Synthesis and hybridization properties of the conjugates of oligonucleotides and stabilization agents. Part 3.

New compounds having tri- or pentamethylenamine linker functions were synthesized. These derivatives were covalently attached through the 5'-phosphoramide linkage to heptanucleotide pd(CCAAACA). Complementary complexes of the octanucleotide pd(TGTTTGGC) and above oligonucleotide conjugates were tested for their thermodynamic response.

Dynamic, thermodynamic, and kinetic basis for recognition and transformation of DNA by human immunodeficiency virus type 1 integrase.

Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN.

Synovial fluid from patients with rheumatoid arthritis inhibits neutrophil apoptosis: role of adenosine and proinflammatory cytokines.

Objective: In synovial fluid (SF) from patients with rheumatoid arthritis (RA), neutrophils are exposed to proinflammatory mediators endowed with either anti-apoptotic or pro-apoptotic properties. We investigated neutrophil apoptosis in the presence of SF from 11 RA patients.

Methods: SF was obtained from affected knees of 11 patients with RA.

Cytotoxicity of the venom of Pelagia noctiluca forskål (Cnidaria: Scyphozoa).

The activity of Pelagia noctiluca venom was never assessed on cultured cells; therefore, we have evaluated on V79 cells the cytotoxicity, genotoxicity and ATP depletion induced after treatment. Venom did not cause alteration on cell DNA, but showed remarkable cytotoxic properties. With the highest nematocyst concentration (150,000 nematocyst/ml) 74 and 39% cells survived after 1 and 3 h, respectively, when evaluated by Trypan blue.

Delayed neutrophil apoptosis induced by synovial fluid in rheumatoid arthritis: role of cytokines, estrogens, and adenosine.

The fate of neutrophils at sites of inflammation, where these cells are likely exposed to both anti- and proapoptotic influences, needs to be clarified. To investigate this issue, we studied the survival of neutrophils in the presence of articular fluids from RA joints before and after immune complex activation. Eight of eleven samples of RA synovial fluid studied were found to inhibit spontaneous and immune complex-stimulated neutrophil apoptosis.

Inhibition of neutrophil O(2)(-) production by unsymmetrical methylene derivatives of benzopyrans: their use as potential antiinflammatory agents.

Some unsymmetrical derivatives of benzopyrans 9 were synthesized and tested to verify their PKC inhibitory activity. For this purpose, the Mannich bases of 7-hydroxycoumarins 6 were treated with 2-(dialkylamino)benzopyran-4-ones or 3-(dialkylamino)naphtho[2,1-b]pyran-1-ones 8 in the presence of acetic or propionic anhydride, yielding compounds 9. Human neutrophils stimulated with either PMA and f-MLF were used as the cellular model.

HPLC determination of adenosine in human synovial fluid.

A high-performance liquid chromatographic method has been developed for the quantitative determination of adenosine in human synovial fluid. The method is simple, rapid and, overall, selective. No interference with the components of the biological matrix was observed in these chromatographic conditions.

Effects of mono-, di- and tri-hydroxybenzoic acids on the nitrosation of propranolol: structure-activity relationship.

Nitrosation of propranolol under standard conditions recommended by the World Health Organization (10mM propranolol hydrochloridre, 40mM sodium nitrite, pH 3.5) was performed in the absence and in the presence of benzoic acid and of twelve mono-, di- and tri-hydroxybenzoic acids, added to the nitrosation mixture in concentrations ranging from 2 to 40mM, in order to examine their effect on the nitrosation reaction. The yield of N-nitrosopropranol (NOP) was reduced by benzoic acid and, with potency decreasing in the following order, by 2,3,4-tri-hydroxybenzoic acid>/=3,4-tri-hydroxybenzoic acid>2,5-di-hydroxybenzoic acid>2,3-di-hydroxybenzoic acid>3-hydroxybenzoic acid>2-hydroxybenzoic acid>3,4,5-tri-hydroxybenzoic acid>4-hydroxybenzoic acid; their inhibiting effect was concentration-dependent.

Synthesis of ionones and carvone analogues: olfactory properties and preliminary toxicity assays.

Vilsmeier reagents react with alpha/beta-ionones and carvone to produce aldehydes 7-11 in a one-step procedure. The indene derivative 11, which came from the double iminoalkylation of carvone and ring closure with the elimination of dimethylamine, was practically odourless, while all the others had peculiar odours which were very different from the starting material. The cytotoxicity data of 9 and 10, which are the most promising potential perfume ingredients, are also reported.

Interaction of oligonucleotides conjugated to substituted chromones and coumarins with HIV-1 reverse transcriptase.

Ten different pyranone-related substituents (chromones or coumarins) were covalently linked to the 5' end of various oligonucleotides (ODN). The interaction of these compounds with human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) was analyzed. A different behavior was found to depend on the structure of the oligonucleotide derivatives.

N,N-dialkylaminosubstituted chromones and isoxazoles as potential anti-inflammatory agents.

The ability of some N,N-dialkylaminosubstituted chromones and isoxazoles to inhibit the protein kinase C (PKC) dependent signal transduction pathway was tested. As a cellular model, human neutrophils stimulated with either phorbol myristate acetate (PMA) or formylmethionine-leucine-phenylalanine (f-MLF) were used. The efficiency of the compounds was established by their capacity to reduce the O2- production by activated human neutrophils.

Simultaneous HPLC determination of multiple components in a commercial cosmetic cream.

