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Biomedical subjects

F Montero

Publications and source records attributed to F Montero.

At least 19 recordsLinked to original sources
PubMed2003

Initial stages of microbiologically influenced tarnishing on titanium after 20 months of immersion in freshwater.

This paper studies the initial stages of iridescent tarnishes on titanium heat exchanger tubes in contact with running freshwater on the river Tagus in Spain for up to 20 months. Electrochemical impedance spectroscopy (EIS), scanning electron microscopy [(SEM with energy dispersive X-ray (EDX)] and X-ray photoelectron spectroscopy (XPS) in conjunction with argon-ion sputtering were the techniques used. The EIS data indicated a capacitive behavior, showing a semicircle that was better defined as the experimental time increased, indicating a decreasing tarnishing resistance of titanium. XPS and EDX results indicated that the main elements identified were calcium, phosphorus, nitrogen, and iron. The amount of these elements was higher on the tarnished titanium specimens than on the untarnished specimens. SEM analysis showed the presence of diatoms in the iridescent tarnishes on titanium tubes.

Animals↗
PubMed2001

Sequence distribution and intercooperativity detection for two ligands simultaneously binding to DNA.

A method for detecting and quantifying the cooperativity in the simultaneous binding of two ligands, A and B, to DNA (intercooperativity; omega(AB)) is proposed. This involves the determination of an apparent affinity constant K(app) for one of the ligands (A) in the limit of its null saturation (nu(Alpha) --> 0), in the presence of the second one (B). A definition for this constant is given and an expression is derived corresponding to a simple model of competitive binding to an unbranched three-state homogeneous polar lattice with nearest-neighbor interactions (Markov chain). The ratio between the apparent and intrinsic affinity constants of one ligand in the maximum saturation limit of the other one becomes omega(2)(AB), and thus can be graphically obtained from K(app)(A) vs nu(B) plots. All the frequencies that define the sequence distribution of ligands can be easily calculated by introducing some generalized statistical weights for the free lattice monomer in a standard sequence generating function procedure. A model of fluorescence quenching emission is obtained from such frequencies under the hypothesis of a short-range electron transfer mechanism of the deactivation; it confirms, as suggested by the binding model, an outstanding influence of the intercooperativity on the distribution.

Binding Sites↗
PubMed2001

Flow cytometric microsphere-based immunoassay: analysis of secreted cytokines in whole-blood samples from asthmatics.

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-gamma (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-gamma than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.

Adult↗
PubMed2000

A ruthenium probe for cell viability measurement using flow cytometry, confocal microscopy and time-resolved luminescence.

The capability of the new luminescent probe (dibenzo[h,j] dipyrido[3,2-a:2',3'-c]phenazine)bis(2,2'-bipyridine)ruthenium(II) dication, (RB2Z), to discriminate live and dead cells has been tested on rat hepatocytes and mouse lymphocytes. RB2Z-stained cells were analyzed using flow cytometry, fluorescence (confocal) microscopy and time-resolved luminescence measurements. The established viability probes propidium iodide (PI) and SYTOX green (SG) were used as controls. The intense luminescence of RB2Z at 606 nm is localized in the nucleus of nonviable cells. Viability measurements by flow cytometry and fluorescence microscopy using RB2Z as dead-cell marker yield the same results as PI and SG. The luminescence lifetime of RB2Z also displays sensitivity to cell viability (0.45 and 0.82 microsecond in presence of fully viable and dead cells, respectively). This ruthenium complex is photostable under laser sources and its 200 nm Stokes shift facilitates multicolor labeling experiments in flow cytometry and fluorescence microscopy. Unlike the currently available probes, the long-lived excited state of RB2Z also allows assays based on luminescence decay measurements.

Animals↗
PubMed1999

Generalization of the theory of transition times in metabolic pathways: a geometrical approach.

Cell metabolism is able to respond to changes in both internal parameters and boundary constraints. The time any system variable takes to make this response has relevant implications for understanding the evolutionary optimization of metabolism as well as for biotechnological applications. This work is focused on estimating the magnitude of the average time taken by any observable of the system to reach a new state when either a perturbation or a persistent variation occurs. With this aim, a new variable, called characteristic time, based on geometric considerations, is introduced. It is stressed that this new definition is completely general, being useful for evaluating the response time, even in complex transitions involving periodic behavior. It is shown that, in some particular situations, this magnitude coincides with previously defined transition times but differs drastically in others. Finally, to illustrate the applicability of this approach, a model of a reaction mediated by an allosteric enzyme is analyzed.

Allosteric Regulation↗
PubMed1999

METATOOL: for studying metabolic networks.

