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.

Evaluation of (arene)Ru(II) complexes of curcumin as inhibitors of dipeptidyl peptidase IV.

Curcumin, the main component of Curcuma longa, shows an anti-hyperglycemic effect and improved insulin sensitivity. This action may be attributed at least in part to its anti-inflammatory properties and also to its possible interaction with dipeptidyl peptidase-4 (DPPIV), the enzyme that the conversion of glucagon-like peptide-1 (GLP-1), responsible for glucose tolerance into inactive GLP-1. In this work we evaluated the inhibitory activities of a series of different arene-Ru(II)-curcumin complexes on bovine kidney dipeptidyl peptidase-4 (DPPIV). We studied also the interaction of these inhibitors on the enzyme with fluorescence studies displaying the binding poses with molecular docking studies. Specifically organometallic ruthenium(II) complexes of general formula [(η(6)-arene)Ru(curcuminato)Cl], with arene being p-(i)PrC6H4Me (1), C6H6 (2), and C6Me6 (3), were evaluated for their inhibition activity toward the mammalian enzyme. Among them, 2 suppressed DPPIV activities more potently (Ki = 20.2(±0.8) µM) than 1, 3, or free curcumin, and all complexes showed an antioxidant activity as free curcumin. As shown from our docking simulations a putative binding site of the compound 2 was found on subdomains S1 and S2 of DPP-IV, where S1 hydrophobic pocket includes catalytic residues and is the primary determinant of substrate specificity for the enzyme. Collectively, our results demonstrate that the complexation of curcumin with ruthenium(II) could be a promising starting point for the development of curcumin-based DPPIV inhibitors.

Thymoquinone, a potential therapeutic agent of Nigella sativa, binds to site I of human serum albumin.

Thymoquinone (TQ) is the main constituent of Nigella sativa essential oil which shows promising in vitro and in vivo antineoplastic growth inhibition against various tumor cell lines. Because of the increasing interest to test it in pre-clinical and clinical researches for assessing its health benefits, we here evaluate the interactions between TQ and human serum albumin (HSA), a possible carrier of this drug in vivo. Binding to HSA was studied using different spectroscopic techniques. Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopies suggest that the association between TQ and HSA does not affect the secondary structure of HSA. Using fluorescence spectroscopy, one mole of TQ was found to bind one mole of HSA with a binding constant of 2.39 +/- 0.2 10(4)M(-1). At 25 degrees C (pH 7.4), van't Hoff's enthalpy and entropy that accompany the binding were found to be -10.24 kJ/mol(-1) and 45 J/mol(-1)K(-1) respectively. The thermodynamic analysis of the TQ-HSA complex formation shows that the binding process is enthalpy driven and spontaneous, and that hydrophobic interactions are the predominant intermolecular forces stabilizing the complex. Furthermore, displacement experiments using warfarin and ibuprofen indicate that TQ could bind to site I of HSA, which is also in agreement with the results of the molecular modeling study.

Complex of dipeptidyl peptidase II with adenosine deaminase.

Dipeptidyl peptidase II (DPPII) from bovine kidney cortex and lung was purified to the electrophoretically homogeneous state. The molecular and catalytic characteristics of the enzyme were determined. It was revealed that DPPII preparations possess adenosine deaminase (ADA) activity at all purification steps. For the first time, the ADA-binding ability of DPPII has been shown similar to the well-known ADA-binding enzyme, DPPIV. The dissociation constant of the DPPII-ADA complex was estimated using a resonant mirror biosensor (80 nM), fluorescence polarization (60 nM), and differential spectroscopy (36 nM) techniques. The data demonstrate that DPPII can form a complex with ADA, but with one order of magnitude higher dissociation constant than that of DPPIV (7.8 nM).

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.

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain.

