Reduction of hypervalent states of myoglobin and hemoglobin to their ferrous forms by thymoquinone: the role of GSH, NADH and NADPH.

The reactivity of thymoquinone towards different redox states of hemoglobin and myoglobin in the presence of GSH, NADH, and NADPH was evaluated by optical spectral analysis. Thymoquinone reduces the ferryl forms (HbIV/MbIV) of both met-hemoglobin (HbIII) and met-myoglobin (MbIII) to oxy-hemoglobin (HbIIO2) and oxy-myoglobin (MbIIO2) under physiological conditions. The reaction is mediated by the intermediate quinone forms of TQ, that is, glutathionyl-dihydrothymoquinone (DHTQ-GS) and dihydrothymoquinone (DHTQ), formed from direct interaction of TQ with GSH or NADH (NADPH). In vitro incubation of oxidized human erythrocytes with TQ, DHTQ, and the GSH/TQ mixture reduces the intracellular met-Hb at different rates. In the present study, we report that TQ and its reduced derivatives can also prevent lipid peroxidation induced by the MbFeIII/H2O2 system. In this system, lipid peroxidation is induced by MbIV or a putative MbIV/.MbVI composite; it is plausible that the antioxidant function of TQ derivatives is related to their ability to reduce these oxidizing species. This is of particular biological significance, as natural quinones may participate in reducing processes that lead to recovery of hemoglobin and myoglobin during oxidative stress.

Nonenzymatic reduction of thymoquinone in physiological conditions.

Thymoquinone (TQ) is the bioactive constituent of the volatile oil of Nigella sativa L. and has been shown to exert antioxidant antineoplastic and anti-inflammatory effects. During the study of its possible mechanism of action, we found that TQ reacts chemically (i.e. nonenzymatically) with glutathione (GSH), NADH and NADPH. A combination of liquid chromatography/UV-Vis spectrophotometry/Mass spectrometry analyses was used to identify the products of these reactions. The reaction that occur in physiological conditions indicates the formation of only two products, glutathionyl-dihydrothymoquinone after rapid reaction with GSH, and dihydrothymoquinone (DHTQ) after slow reaction time with NADH and NADPH. Measurement of the antioxidant activity of reduced compounds against organic radicals such as 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) also revealed a potential scavenging activity for glutathionyl-dihydrothymoquinone similar to that of DHTQ. Under our experimental conditions, TQ shows lower scavenging activities than glutathionyl-dihydrothymoquinone and DHTQ; it is very interesting to observe that the reduced compounds apparently show an antioxidant capacity equivalent to Trolox. The results indicate a possible intracellular nonenzymatic metabolic activation of TQ dependent on GSH, NADH or NADPH that may represent a "cellular switch" able to modulate cellular antioxidant defences.

Phytochemistry, micromorphology and bioactivities of Ajuga chamaepitys (L.) Schreb. (Lamiaceae, Ajugoideae): Two new harpagide derivatives and an unusual iridoid glycosides pattern.

Ajuga chamaepitys (L.) Schreb, well-known as Camaepitium or Ground Pine, is an annual herb typical of the Mediterranean area accounting several uses in the traditional medicine. In this work we have, analyzed the plant iridoid fraction together with the essential oil composition and study of the plant indumentum. Finally, we assayed the polar extracts and essential oil obtained from the aerial parts for antioxidant activity and cytotoxicity on tumor cells. The analysis of the monoterpene glycosides allowed us to isolate from roots and aerial parts and to structurally elucidate by NMR and MS the following compounds: ajugoside (1), reptoside (2), 8-O-acetylharpagide (3), harpagide (4), 5-O-ß-d-glucopyranosyl-harpagide (5), asperulosidic acid (6), deacetyl asperulosidic acid (7) and 5-O-ß-d-glucopyranosyl-8-O-acetylharpagide (8), among which 5 and 8 were two new natural products. Chemotaxomic relevance of these constituents was discussed. The chemical analysis of A. chamaepitys essential oil by GC-FID and GC-MS showed ethyl linoleate (13.7%), germacrene D (13.4%), kaurene (8.4%), ß-pinene (6.8%), and (E)-phytol (5.3%) as the major volatile components. The micromorphological and histochemical study showed that iridoids and essential oil are mainly produced in the type III capitates and peltate trichomes of leaves and flowers. Biological evaluations of A. chamaepitys polar extracts and essential oil showed that the former were more potent as radical scavengers than the latter. MTT assay revealed that essential oil and ethanolic extracts were moderately cytotoxic on tumor cells with IC50 of 36.88 and 59.24µg/mL on MDA-MB 231 cell line, respectively, and IC50 of 60.48 and 64.12µg/mL on HCT116, respectively.

