Synchronization and entrainment of coupled circadian oscillators.

Circadian rhythms in mammals are controlled by the neurons located in the suprachiasmatic nucleus of the hypothalamus. In physiological conditions, the system of neurons is very efficiently entrained by the 24 h light-dark cycle. Most of the studies carried out so far emphasize the crucial role of the periodicity imposed by the light-dark cycle in neuronal synchronization. Nevertheless, heterogeneity as a natural and permanent ingredient of these cellular interactions seemingly plays a major role in these biochemical processes. In this paper, we use a model that considers the neurons of the suprachiasmatic nucleus as chemically coupled modified Goodwin oscillators, and introduce non-negligible heterogeneity in the periods of all neurons in the form of quenched noise. The system response to the light-dark cycle periodicity is studied as a function of the interneuronal coupling strength, external forcing amplitude and neuronal heterogeneity. Our results indicate that the right amount of heterogeneity helps the extended system to respond globally in a more coherent way to the external forcing. Our proposed mechanism for neuronal synchronization under external periodic forcing is based on heterogeneity-induced oscillator death, damped oscillators being more entrainable by the external forcing than the self-oscillating neurons with different periods.

Surface-enhanced Raman and steady fluorescence study of interaction between antitumoral drug 9-aminoacridine and trypsin-like protease related to metastasis processes, guanidinobenzoatase.

Fluorescence spectroscopy and surface-enhanced Raman spectroscopy (SERS) were applied to study the interaction of the antitumoral drug 9-aminoacridine (9AA) with a trypsin-like protease guanidinobenzoatase (GB) extracted from a mouse Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission was detected in the emission fluorescence spectra at certain drug and enzyme concentrations. A SERS study was accomplished on silver colloids at several excitation wavelengths in order to obtain more information about the interaction mechanism. The results derived from Raman spectroscopy indicated that 9AA in the amino monomeric form may interact with the enzyme by means of two different bonds: an ionic bond with a negatively charged amino acid and a ring stacking interaction with an aromatic residue placed in the catalytic site of GB. This interaction mechanism was responsible for a strong exciplex emission detected at a longer wavelength than the expected value of the normal fluorescence emission. Moreover, the GB concentration dependence of the interaction suggested that the drug was sensitive to the quaternary structure of the enzyme.

Interaction of the antitumor drug 9-aminoacridine with guanidinobenzoatase studied by spectroscopic methods: a possible tumor marker probe based on the fluorescence exciplex emission.

Fluorescence spectroscopy, surface-enhanced Raman spectroscopy (SERS), and analytical centrifugation are applied in this work to study the interaction of the antitumor drug 9-aminoacridine (9AA) with a trypsin-like protease, guanidinobenzoatase (GB), extracted from an Erlich tumor. As a consequence of this interaction, a strong 9AA exciplex emission can be detected at a certain drug and enzyme concentration. The 9AA exciplex emission was also studied for 9AA interacting with others serin proteases: alpha-chymotrypsin, trypsin, and penicillin G-acylase (PGA), as well as with bovine serum albumin (BSA) in order to obtain information about the active center of GB. We have found that the exciplex 9AA emission may be induced by a ring-stacking interaction between the monomeric drug, under the amino form, and an aromatic residue placed in the catalytic site of the protein. The results derived from Raman spectroscopy corroborate this interaction mechanism, as demonstrated by the existence of typical protonated amino 9AA marker bands as well as an important modification of the ring vibrations, thus indicating the existence of an interaction through ring stacking. The analytical centrifugation technique was applied to study the GB association in aqueous solution, demonstrating that the 9AA/GB interaction depends on the enzyme quaternary structure. An interaction of 9AA with an associate form of GB, which may be the actual enzyme active form, is suggested.

Essential role of the concentration of immobilized ligands in affinity chromatography: purification of guanidinobenzoatase on an ionized ligand.

Guanidinobenzoatase, a plasma protein with possible application as a 'tumor marker', has been fully purified by one-step affinity chromatography. The affinity matrix was prepared by 'controlled' immobilization of an enzyme inhibitor (agmatine) onto commercial agarose gels containing carboxyl moieties activated as N-hydroxysuccinimide esters. In this way, agmatine becomes immobilized through an amido bond and preserves an ionized guanidino moiety. Different matrices with different concentration of ligands were prepared in order to evaluate their properties as affinity supports. Interestingly, matrices with a very low concentration of immobilized ligands (2 microlmol/ml, corresponding to the modification of only 5% of active groups in the commercial resins) exhibited a low capacity for unspecific adsorption of proteins (as anion-exchange resins) and displayed also a high capacity for specific adsorption of our target protein. On the other hand, when affinity matrices possessed a moderate concentration of agmatine (10 micromol/ml of gel or higher), two undesirable phenomena were observed: (a) the matrix behaves as a very good anionic exchange support able to non-specifically adsorb most of plasma proteins and (b) the specific adsorption of our target protein becomes much lower. The latter phenomenon could be due to steric hindrances promoted by the interaction between each individual immobilized ligand and the corresponding binding pocket in the target protein. These hindrances could also be promoted by the presence of a fairly dense layer of immobilized ligands covering the support surface, thus preventing interactions between immobilized ligands and partially buried protein-binding pockets. In this way, a successful affinity purification (23.5% yield, x220 purification factor, a unique electrophoretic band) could be achieved by combination of three approaches: (i) the use of affinity matrices possessing a very low density of immobilized ligands, (ii) performing affinity adsorption at high ionic strength and (iii) performing specific desorption with substrates or substrate analogues.