The effect of oxidative stress induced by neurotoxic metal ions on the properties of the brain 20S proteasome or multicatalytic proteinase complex (MPC) has been studied. Exposure of the 20S proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. T-L activity showed gradual activation by both iron ions but inhibition by Cu(II) and Zn(II). ChT-L and PGPH activities were inhibited whereas BrAAP activity was widely activated by all the tested metal salts except for zinc ions. Moreover, the exposure to ferrous salt increased the degradation rate of casein. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions. In addition, modifications induced by ferrous salt on the catalytic subunits were also supported by western blot analyses performed using anti-X, anti-Y and anti-Z antibodies. The results obtained clearly indicate that metal-catalyzed oxidation strongly affects the functions of the brain 20S proteasome, even though the catalytic subunits seem to be differently influenced by oxidative phenomena.

Interaction of adenosine deaminase with inhibitors. Chemical modification by diethyl pyrocarbonate.

The interaction of adenosine deaminase (adenosine aminohydrolase, ADA) from bovine spleen with inhibitors--erythro-9-(2-hydroxy-3-nonyl)adenine, erythro-9-(2-hydroxy-3-nonyl)-3-deazaadenine, and 1-deazaadenosine--was investigated. Using selective chemical modification by diethyl pyrocarbonate (DEP), the possible involvement of His residues in this interaction was studied. The graphical method of Tsou indicates that of six His residues modified in the presence of DEP, only one is essential for ADA activity. Inactivation of the enzyme, though with low rate, in complex with any of the inhibitors suggests that the adenine moiety of the inhibitors (and consequently, of the substrate) does not bind with the essential His to prevent its modification. The absence of noticeable changes in the dissociation constants of any of the enzyme-inhibitor complexes for the DEP-modified and control enzyme indicates that at least the most available His residues modified in our experiments do not participate in binding the inhibitors--derivatives of adenosine or erythro-9-(2-hydroxy-3-nonyl)adenine.

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.

3'-deoxyribofuranose derivatives of 1-deaza and 3-deaza-adenosine and their activity as adenosine deaminase inhibitors.

2,6-Dichloro-1-deazapurine and 2,6-dichloro-3-deazapurine were coupled with 1,2-O-diacetyl-5-O-benzoyl-3-deoxy-beta-D-ribofuranose. Deprotection of the obtained compounds and reaction with liquid ammonia gave the desired 2-chloroadenine nucleosides, which were dechlorinated to afford the corresponding 1-deaza and 3-deazaadenosine derivatives. Biological studies performed on ADA from calf intestine showed that these new nucleosides are inhibitors of the enzyme.

Purification and partial characterization of glyoxalase I from bovine brain.

Glyoxalase I was purified to homogeneity from bovine brain using affinity chromatography on S-hexylglutathione-Sepharose 6B with a yield of 22%. The enzyme was a dimer (44,000 Daltons) composed of, apparently, identical subunits (22,000 Daltons), as shown by SDS electrophoresis, and contained one mole of Zn2+/monomer. The active site metal ion, Zn2+, was removed by dialysis against EDTA, but the activity of the apoenzyme obtained was not completely restored after addition of Co2+ and Zn2+ (<25%), while a recovery of 50% was obtained after addition of Mg2+. The enzyme was inhibited by S-bromobenzylglutathione and S-p-nitrobenzylglutathione with a Ki value of 21 microM and 32 microM, respectively. The highest dissociation constant observed for the brain enzyme with respect to that reported for human erythrocytes, or other mammalian forms of enzyme could be related to a tissue-specific dependence of the glyoxalase I activity.

Functional residues on the enzyme active site of glyoxalase I from bovine brain.

