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Biomedical subjects

F Blasco

Publications and source records attributed to F Blasco.

At least 19 recordsLinked to original sources
PubMed2005

Investigation of laser-irradiated Ar cluster dynamics from K-shell x-ray emission measurements.

Intense (up to a few 10(17) W/ cm2) femtosecond (down to 40 fs) laser pulses are focused onto a partially clusterized argon gas jet. The target was previously characterized and optimized in order to get a homogeneous and dense jet of clusters with a well controlled size. The interaction leads to x-ray emission that is absolutely calibrated and spectrally resolved using a high resolution time-integrated spectrometer in the K-shell range (from 2.9 to 4.3 keV). X-ray spectra are investigated as a function of different laser temporal parameters such as the nanosecond prepulse contrast, the laser pulse duration, and the femtosecond delay between two different laser pulses. The cluster size ranges from 180 to 350 angstroms and irradiation by laser pulses with both linear and circular polarization is investigated. The experimental results are discussed in terms of the laser-cluster interaction dynamics. They are compared with the predictions of collision-dominated nanoplasma models. However, further interaction processes are required in order to explain the observed characteristic lines demonstrating highly charged ions up to Ar16+.

Journal Article↗
PubMed2005

[Expert opinion on the basic surgical technique for laparoscopic ventral hernia repair].

Laparoscopic ventral hernia repair is currently the subject of intense debate, even though it provides a series of advantages over open surgery and is feasible and safe. Various studies have shown this technique to be as effective as open repair with a lower recurrence rate. Despite the excellent results of laparoscopic repair of ventral hernias, there are numerous controversies associated with this procedure. These controversies concern the indications and contraindications of the procedure, and technical aspects such as how to create the pneumoperitoneum, perform adhesiolysis, manage the hernia sac, and insert and fix the mesh to the anterior abdominal wall. Also controversial are outcome, complications related to postoperative seroma, and which type and size of mesh should be used. The present article aims primarily to address many of these issues, based on the experience of distinct surgeons with expertise in this approach, in order to provide data to establish a consensus on how laparoscopic ventral hernia repair should be performed.

Hernia, Ventral↗
PubMed2004

CYP175A1 from Thermus thermophilus HB27, the first beta-carotene hydroxylase of the P450 superfamily.

The biological function of thermostable P450 monooxygenase CYP175A1 from Thermus thermophilus HB27 was studied by functional complementation in Escherichia coli. The gene product of CYP175A1 added hydroxyl groups to both beta rings of beta-carotene to form zeaxanthin (beta,beta-carotene-3,3'-diol) in E. coli, which produces beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. In addition, spectroscopic methods revealed that E. coli carrying CYP175A1 and the cDNA of the Haematococcus pluvialis carotene ketolase was able to synthesise hydroxyechinenone. The predicted amino acid sequence of the enzyme from T. thermophilus does not show substantial similarity with other known beta-carotene hydroxylases, but 41% with the cytochrome P450 monooxygenase from Bacillus megaterium (CYP102A1, P450 BM3). It is concluded that CYP175 A1 represents a new type of beta-carotene hydroxylase of the P450 superfamily.

Cytochrome P-450 Enzyme System↗
PubMed2004

Expression profiles of a human pancreatic cancer cell line upon induction of apoptosis search for modulators in cancer therapy.

We analyzed the differential gene expression in the pancreatic cancer cell line NP-18 upon induction of apoptosis caused by cyclin-dependent kinase inhibition triggered by either overexpression of the tumor suppressor gene p16(INK4A)using an adenoviral construction or incubation with the chemical inhibitors, roscovitine or olomoucine. Screening was performed using cDNA arrays from Clontech that allowed the determination of the expression of 1,176 genes specifically related with cancer. The analysis was carried out using the Atlas Image 2.01 (Clontech) and GeneSpring 4.2 (Silicon Genetics) softwares. Among the differentially expressed genes, we chose for further validation histone deacetylase 1 (HDAC1), von Hippel Lindau and decorin as upregulated genes, and Sp1, hypoxia-inducible factor-1 alpha and DNA primase as downregulated genes. The changes in the expression of these genes to mRNA were validated by quantitative RT-PCR and the final translation into protein by Western blot analysis. Inhibition of HDAC activity, Sp1 binding and DNA primase expression led to an increase in the level of apoptosis, both in parental cells and in doxorubicin-resistant cells. Therefore, these proteins could constitute possible targets to develop modulators in cancer chemotherapy that would increase or restore apoptosis.

Adenoviridae↗
PubMed2002

Photon acceleration of ultrashort laser pulses by relativistic ionization fronts.

We present experimental results from the collision of weak ultrashort pulses with relativistic ionization fronts in copropagation and counterpropagation. The observed frequency upshifts of the probe pulses provide not only information about the electron density of the ionization front but also reveal the fine structure of the front. The connection between the correlation lengths for copropagation and counterpropagation and the longitudinal and transverse dimensions of the ionization front is also demonstrated thus showing the feasibility of using the frequency upshift experienced by short probe pulses to fully characterize relativistic ionization fronts and other relativistic coherent structures in laser-produced plasmas.

Journal Article↗
PubMed2002

Hot-electron influence on L-shell spectra of multicharged Kr ions generated in clusters irradiated by femtosecond laser pulses.