Effect of mercuric ions on human erythrocytes. Relationships between hypotonic swelling and cell aggregation.

This study was undertaken to verify the hypothesis that the haemolytic effect of mercuric ions on human erythrocytes is strongly decreased under swelling conditions (relative to isotonic suspensions). In fact, interaction of Hg2+ with swollen erythrocytes yields a rapid and cooperative cell aggregation, a phenomenon that appears to prevent penetration of mercuric ions into the cells and, accordingly, to avoid any haemolytic effect induced by the Hg2+ entrance. Since in vivo erythrocytes undergo big shape changes (swelling being a kind of shape modification) related to mechanical or (in some animals) osmotic stresses, the reported observations turn out to be also of some relevance for the understanding of certain toxicological effects of mercuric ions.

Encapsulation of proteins into human erythrocytes: a kinetic investigation.

Moderate osmotic shocks of human erythrocytes by hypotonic dialysis (0.06 mosmol/kg) induce cell swelling and formation of pores, without causing apparent lysis. Using 125I-labeled macromolecules of different molecular weight and net charge, we followed the kinetics and efficiency of their encapsulation into erythrocytes. After a 20-30 min period of cell dialysis, macromolecules of up to 50 kDa begin diffusing into the swollen cells by a process which can be described by a first-order two-compartment kinetics. Adsorption to the external cell surface was insignificant, while adsorption to the inner membrane surface was substantial (15-20%) only for positively charged proteins, at physiological pH. After resealing, pores of a 12-14 kDa cut-off might remain open allowing some release of entrapped material (20-30%), depending on the final cytocrit, while the remaining might be associated with inner membrane or cytosolic components. Although the method of hypotonic dialysis is known to affect minimally the biophysical and immunological properties of red blood cell membranes, the interaction of encapsulated material with cell constituents would need to be further assessed when considering red cells as macromolecular carriers.

Tryptophan environment in adenosine deaminase. I. Enzyme modification with N-bromosuccinimide in the presence of adenosine and EHNA analogues.

Adenosine deaminase from bovine cerebral hemisphere (white and gray matter) and spleen was treated with N-bromosuccinimide, a reagent known to oxidize selectively tryptophan residues in proteins. Spectrally observable tryptophan modification was accompanied by enzyme inactivation. Tsow graphics revealed that two Trps are essential for the activity of enzyme from both tissues. Enzyme inhibitors and substrate analogues, derivatives of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and adenosine, were able to protect Trp against modification, and this effect correlated in general with the enzyme activity protection. In the presence of adenosine deaza analogues (the noninhibitor tubercidin among them) only two Trps were modified in the fully inactivated enzyme. In the presence of EHNA and its deaza analogues, full inactivation of the enzyme was accompanied by the modification of four Trps. The obtained data confirm the previous hypothesis about the presence on the enzyme of different binding sites for adenosine and EHNA derivatives that are responsible for the different effects on the enzyme conformation elicited by the corresponding derivatives. Moreover, these data allow us to suggest that Trp residues, still unidentified by X-ray analysis, are essential for the functioning of the enzyme.

Adenosine deaminase from Saccharomyces cerevisiae: kinetics and interaction with transition and ground state inhibitors.

Several adenosine analogs, such as coformycin, 2'-deoxycoformycin and erythro-9-(3-nonyl-p-aminobenzyl)adenine (EHNA), which are strong inhibitors of mammalian adenosine deaminase, are much weaker inhibitors of the Saccharomyces cerevisiae enzyme. The specificity of the yeast enzyme is more restricted than that of mammalian adenosine deaminase, particularly towards the ribose moiety and around position 6 and 1 of the substrate. The sulphydryl group appears to be more masked in the yeast than in the mammalian enzyme. The kinetic effects of pH with adenosine substrate and with the inhibitor purine riboside are reported. The findings on specificity and pH kinetic effects can be interpreted in a model involving proton transfer from the -SH group of the enzyme to the N-1 atom of the substrate.

Binding of hirudin to human alpha, beta and gamma-thrombin. A comparative kinetic and thermodynamic study.