MOTIVATION: To reconstruct metabolic pathways from biochemical and/or genome sequence data, the stoichiometric and thermodynamic feasibility of the pathways has to be tested. This is achieved by characterizing the admissible region of flux distributions in steady state. This region is spanned by what can be called a convex basis. The concept of 'elementary flux modes' provides a mathematical tool to define all metabolic routes that are feasible in a given metabolic network. In addition, we define 'enzyme subsets' to be groups of enzymes that operate together in fixed flux proportions in all steady states of the system. RESULTS: Algorithms for computing the convex basis and elementary modes developed earlier are briefly reviewed. A newly developed algorithm for detecting all enzyme subsets in a given network is presented. All of these algorithms have been implemented in a novel computer program named METATOOL, whose features are outlined here. The algorithms are illustrated by an example taken from sugar metabolism. AVAILABILITY: METATOOL is available from ftp://bmsdarwin.brookes.ac. uk/pub/software/ibmpc/metatool. SUPPLEMENTARY INFORMATION: http://www. biologie.hu-berlin.de/biophysics/Theory/tpfeiffer/metatoo l.html

Algorithms↗
PubMed1997

Transient times in linear metabolic pathways under constant affinity constraints.

In the early seventies, Easterby began the analytical study of transition times for linear reaction schemes [Easterby (1973) Biochim. Biophys. Acta 293, 552-558]. In this pioneer work and in subsequent papers, a state function (the transient time) was used to measure the period before the stationary state, for systems constrained to work under both constant and variable input flux, was reached. Despite the undoubted usefulness of this quantity to describe the time-dependent features of these kinds of systems, its application to the study of chemical reactions under other constraints is questionable. In the present work, a generalization of these magnitudes to linear metabolic pathways functioning under a constant-affinity constraint is carried out. It is proved that classical definitions of transient times do not reflect the actual properties of the transition to the steady state in systems evolving under this restriction. Alternatively, a more adequate framework for interpretation of the transient times for systems with both constant and variable input flux is suggested. Within this context, new definitions that reflect more accurately the transient characteristics of constant affinity systems are stated. Finally, the meaning of these transient times is discussed.

Kinetics↗
PubMed1997

Network organization of cell metabolism: monosaccharide interconversion.

The structural properties of carbohydrate metabolism are being studied. The present contribution focuses mainly on those processes involving the transfer of carbon fragments among sugars. It is shown how enzymatic activities fix the way the system self-organizes stoichiometrically at the steady state. It is proven that there exists a specific correspondence between the set of all possible enzymic activities, the activity set, and the set of stoichiometrically compatible flux distributions through the pathway. On the one hand, there are enzymic activities that do not allow a stoichiometrically feasible coupling at the steady state of the reactions involved in the conversion. On the other hand, there are enzymic activities that are related to one or more flux distributions at the steady state (i.e. with one or several rate vectors respectively). For this latter group, it can be demonstrated that the structure of the system depends on other non-structural factors, such as boundary constraints and the kinetic parameters. As a consequence, it is suggested that this kind of metabolic process must be viewed as a complex reaction network instead of a sequential number of steps. Some implications of these derivations are illustrated for the particular conversion of CO2 --> C3. General remarks are also discussed within the framework of network models of cell metabolism.

Cells↗
PubMed1997

[Exposure to lead among the population of Barcelona: chronologic trends from 1984 to 1995].

BACKGROUND: This study was designed to determine current lead exposure in the Barcelona population and to evaluate the changes occurred during the last 10 years. Blood lead concentration was investigated in a random sample of 694 healthy subjects (age range: 0-65 years). PATIENTS AND METHODS: Adults were random selected from a group of blood donors. Samples of children analysed were selected from subjects with a preoperatory analyses without any disease that could modify blood lead levels. Lead levels were determined by atomic absorption spectrometry. RESULTS: Blood lead concentration was 4.06 +/- 1.4 micrograms/dl in umbilical cord, 8.9 +/- 2.9 micrograms/dl in the paediatric population and 7.8 +/- 4.2 micrograms/dl in the total of adults analyzed. There was statistical differences between the younger subjects and the older population. In 1984 the results found were 18.6 +/- 6.6 micrograms/dl. CONCLUSIONS: The results obtained show that in the last 10 years a reduction on the blood lead levels was occurred. This reduction is parallel with a diminish in the lead petrol concentration in the ambient air.

Adolescent↗
PubMed1997

Theoretical approaches to the evolutionary optimization of glycolysis--chemical analysis.