Bovine brain glyoxalase I was investigated in order to identify amino acid residues essential for its catalytic activity. This enzyme is a 44-kDa dimeric protein which exhibits a characteristic intrinsic fluorescence, with an emission peak centered at 342 nm. The total of eight tryptophan residues/molecule was estimated by using a fluorescence titration method. Low values of Stern Volmer quenching constants for the quenchers used indicated that the tryptophan residues are relatively buried in the native molecule. Similar results were obtained for glyoxalase I, purified from yeast and human erythrocytes. The activity of bovine brain glyoxalase I was found to be particularly sensitive to 2,3-butanedione and diethylpyrocarbonate, selective reagents for arginine and histidine residues, respectively. A minor effect was observed by treatment of the enzyme with other amino acid-specific reagents. A protective effect of the competitive inhibitor S-hexylglutathione was observed for all reagents used, indicating the presence of modified amino acids in or near the enzyme active site.

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.

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: functional implications and different classes of inhibitors.

Adenosine deaminase (ADA) is an enzyme of the purine metabolism which catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme has been found in a wide variety of microorganisms, plants, and invertebrates. In addition, it is present in all mammalian cells that play a central role in the differentiation and maturation of the lymphoid system. However, despite a number of studies performed to date, the physiological role played by ADA in the different tissues is not clear. Inherited ADA deficiency causes severe combined immunodeficiency disease (ADA-SCID), in which both B-cell and T-cell development is impaired. ADA-SCID has been the first disorder to be treated by gene therapy, using polyethylene glycol-modified bovine ADA (PEG-ADA). Conversely, there are several diseases in which the level of ADA is above normal. A number of ADA inhibitors have been designed and synthesized, classified as ground-state and transition-state inhibitors. They may be used to mimic the genetic deficiency of the enzyme, in lymphoproliferative disorders or immunosuppressive therapy (i.e., in graft rejection), to potentiate the effect of antileukemic or antiviral nucleosides, and, together with adenosine kinase, to reduce breakdown of adenosine in inflammation, hypertension, and ischemic injury.

Isolation and characterization of bovine thymus multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC or proteasome) from bovine thymus was isolated and purified to homogeneity applying a protocol utilizing ion exchange and gel permeation chromatography as major purification tools. The purified complex shows molecular properties that are common for proteasomal molecules (high molecular mass, multisubunit organization, and multiple proteolytic activities) even though a peculiar subunit composition and the presence of specific regulatory mechanisms affecting the assembled proteolytic activities suggest a specialized function for this complex. Thymus proteasome is characterized by the presence of LMP2, LMP7, and LMP10 (MECL1) subunits, which replace the X, Y, and Z subunits. Since a similar complex was previously isolated in bovine spleen, it appears that the proteasomal population containing the LMP subunits is characteristic for organs involved in immune response. Both the thymus and spleen proteasomes are characterized by a marked efficiency in cleaving peptide bonds after branched-chain and aromatic amino acids, indicating that this proteasomal population is most likely involved in intracellular processing of class I antigenic peptides and is an example of an "in vivo" functioning immunoproteasome. However, in spite of several similarities, the complexes isolated from the two lymphoid organs do not show superimposable functional properties, which suggests the presence of organ-specific regulatory mechanisms affecting each of the proteolytic components assembled in the complex.

Multimeric self-assembly equilibria involving the histone-like protein H-NS. A thermodynamic study.

The thermodynamic parameters affecting protein-protein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by "large zone" gel-permeation chromatography. The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters. On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.

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.

Inhibition studies on membrane adenosine deaminase from human placenta.

The ecto form of adenosine deaminase isolated from human placental membrane was tested towards its sensitivity against adenosine deaminase inhibitors, such as aza and deaza analogues of adenosine and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Ki values of the inhibitors observed were similar to these obtained for the small form of adenosine deaminase purified from human erythrocytes, indicating that the presence of the binding protein on placental adenosine deaminase does not produce alteration in the binding of these inhibitors on the enzyme active site. The inhibition rate of 2'-deoxycoformycin, one of the most potent ADA inhibitors is affected by the presence of the binding protein on human placental adenosine deaminase, that probably modulates the rearrangement of the active site produced by the binding with this tight-binding inhibitor.

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.