Thermodynamic parameters for the binding of hirudin to human alpha, beta and gamma-thrombin have been determined between pH 5.0 and 9.0, and from 10 degrees C to 40 degrees C; kinetic data for the association and dissociation of the proteinase-inhibitor complex were obtained at pH 7.5 and 21 degrees C. These results have been analysed in parallel with the inhibitor-binding properties of human alpha, beta and gamma-thrombin for the bovine basic pancreatic trypsin inhibitor (Kunitz-type inhibitor; BPTI). For the purpose of an homogeneous comparison, values of the apparent association equilibrium constant for BPTI binding to human gamma-thrombin have been determined between pH 5.0 and 9.0, at 21 degrees C. The different binding behaviour of hirudin and BPTI with respect to human alpha, beta and gamma-thrombin has been related to the inferred stereochemistry of the proteinase-inhibitor contact regions. In particular, whereas the beta and gamma-loops play an appreciable role in the stabilization of the enzyme-hirudin complexes, they contribute to impairment of the adduct formation for the proteinase/BPTI system.

Interaction of Hsp90 with 20S proteasome: thermodynamic and kinetic characterization.

The proteasome and heat shock proteins have been found in the centrosome. The evidence of their copurification reported by several studies suggests that they form stable complex. In addition, Hsp90 is involved in the loading of proteasome-generated antigenic peptides to the class I major histocompatibility complex. In this article, we report a detailed thermodynamic and kinetic characterization of the Hsp90-20S proteasome interaction, using a surface plasmon resonance technique. The modulation exerted by protons in solution has been investigated, and the results have been discussed, taking into account structural motifs characterizing the binding interface between the two macromolecules.

Peroxynitrite-mediated oxidation of fibrinogen inhibits clot formation.

The clotting activity of human fibrinogen was fully inhibited in vitro by peroxynitrite. The decrease of activity followed an exponential function and the concentration of peroxynitrite needed to inhibit 50% of fibrinogen clotting was 22 microM at 25 degrees C. The oxidative modification(s) induced by the peroxynitrite system (i.e. ONOO-, ONOOH and ONOOH*) appeared specifically to affect fibrin clot formation (through the inhibition of fibrinogen polymerization) since the interaction of peroxynitrite-modified fibrinogen with thrombin appeared to be unaffected. The addition of NaHCO3 decreased the peroxynitrite effect on fibrinogen clotting, suggesting that the reactive species formed by the reaction of CO2 with peroxynitrite are less efficient oxidants of peroxynitrite itself. Similar effects were observed after addition of bilirubin, which also exerted a significant protection against peroxynitrite-mediated modification of fibrinogen.

Purine and 1-deazapurine ribonucleosides and deoxyribonucleosides: synthesis and biological activity.

A series of 6-(hydroxylamino)purine and -1-deazapurine nucleosides were synthesized and tested for their antitumor and adenosine deaminase inhibitory activity. All the examined molecules displayed an in vitro activity comparable to that of the reference compounds 6-(hydroxylamino)-9-beta-D-ribofuranosylpurine (HAPR) and ara-A, their ID50 ranging from 0.9 microM to approximately 100 microM. The 6-hydroxylamino derivatives of 1-deazapurine 9, 12, and 17 and also the blocked compound 13 are inhibitors of ADA whereas the purine derivatives 4 and 6 and the nitro compounds 11 and 16 are resistant to the enzyme. 7-(Hydroxylamino)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3H-imi dazo[4,5- b]pyridine, the less cytotoxic but the most active ADA inhibitor in the series (Ki = 2.7 x 10(-7)), greatly potentiates the antitumor activity of ara-A in vitro.

Adenosine deaminase inhibitors: synthesis and structure-activity relationships of 2-hydroxy-3-nonyl derivatives of azoles.

A series of erythro-1-(2-hydroxy-3-nonyl)azole derivatives have been synthesized and evaluated for adenosine deaminase (ADA) inhibitory activity, in order to introduce simplifications in the ADA inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 1a). The synthesis of most of the reported compounds was achieved by reaction of 2-bromo-3-nonanone with the suitable azole followed by reduction of the carbonyl group to give a diastereoisomeric mixture of N-substituted (2-hydroxy-3-nonyl)azoles. Separation of diastereoisomers was achieved by HPLC or by preparative TLC plates. The results of the enzymatic test indicate that the nitrogen in the 3-position, and secondly, the nitrogen in the 5-position are very important for the interaction of the azole ring with the inhibitory site on the enzyme. In fact, the pyrazole and the 2-substituted 1,2,3-triazole derivatives (10 and 15, respectively) are nearly inactive, whereas the erythro-1-(2- hydroxy-3-nonyl)-1,2,4-triazole (18e) was the most potent ADA inhibitor in the series with Ki = 0.3 microM.