In the first part of this work [Heinrich, R., Montero, F., Klipp, E., Waddell, T. G. & Melendez-Hevia, E. (1997) Eur. J. Biochem. 243, 191-201] the kinetic and thermodynamic constraints under which an optimal glycolysis must be designed have been analysed. In this second part, we present a chemical analysis of the glycolytic pathway in order to determine if its design is chemically optimized according the possibilities that a glycolytic design can have. Our results demonstrate that glycolysis in modern-day cells (from glucose to lactate) has an optimized design for maximizing the flux of ATP production, and a thermodynamic profile which guarantees a high kinetic efficiency. We also discuss some cases of paleometabolism for this pathway as alternative metabolic pathways, less optimized, that exist in some bacteria. Our analysis relates mainly to metabolism designed under constant chemical affinity (substrates and products of the pathway constant), where the target of optimization can be the flux of ATP production. We also discuss the case of an externally imposed input flux, whose target of optimization is the stoichiometric yield of ATP.

Adenosine Triphosphate↗
PubMed1997

Theoretical approaches to the evolutionary optimization of glycolysis: thermodynamic and kinetic constraints.

It is analyzed whether the structural design of contemporary glycolysis can be explained theoretically on the basis of optimization principles originating from natural selection during evolution. Particular attention is paid to the problem of how the kinetic and thermodynamic properties of the glycolytic pathway are related to its stoichiometry with respect to the number and location of ATP-coupling sites. The mathematical analysis of a minimal model of unbranched energy-converting pathways shows that the requirement of high ATP-production rate favours a structural design that includes not only ATP-producing reactions (P-sites) but also ATP-consuming reactions (C-sites). It is demonstrated that, at fixed overall thermodynamic properties of a chain, the ATP-production rate may be enhanced by kinetic optimization. The ATP-production rate is increased if the C-sites are concentrated at the beginning and all the P-sites at the end of the pathway. An optimum is attained, which is characterized by numbers of coupling sites corresponding to those found in glycolysis. Various extensions of the minimal model are considered, which allow the effects of internal feedback-regulations, variable enzyme concentrations, and the symmetric branching of glycolysis at the aldolase step to be considered.

Adenosine Triphosphate↗
PubMed1997

Sequence specificity, enantiospecificity and polyelectrolyte effect in the binding to DNA of a 6-(2-pyridyl)phenanthridine chiral photonuclease.

In order to establish the basis for the rational design of a novel family of intercalating chiral photonuclease drugs aimed at photochemotherapy, namely N, N'-dialkylated 6-(2-pyridinium)phenanthridinium (pyp) dications, a detailed investigation of the DNA binding of the dq2pyp member (where dq2 stands for -CH2CH2-), was conducted. The study addresses the sequence- and enantiospecificity, as well as polyelectrolyte effects in the drug-DNA interaction. Binding isotherms with synthetic polynucleotides, forcefield calculations, affinity chromatography in a DNA-cellulose stationary phase and salt-dependent equilibrium and kinetic studies with DNA were used. dq2pyp shows a strong preference for alternating GC over AT base pairs; binding to homopolymeric DNA is weak (< 3 x 10(4) M-1). Affinity chromatography shows enantiospecific binding of dq2pyp to DNA. The polyelectrolyte contribution to the binding free energy are shown to be relatively important (-4.8 kcal/nmol out of an overall value of -7.2 kcal/mmol at 10.2 mM Na+). The slope of the logkd (dissociation rate constant) vs. log[Na+] plot (0.7) agrees with the values predicted from counterion condensation theory for a dicationic intercalator, giving further support to such a DNA binding mode for dq2pyp. The relatively high kinetic dissociation constants (logkd = 0.70log[Na+] + 3.79) in comparison with those of propidium (two orders of magnitude larger at any Na+ concentration) seems to originate from the absence of amino groups in dq2pyp. The kinetic association constants (logka = -1.06log[Na+] + 5.53) are twice these of propidium, probably due to the less restrictive positioning of dq2pyp at the intercalation site. The kinetic studies support a mechanism of intercalation in which the drug forms a pre-equilibrium outside the complex followed by the intercalation of the drug. Molecular modelling is used throughout to rationalize all the experimental data, as well as to propose new candidates with improved DNA affinity and residence time.

Base Sequence↗
PubMed1996

The metabolic productivity of the cell factory.

It is widely accepted that some performance function has been optimized during the evolution of metabolic pathways. One can study the nature of such a function by analogy with the industrial manufacturing world, in which there have been efforts over recent decades to optimize production chains, and in which it is now accepted that fluxes are not the only important system variables that determine process efficiency, because inventory turnover must also be considered. Inspired by the parallels between living cells and manufacturing factories, we propose that fluxes and transit time may have simultaneously been major targets of natural selection in the optimization of the design, structure and kinetic parameters of metabolic pathways. Accordingly we define the ratio of flux to transit time as a performance index of productivity in metabolic systems: it measures the efficiency with which stocks are administered, and facilitates comparison of a pathway in different steady states or in different tissues or organisms. For a linear chain of two enzymes, at a fixed total equilibrium constant, we have analysed the variation of flux, transit time and productivity index as functions of the equilibrium constants of the two steps. The results show that only the productivity index has a maximum, which represents a good compromise in optimizing flux and transit time. We have extended control analysis to the productivity index and derived the summation theorem that applies to it. For linear chains of different length with maximum productivity index values, the distribution of control coefficients with regard to the three parameters has a characteristic profile independent of the length of the chain. Finally, this control profile changes when other variables are optimized, and we compare the theoretical results with the control profile of the first steps of glycolysis in rat liver.

