2): 351-7, 2014 Feb 28.
Article in English | MEDLINE | ID: mdl-24512352

ABSTRACT

Palytoxin ranks among the most potent marine biotoxins. Its lethality was well known to native Hawaiians that used to smear a "moss" containing the toxin on their spears to cause instant death to their victims. Human intoxications due to exposure to palytoxin and to its many congeners have been reported worldwide. Currently, palytoxins constitute the main threat to public health across the Mediterranean Sea. In the present work we report on the isolation and stereostructural determination of a new palytoxin analogue from a Hawaiian Palythoa tuberculosa sample. This new toxin is a stereoisomer of 42-hydroxypalytoxin isolated from Palythoa toxica. The whole absolute configuration of this latter toxin is also reported in the paper. Interestingly, the two 42-hydroxypalytoxins do not share the same biological activity. The stereoisomer from P. tuberculosa showed cytotoxicity toward skin HaCaT keratinocytes approximately 1 order of magnitude lower than that of 42-hydroxypalytoxin from P. toxica and about 2 orders of magnitude lower than that of palytoxin itself. This finding holds the prospect of interesting structure-activity relationship evaluations in the future.


Subject(s)
Acrylamides/pharmacology , Anthozoa/chemistry , Marine Toxins/chemistry , Acrylamides/chemistry , Animals , Chromatography, High Pressure Liquid , Cnidarian Venoms/chemistry , Cnidarian Venoms/pharmacology , Hawaii , Humans , Keratinocytes/drug effects , Marine Toxins/toxicity , Mediterranean Sea , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Pyrans/chemistry , Pyrans/pharmacology , Stereoisomerism , Structure-Activity Relationship
2.
Environ Sci Technol ; 47(4): 2034-42, 2013 Feb 19.
Article in English | MEDLINE | ID: mdl-23339823

ABSTRACT

Palytoxins are potent marine biotoxins that have recently become endemic to the Mediterranean Sea, and are becoming more frequently associated with seafood. Due to their high toxicity, suitable methods to quantify palytoxins are needed. Thus, we developed an indirect sandwich ELISA for palytoxin and 42-hydroxy-palytoxin. An intralaboratory study demonstrated sensitivity (limit of detection, LOD = 1.1 ng/mL; limit of quantitation, LOQ = 2.2 ng/mL), accuracy (bias of 2.1%), repeatability (RSDr = 6% and 9% for intra- and interassay variability, respectively) and specificity: other common marine toxins (okadaic acid, domoic acid, saxitoxin, brevetoxin-3, and yessotoxin) do not cross-react in this assay. It performed well in three different matrices: observed LOQs were 11.0, 9.6, and 2.4 ng/mL for mussel extracts, algal net samples and seawater, respectively, with good accuracy and precision. The LOQ in seafood is 11 µg palytoxin/kg mussel meat, lower than that of the most common detection technique, LC-MS/MS.


Subject(s)
Acrylamides/analysis , Environmental Monitoring/methods , Acrylamides/immunology , Animals , Antibody Affinity , Cnidarian Venoms , Enzyme-Linked Immunosorbent Assay , Reference Standards
3.
Cell Death Dis ; 3: e282, 2012 Mar 15.
Article in English | MEDLINE | ID: mdl-22419111

ABSTRACT

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ(m)) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.


Subject(s)
Electron Transport Complex IV/antagonists & inhibitors , Mitochondria/drug effects , tat Gene Products, Human Immunodeficiency Virus/pharmacology , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Brain/drug effects , Brain/enzymology , Cytochromes c/metabolism , Electron Transport Complex IV/metabolism , Humans , Ion Transport , Liver/drug effects , Liver/enzymology , Membrane Potential, Mitochondrial , Mice , Mice, Inbred BALB C , Mitochondria/enzymology , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Myocardium/enzymology , Oxidative Phosphorylation , Permeability , Proto-Oncogene Proteins c-bcl-2/metabolism , tat Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/physiology
4.
Cell Mol Life Sci ; 62(3): 355-61, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15723170

ABSTRACT

The human HIV transactivator protein Tat is essential for efficient viral transcription that occurs by a complex mechanism involving interaction of Tat with the TAR RNA element. This interaction appears to require the mediation of a cellular protein, cyclin T1. However, the possibility that Tat and TAR associate in a binary Tat-TAR complex has been little investigated. Using a chemically synthesized active Tat protein, the kinetic and equilibrium parameters of its interaction with TAR were determined by surface plasmon resonance technology. Independently of partner and method of immobilization onto the sensor chip, the association (k(a) = 5-9 x 10(5) M(-1) s(-1)) and dissociation rate constants (k(d) = 1.7-4.3 x 10(-3) s(-1)) yielded similar equilibrium dissociation constants (K(d) = 2-8 nM). A truncated peptide encompassing residues 30-86 of Tat did not bind to TAR at all. We conclude that Tat can form a high-affinity complex with TAR in the absence of cyclin T1 and that the N-terminal domain of Tat is essential for this interaction, suggesting a conformational link between this domain and the basic domain of Tat. These results are important in our quest for developing therapeutic compounds that impair viral replication.