Adenosine deaminase inhibitors: synthesis and structure-activity relationships of imidazole analogues of erythro-9-(2-hydroxy-3-nonyl)adenine.

A series of erythro-1-(2-hydroxy-3-nonyl)imidazole derivatives have been synthesized and evaluated for adenosine deaminase (ADA) inhibitory activity, in order to introduce simplifications in the ADA inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 1a) and 3-deaza-EHNA (1c). Opening the pyrimidine or pyridine ring of EHNA or 3-deaza-EHNA respectively led to compounds which are still ADA inhibitors. The most potent compound was erythro-1-(2-hydroxy-3-nonyl)imidazole-4-carboxamide (5, Ki = 3.53 x 10(-8) M), which provided potential donor and acceptor sites for hydrogen bonding. Lack of one of this sites could account for the order of potency of all compounds examined in this series. Opening the same ring in adenosine and in 3-deazaadenosine led to fully inactive compounds. These results support the hypothesis of the existence, at or near the enzyme active site, of a hydrophobic region able to bind the erythro-nonyl moiety.

Adenosine deaminase inhibitors. Synthesis of deaza analogues of erythro-9-(2-hydroxy-3-nonyl)adenine.

Structural analogues of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), in which the adenine moiety of the molecule was modified, were prepared in order to investigate the structural requirement of EHNA as an inhibitor of adenosine deaminase (ADA). Thus, 1- and 3-deaza-EHNA and their 6-deamino analogues were synthesized and evaluated as inhibitors of ADA from calf intestine. Inhibition studies indicated that isosteric substitution of pyrimidine nitrogens by carbons could be tolerated at the enzymatic binding site. In fact, 3-deaza-EHNA was found to have an inhibitory activity comparable to EHNA itself, and 1-deaza-EHNA, though less potent, is a good inhibitor. The 6-amino group gives an important contribution to the enzymatic binding if the N1 nitrogen is also present, conferring on the compound the characteristic of a semitight inhibitor.

Adenosine deaminase inhibitors. Synthesis and biological activity of deaza analogues of erythro-9-(2-hydroxy-3-nonyl)adenine.

Two new deaza analogues of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 1), 7-deaza-EHNA (6) and 1,3-dideaza-EHNA (11), were synthesized and evaluated for adenosine deaminase (ADA) inhibitory activity and compared with EHNA, 1-deaza-EHNA (2), and 3-deaza-EHNA (3). Substitution of a methine group for a nitrogen atom in the 7-position of the purine moiety of EHNA produces a dramatic drop in the inhibitory activity (Ki = 4 X 10(-4) M) whereas compounds 2 and 3 are still good inhibitors (Ki = 1.2 X 10(-7) M and 6.3 X 10(-9) M respectively). EHNA and its deaza analogues so far synthesized were also tested in vitro for their antiviral and antitumor activity in a range of cellular systems. EHNA and 1-deaza-EHNA are equiactive as inhibitors of human respiratory syncytial virus (HRSV) replication (MIC = 6.25 micrograms/mL) while the other compounds are inactive. On the other hand, all the examined compounds displayed an antitumor activity comparable to that of the reference compound 1-beta-D-arabinofuranosyladenine (ara-A), 7-deaza-EHNA being the most active of all. The results obtained showed that there is no correlation between adenosine deaminase inhibition and antiviral or antitumor activity in this series of compounds. 3-Deaza-EHNA, the most active inhibitor of ADA among the EHNA deaza analogues, greatly potentiates the antitumor activity of ara-A in vitro. In vivo activity was observed only when the two compounds were used in combination.

Synthesis and biological evaluation of N6-cycloalkyl derivatives of 1-deazaadenine nucleosides: a new class of anti-human immunodeficiency virus agents.