Animals↗
PubMed1995

DNA photocleavage by novel intercalating 6-(2-pyridinium)phenanthridinium viologens.

A new type of DNA-intercalating viologen dications, derived from the N,N'-dialkyl-6-(2-pyridyl)phenanthridine structure (in which dialkyl is -CH2CH2-,-CH2CH2CH2-, or (-CH3)2, abbreviated dq2pyp, dq3pyp, and Me2pyp, respectively), are able to produce frank strand breaks in supercoiled plasmid DNA upon irradiation with visible light. The amount of photocleavage is similar for the three drugs. The observed DNA photosensitization appears to follow a single-strand cleavage model, as shown by a kinetic analysis of the reaction with dq2pyp. The photodynamic action of the drugs seems to be initiated by a light-induced electron transfer reaction from the nucleobases, given the singlet excited-state redox potentials (ca. + 2.1 V vs. SHE) and the low quantum yields of singlet molecular oxygen production of the drugs (0.1-0.2 in aerated D2O).

DNA, Superhelical↗
PubMed1995

Stereospecific DNA binding of luminescent atropisomeric viologens.

Binding to B-DNA of one enantiomer of dq3pyp, --a member of a novel family of intercalating luminescent viologens derived from N,N'-dialkyl-6-(2-pyridyl)phenanthridine, in which dialkyl is -(CH2)2- (dq2pyp), -(CH2)3- (dq3pyp), or (-CH3)2 (Me2pyp)--, is strongly favored compared to its estable atropisomer. Evidence of the stereospecific interaction has been gained by resolution of a rac-dq3pyp mixture upon dialysis in the presence of DNA, monitored by circular dichroism. Molecular modeling suggests that the S enantiomer of both dq3pyp and dq2pyp is the one preferred for the binding. No similar resolution has been achieved with rac-dq2pyp due to the lower barrier to interconversion of its atropisomers, as shown by 1H-NMR. The identical binding energy of the two modeled diastereoisomeric Me2pyp-DNA complexes discard enantiospecific interaction of this drug. Affinity chromatography on DNA-cellulose allows isolation of the pure enantiomers of dq3pyp.

Circular Dichroism↗
PubMed1995

Interaction with DNA of photoactive viologens based on the 6-(2- pyridinium)phenanthridinium structure.

A new type of DNA-interacting violgens derived from the N,N'-dialkyl 6-(2-pyridinium)-phenanthridinium structure (in which dialkyl is -CH2CH2-,-CH2CH2CH2-, or (-CH3)2) have been synthesized. Electronic spectroscopy, steady-state and time-resolved fluorescence, cyclic voltammetry, binding isotherms, viscosity titrations, and molecular modeling techniques were employed to characterize the structural, photophysical and redox properties of the novel drugs as well as the corresponding drug-DNA complexes. The viologens display significant visible absorption (up to ca. 490 nm), and a rather intense luminescence (phi cm from 0.06 to 0.20 at 491-565 nm wavelength maxima) which is efficiently quenched by DNA. The calculated redox potentials of these drugs in their singlet excited state (+2.1 V vs. SHE) predict a large driving force for a photoelectron transfer reaction from the nucleobases to the drugs. Photochemical measurements of the viologens in the presence of mononucleotides, nucleosides, and deoxyribose indicate that the observed fluorescence quenching occurs indeed by electron transfer from the DNA bases rather than the sugar phosphate backbone. Large association constants to double helical DNA (in the order of 10(5) M-1) have been evaluated from the absorbance-based binding isotherms. Viscosimetry supports intercalation of the drugs into the DNA helix. Computer simulations (molecular mechanics of d(CGCGCG)2-drug complexes) confirm the intercalative nature of the binding and provide finer details about the geometry of the different viologen-DNA complexes. Molecular modeling has also revealed a stereoselective interaction of the enantiomeric drug conformers with the chiral DNA helix. A DNA-targeted drug design of future generations of these ligands in order to improve and/or modulate their photochemical, redox, and nucleic acid binding properties appears to be possible by a careful selection of the N,N'-dialkylating chain and/or the substituents on the azaheterocyclic moieties.

Base Sequence↗