A high-performance liquid chromatographic method for the simultaneous determination of magnesium ascorbyl phosphate (I), imidazolidinylurea (II), a mixture of methyl-(III), ethyl-(IV), propyl-(V), butyl-(VI) parabens dissolved in phenoxyethanol, and ascorbyl palmitate (VII), was studied by using a cyano-propyl column and a methanol gradient at 220 and 240 nm. Calibration curves were found to be linear in the 0.05-5 mg ml(-1) range (compounds I, II, VII) and 0.

HIV-1 reverse transcriptase is capable of elongating derivatives of sequence specific noncomplementary oligodeoxynucleotides.

We have carried out a comparison of KM and Vmax values for various primers in the polymerization reaction catalyzed by the HIV-1 RT. The affinity of RT for complementary d(pT)6 containing two different 5'-end pyranone derivatives was 2-3 orders of magnitude higher (KM = 3-15 nM) than that of d(pT)6 (KM = 12.6 mM).

The effect of benzenediols and benzenetriols on the nitrosation of propranolol depends on the position of hydroxyl groups on the benzene ring.

Nitrosation of propranolol under the standard conditions recommended by the World Health Organization (10 mM propranolol hydrochloride, 40 mM sodium nitrite, pH 3.5) was carried out in the absence and in the presence of phenol, benzenediols and benzenetriols added to the nitrosation mixture in concentrations ranging from 2 to 40 mM. The yield of N-nitrosopropranolol (NOP) was reduced, with potency decreasing in the following order, by 1,2-benzenediol > 1,2,3-benzenetriol > 1,4-benzediol; their inhibiting effect was dose-dependent.

Synthesis and hybridization properties of the conjugates of oligonucleotides and stabilization agents--II.

New pyranone derivatives having tri- or pentamethylenamine linker functions were synthesized. These derivatives were covalently attached through the 5'-phosphoramide linkage to heptanucleotide pd(CCAAACA). Complementary complexes of the octanucleotide pd(TGTTTGGC) and above oligonucleotide conjugates were tested for their thermodynamic response.

Anti-PKC activity of some 3-(dialkylamino)-1H-naphtho [2,1-B] pyran-1-ones.

Some 3-(dialkylamino)-1H-naphtho [2,1-b] pyran-1-ones were tested to evaluate their ability to inhibit Protein Kinase C (PKC) activation. The model consisted in the detection of the superoxide anion in activated neutrophils. Naphthopyrans carrying 3-(diethylamino), 3-(1-piperidinyl) and 3-[bis(2-methoxyethyl)amino] groups emerged as the most active ones.

Characterization and quantitation of the active polynucleotide fraction (PDRN) from human placenta, a tissue repair stimulating agent.

The polydeoxyribonucleotide (PDRN) fraction is an extract which forms the active component in a new formulation of the drug Placentex (a tissue repair stimulating agent), obtained from human placenta through an original proprietory extraction method. From a comparison of the UV, NMR and IR spectra of this fraction (before and after nuclease treatment) with that of a similar standard (Sigma D1501), it was shown that the active substances in the PDRN fraction mainly consist of a mixture of DNA fragments. By gel electrophoresis, the molecular weights of the DNA fragments were shown to range from 50 to 2000 base pairs.

Nitrosation of propranolol under simulated gastric conditions.

Nitrosation of propranolol, a beta-adrenergic blocking drug previously found to react with nitrite in HCl solution yielding a genotoxic nitrosamine, was examined under simulated gastric conditions. In the presence of a low concentration of nitrite (2.9 mM) and a therapeutic gastric concentration of the drug (5.

Low clastogenic activity in vivo of the N-nitroso derivatives of 5 beta-adrenergic-blocking drugs proved to be potent genotoxins in vitro.

The N-nitroso derivatives formed by the in vitro reaction of 5 beta-adrenergic-blocking drugs with sodium nitrite and previously found to induce DNA fragmentation and repair in primary cultures of both rat and human hepatocytes were tested for their ability to exert a clastogenic effect in vivo. In partially hepatectomized rats given by gavage a single dose of 1000 mg/kg, all 5 N-nitroso derivatives caused a statistically significant increase in the frequency of micronucleated hepatocytes, the clastogenic potencies of NO-propranolol, NO-metoprolol, and NO-nadolol being slightly greater than those of NO-atenolol and NO-sotalol. In contrast, none of them produced a significant increase in the frequency of micronucleated polychromatic erythrocytes in the bone marrow and the spleen.

Synthesis in solution of oligodeoxynucleotides and some their 5'- and 3'-linked derivatives.

Antisense oligodeoxynucleotides (ODNs) and their derivatives are highly interesting tools to regulate gene expression and promising drugs for antiviral and anticancer therapy. In view of performing more extensive pharmacological trials requiring relatively great amounts of ODNs, we used a method of ODN synthesis derived from the phosphotriester approach which allow to prepare ODNs covalently linking cholesteryl residue (in 5' or 3') and phenazinium group (in 5'). HPLC purifications are discussed.

In vitro and in vivo evaluation of the genotoxicity of N-nitrosooxprenolol.

N-nitrosooxprenolol (NO-oxprenolol) might be formed in the stomach of patients taking the beta-adrenergic blocking drug, oxprenolol. This nitroso derivative has previously been shown to induce DNA damage and repair in both rat and human cultured hepatocytes. The results of the present study show that in the presence of co-cultured rat hepatocytes, 0.

HPLC analysis of theophylline: bioequivalence study of two sustained-release formulations at steady state.

Theophylline is at present one of the standard drugs used for asthma and obstructive respiratory diseases but still presents clinical problems due to its narrow therapeutic range. To provide additional studies in this important field, the in vivo bioavailability of Bronchoretard, a theophylline sustained-release preparation, not available at present in Italy, has been compared with that of Diffumal, formulation of large consumption. An isocratic reverse-phase HPLC method to assess the theophylline in plasma is described.