Subject(s)
Gene Products, tat/metabolism , RNA, Viral/metabolism , Gene Products, tat/chemistry , Humans , Immobilization , Kinetics , Protein Array Analysis , Protein Binding/physiology , RNA, Viral/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Streptavidin/chemistry , Structure-Activity Relationship , Surface Plasmon Resonance , Time Factors
5.
J Virol ; 75(2): 661-71, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11134280

ABSTRACT

N-terminal modifications of the chemokine RANTES bind to C-C chemokine receptor 5 (CCR5) and block human immunodeficiency virus type 1 (HIV-1) infection with greater efficacy than native RANTES. Modified RANTES compounds induce rapid CCR5 internalization and much slower receptor reexpression than native RANTES, suggesting that receptor sequestration is one mode of anti-HIV activity. The rates of CCR5 internalization and reexpression were compared using the potent n-nonanoyl (NNY)-RANTES derivative and CD4(+) T cells derived from donors with different CCR5 gene polymorphisms. NNY-RANTES caused even more rapid receptor internalization and slower reexpression than aminooxypentane (AOP)-RANTES. Polymorphisms in the promoter and coding regions of CCR5 significantly affected the receptor reexpression rate after exposure of cells to NNY-RANTES. These observations may be relevant for understanding the protective effects of different CCR5 genotypes against HIV-1 disease progression.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Chemokine CCL5/analogs & derivatives , Chemokine CCL5/pharmacology , Receptors, CCR5/genetics , Receptors, CCR5/metabolism , Adult , Blood Donors , Chemokine CCL5/metabolism , Genotype , Humans , Ligands , Receptors, CCR5/chemistry
6.
J Med Chem ; 43(12): 2356-61, 2000 Jun 15.
Article in English | MEDLINE | ID: mdl-10882361

ABSTRACT

Analogues of bombesin which incorporate dipeptide or turn mimetics have been synthesized. One of them (compound 11) containing a seven-membered lactam ring revealed a good affinity for GRP/BN receptors on rat pancreatic acini (K(i) value of 1.7 +/- 0.4 nM) and on Swiss 3T3 cells (K(i) value of 1.0 +/- 0.2 nM). On the basis of this observation, antagonists containing the same dipeptide mimic were obtained by modification of the C-terminal part of the bombesin analogues. The most potent constrained compounds (15 and 17) were able to antagonize 1 nM bombesin-stimulated amylase secretion from rat pancreatic acini with high potency (K(i) = 21 +/- 3 and 3.3 +/- 1.0 nM, respectively) and 10(-7) M bombesin-stimulated ¿(3)Hthymidine incorporation into Swiss 3T3 cells (K(i) = 7.8 +/- 2. 0 and 0.5 +/- 0.1 nM, respectively).


Subject(s)
Bombesin/analogs & derivatives , Bombesin/chemical synthesis , Oligopeptides/chemical synthesis , Receptors, Bombesin/agonists , Receptors, Bombesin/antagonists & inhibitors , 3T3 Cells , Amylases/metabolism , Animals , Bombesin/chemistry , Bombesin/pharmacology , In Vitro Techniques , Male , Mice , Molecular Mimicry , Oligopeptides/chemistry , Oligopeptides/metabolism , Oligopeptides/pharmacology , Pancreas/drug effects , Pancreas/metabolism , Rats , Rats, Wistar , Receptors, Bombesin/metabolism , Structure-Activity Relationship , Thymidine/metabolism
7.
J Pept Res ; 53(3): 275-83, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10231715

ABSTRACT

Bombesin receptor antagonists are potential therapeutic agents due to their ability to act as inhibitors of cellular proliferation. On the basis of our hypothesis concerning the mechanism of action of gastrin associating an activating enzyme to the receptor and on the results reported in the literature, we have synthesized bombesin analogs which have been modified in the C-terminal part. Potent bombesin receptor antagonists were obtained by replacement of Leu-13 with a statyl residue or with a residue bearing an hydroxyl group in place of the carbonyl function of Leu-13. Several inhibitors were able to recognize the bombesin receptor on rat pancreatic acini and antagonized bombesin stimulated amylase secretion in the nanomolar range. These compounds were also able to recognize the bombesin receptor and to inhibit [3H] thymidine incorporation in 3T3 cells with the same potency.


Subject(s)
Organophosphorus Compounds/chemical synthesis , Receptors, Bombesin/antagonists & inhibitors , 3T3 Cells , Amylases/metabolism , Animals , Dose-Response Relationship, Drug , Inhibitory Concentration 50 , Kinetics , Mice , Models, Chemical , Pancreas/drug effects , Rats , Thymidine/metabolism
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