A series of 1-deazaadenine nucleosides with the N6 nitrogen unsubstituted or bearing methyl or cycloalkyl substituents, with or without a chloro group in the 2-position, and with the glycosylic moiety being ribose (1-16), 2'-deoxyribose (17-32), or 2', 3'-dideoxyribose (33-48) were designed and synthesized starting from 5,7-dichloro-3H-imidazo[4,5-b] pyridine (50). These compounds were evaluated for their in vitro activity against human immunodeficiency virus type-1 (HIV-1) and herpes simplex virus type-1 (HSV-1). In addition they were tested for their ability to inhibit adenosine deaminase (ADA) from calf intestine. While the parent compounds 1-deazaadenosine (9), 2'-deoxy-1-deazaadenosine (25), and 2',3'-dideoxy-1- deazaadenosine (41) and the corresponding 2-chloro derivatives were inactive, nucleosides bearing cycloalkyl substituents on N6 exhibited moderate to good anti-HIV-1 activity, compared to 2',3'-dideoxyadenosine, with the degree and pattern of improvement depending on the structure of the sugar moiety. In general, 2'-deoxy- and 2',3'-dideoxy derivatives were more potent compounds than the corresponding ribose nucleosides. Compounds bearing a 6-cycloheptyl or cyclooctylamine were the most active in every series. The presence of a chloro group in the 2-position improved both activity and therapeutic index in every series, the most active compound being 2'-deoxy-2-chloro-N6-cycloheptyl-1-deazaadenosine (23; ED50 = 0.2 microM). On the other hand, most of these derivatives were inactive as anti-HSV-1 agents, showing a high degree of virus selectivity. The 1-deazaadenine derivatives were not substrates of adenosine deaminase, and some of them proved to be good inhibitors of the enzyme. However, the ADA inhibitory activity does not account for the antiviral potency since increased lipophilicity and steric hindrance of substituents resulted in derivatives much less active than the parent compounds.

Acid-stable serine proteinase inhibitors in the urine of Alzheimer disease subjects.

A comparative study of the levels of acid-stable proteinase inhibitors (kallikrein and trypsin inhibitors) in the urine of healthy and Alzheimer subjects, of both sexes, has been performed. A preliminary characterization of the purified inhibitors indicates that the urinary antitryptic activity is accounted for by the presence of the well known Urinary Trypsin Inhibitor (UTI) while an apparently new molecule appears to be responsible for the antikallikrein activity. The urinary levels of kallikrein inhibitors are very similar in healthy and sick subjects while the levels of trypsin inhibitors appear significatively increased in Alzheimer subjects of both sexes. The data presented here support the hypothesis that unpaired proteolytic processes could be involved in the pathogenesis of Alzheimer's disease and suggest that the levels of urinary acid-stable inhibitors may prove to be useful markers of the disease.

Effect of mercuric ions on human erythrocytes. 1. Rate of haemolysis induced by osmotic shock as a function of incubation time.

Incubation of human erythrocytes with mercuric ions for 5 min causes a transient increase in mechanical resistance as measured by osmotic shock. After a longer incubation time (necessary for the metal to cross the cell membrane), cells recover their normal fragility; however, after 10-20 min of exposure to mercuric ions haemolysis occurs in the incubation vessel. In contrast, p-chloromercuriben-zenesulfonate (a compound known to bind mainly to band 3 of the cell membrane without passing into the cell) increases the osmotic resistance of erythrocytes, as observed transiently for mercuric ions, but does not induce haemolysis even at incubation times of 20-30 min. Therefore it seems that the interaction per se of mercuric ions with the membrane does not represent the main damaging event and therefore the toxicological effect of mercuric ions must be mostly related to subsequent processes, such as the interaction of the metal with intracytoplasmic components and/or disruption of the cytoskeleton.

Structure--function relationships in bovine thymus 20S proteasome: a fluorimetric study.

The structure--function relationships occurring on the bovine thymus 20S proteasome, which exhibits the features of an immunoproteasome, have been studied. The investigation has been performed, essentially, using a fluorimetric approach, taking advantage either of the sensitivity of the complex to sodium dodecil sulfate and chaotropic agents (urea and guanidine hydrochloride) or of the presence, on the molecule, of a high number of tryptophan residues. The results obtained indicate that the perturbation or the oxidation of these residues affect the catalytic events taking place on the thymus proteasome and that the functional effects determined by SDS and chaotropic agents most likely occur through a series of progressive structural modifications leading to an inactive molecule. The presence of structural intermediates in the proteasome inactivation process suggests that thymus proteasome is a molecule characterized, at the same time, by structural flexibility (modulation of active sites) and structural stability (maintaining of the quaternary structure) in agreement with its crucial role in the cell life cycle.

Inhibition of adenosine deaminase by deaza derivatives of adenosine and purine riboside.

Deaza analogs of adenosine, where carbon substitutes for nitrogen, are not substrates for adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4); little is reported on their ability to be inhibitors. Among the deaza analogs of adenosine and purine riboside that we have been testing, 1-deazaadenosine proved to be the best inhibitor (Ki = 6.6 X 10(-7)M). 1-deazapurine riboside, 1,3-dedeazadenosine, 3-deazaadenosine have affinity by about two and three order of magnitude lower.