Interaction of antimalarial drug quinacrine with nucleic acids of variable sequence studied by spectroscopic methods.

The interaction of antimalarial drug quinacrine (QA) with polynucleotides is studied by UV-visible absorption, fluorescence and surface-enhanced Raman spectroscopy (SERS). The polynucleotides employed for such a study were calf thymus DNA, poly(A).poly(T), poly(A).poly(U), poly(C).poly(G) and poly(dG-dC).poly(dG-dC). Absorption and fluorescence spectra of QA complexes indicate that an interaction with the biomolecule is taking place, although different interaction mechanisms are probable depending on the sequence. The SERS spectra also reflect spectral changes which depend on the polymer sequence and that can be correlated to those observed by fluorescence, with the advantage of the detailed structural information provided by this vibrational technique. QA interacts with polynucleotides through its diprotonated form and by ring stacking. The strength of such interaction is extremely sequence dependent, thus suggesting different interaction mechanisms in each case. The SERS technique allows the simultaneous study of those polynucleotide moieties that are directly involved in the interaction thanks to the short-range character of the SERS spectroscopy. The interaction of QA with the above nucleic acids lead to a different change in the chain stability and flexibility which is further related to the different denaturation tendency of the polymer in the presence of the metal surface.

Affinity chromatography of plasma proteins (guanidinobenzoatase): use of mimetic matrices and mimetic soluble ligands to prevent the binding of albumin on target affinity matrices.

Serum albumin is the most abundant protein in plasma and it has a high capacity to bind many small compounds and macromolecules. In this way, albumin may promote important interferences during affinity chromatography of plasma proteins. Guanidinobenzoatase (GB) is a very relevant plasma protease that seems to be related to tumoral processes. This enzyme may be adsorbed on tailor-made agmatine-amide-agarose (CH-A) supports (e.g., the ones having 2 micromol of guanidino groups per ml of agarose attached to the support, through a 6 C aliphatic chain). Such tailor-made supports containing a very low concentration of ionized groups are hardly able to adsorb any protein by anion-exchange. However, they are able to strongly adsorb albumin. In order to solve this problem new mimetic affinity matrices have been designed: (i) by using the same ligand immobilized through a different chemical linkage [guanidino groups attached via secondary amino bonds, (AEA)] or (ii) by using slightly different ligands (e.g., 1,8-octanediamine containing a primary amino group instead of a guanidino one) also attached to the support via amido bonds (CH-DAO). Albumin adsorbs on the target and on the two mimetic matrices while GB is mainly adsorbed on the target one. Moreover, the adsorption of albumin on the affinity matrix (CH-A) is very strongly inhibited by the presence of low concentrations of soluble ligands (e.g., 1,8-octanediamine containing two ionized primary amino groups). On the contrary, the adsorption of GB on CH-A is hardly inhibited by the presence of such mimetic soluble ligand. In this way, the former offering of crude GB samples to AEA plus the use of mimetic inhibitors during adsorption of the extract on CH-A completely prevent the undesirable adsorption of albumin. In a such way, an extremely selective adsorption of GB can be performed. Such an improved chromatography procedure allows a very easy affinity purification and detection of GB.

Fluorescence and surface-enhanced Raman study of 9-aminoacridine in relation to its aggregation and excimer emission in aqueous solution and on silver surface.

Fluorescence spectroscopy and surface-enhanced Raman spectroscopy (SERS) have been applied to study the aggregation and excimer emission of 9-aminoacridine (9AA) and 9-aminoacridine hydrochloride (9AA-HCl) in aqueous solution and on silver colloids. The effect of the drug concentration, pH, and chloride concentration on these processes has been investigated. The excimer emission of 9AA is connected to the dimerization of this drug in solution: the formation of 9AA dimers is greatly favored when the drug is under the amino form at neutral and acidic pH, while at alkaline pH the imino 9AA form tends to form large-size aggregates which cannot be excited to render excimer emission. 9AA is adsorbed on the silver surface under two different forms: strongly and weakly attached 9AA, each one corresponding to the different drug tautomers: imino and amino. The interaction of 9AA with silver induces a charge transfer from the adsorbate to the metal leading to a remarkable fluorescence quenching, a basicity decrease of the adsorbed drug and a considerable weakening of the dimer-excimer emission. Furthermore, an attribution of the main Raman features appearing in the SERS spectra has been proposed, providing marker bands for the imino and amino 9AA tautomers, and a mechanism for the molecular dimerization is also suggested.