Strong L-shell x-ray emission has been obtained from Kr clusters formed in gas jets and irradiated by 60-500-fs laser pulses. Spectral lines from the F-, Ne- Na-, and Mg-like charge states of Kr have been identified from highly resolved x-ray spectra. Spectral line intensities are used in conjunction with a detailed time-dependent collisional-radiative model to diagnose the electron distribution functions of plasmas formed in various gas jet nozzles with various laser pulse durations. It is shown that L-shell spectra formed by relatively long nanosecond-laser pulses can be well described by a steady-state model without hot electrons when opacity effects are included. In contrast, adequate modeling of L-shell spectra from highly transient and inhomogeneous femtosecond-laser plasmas requires including the influence of hot electrons. It is shown that femtosecond-laser interaction with gas jets from conical nozzles produces plasmas with higher ionization balances than plasmas formed by gas jets from Laval nozzles, in agreement with previous work for femtosecond laser interaction with Ar clusters.

Journal Article↗
PubMed2001

Electron transfer from heme bL to the [3Fe-4S] cluster of Escherichia coli nitrate reductase A (NarGHI).

We have investigated the functional relationship between three of the prosthetic groups of Escherichia coli nitrate reductase A (NarGHI): the two hemes of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster of the electron-transfer subunit (NarH). In two site-directed mutants (NarGHI(H56R) and NarGHI(H205Y)) that lack the highest potential heme of NarI (heme b(H)), a large negative DeltaE(m,7) is elicited on the NarH [3Fe-4S] cluster, suggesting a close juxtaposition of these two centers in the holoenzyme. In a mutant retaining heme b(H), but lacking heme b(L) (NarGHI(H66Y)), there is no effect on the NarH [3Fe-4S] cluster redox properties. These results suggest a role for heme b(H) in electron transfer to the [3Fe-4S] cluster. Studies of the pH dependence of the [3Fe-4S] cluster, heme b(H), and heme b(L) E(m) values suggest that significant deprotonation is only observed during oxidation of the latter heme (a pH dependence of -36 mV pH(-1)). In NarI expressed in the absence of NarGH [NarI(DeltaGH)], apparent exposure of heme b(H) to the aqueous milieu results in both it and heme b(L) having E(m) values with pH dependencies of approximately -30 mV pH(-1). These results are consistent with heme b(H) being isolated from the aqueous milieu and pH effects in the holoenzyme. Optical spectroscopy indicates that inhibitors such as HOQNO and stigmatellin bind and inhibit oxidation of heme b(L) but do not inhibit oxidation of heme b(H). Fluorescence quench titrations indicate that HOQNO binds with higher affinity to the reduced form of NarGHI than to the oxidized form. Overall, the data support the following model for electron transfer through the NarI region of NarGHI: Q(P) site --> heme b(L) --> heme b(H) --> [3Fe-4S] cluster.

Benzoquinones↗
PubMed2001

The coordination and function of the redox centres of the membrane-bound nitrate reductases.

Under anaerobic conditions and in the presence of nitrate, the facultative anaerobe Escherichia coli synthesises an electron-transport chain comprising a primary dehydrogenase and the terminal membrane-bound nitrate reductase A (NarGHI). This review focuses on recent advances obtained on the structure and function of the three protein subunits of membrane-bound nitrate reductases. We discuss a global architecture for the Mo-bisMGD-containing subunit (NarG) and a coordination model for the four [Fe-S] centres of the electron-transfer subunit (NarH) and for the two b-type haems of the anchor subunit NarI.

Cell Membrane↗
PubMed2001

The diheme cytochrome b subunit (Narl) of Escherichia coli nitrate reductase A (NarGHI): structure, function, and interaction with quinols.

Significant recent advances have been made in studies of the major dissimilatory nitrate reductase (NarGHI) of Escherichia coli. This enzyme is a complex iron-sulfur ([Fe-S]) molybdoenzyme that oxidizes menaquinol or ubiquinol at a periplasmically oriented Q-site (Qp site), and reduces nitrate at a cytoplasmically-oriented molybdo-(bismolybdopterin guanine dinucleotide) (Mo-bisMGD) cofactor. The Qp site, as well as two hemes, termed bL and bH, are localized in a hydrophobic diheme cytochrome b(Narl) that: (i) provides a conduit for electron-transfer from the periplasmically-oriented Qp-site; (ii) provides a membrane anchoring functionality for the membrane-extrinsic subunits (NarGH) that coordinate the Mo-bisMGD (NarG) and four [Fe-S] clusters (NarH); and (iii) helps ensure the separation of sites of H+-yielding and H+-consuming reactions such that enzyme turnover leads to the generation of a proton-electrochemical potential across the cytoplasmic membrane. This minireview focuses on recent advances and future prospects for the diheme cytochrome b subunit (Narl) of NarGHI.

Amino Acid Sequence↗
PubMed2000

Heteronuclear NMR and soft docking: an experimental approach for a structural model of the cytochrome c553-ferredoxin complex.

The combination of docking algorithms with NMR data has been developed extensively for the studies of protein-ligand interactions. However, to extend this development for the studies of protein-protein interactions, the intermolecular NOE constraints, which are needed, are more difficult to access. In the present work, we describe a new approach that combines an ab initio docking calculation and the mapping of an interaction site using chemical shift variation analysis. The cytochrome c553-ferredoxin complex is used as a model of numerous electron-transfer complexes. The 15N-labeling of both molecules has been obtained, and the mapping of the interacting site on each partner, respectively, has been done using HSQC experiments. 1H and 15N chemical shift analysis defines the area of both molecules involved in the recognition interface. Models of the complex were generated by an ab initio docking software, the BiGGER program (bimolecular complex generation with global evaluation and ranking). This program generates a population of protein-protein docked geometries ranked by a scoring function, combining relevant stabilization parameters such as geometric complementarity surfaces, electrostatic interactions, desolvation energy, and pairwise affinities of amino acid side chains. We have implemented a new module that includes experimental input (here, NMR mapping of the interacting site) as a filter to select the accurate models. Final structures were energy minimized using the X-PLOR software and then analyzed. The best solution has an interface area (1037.4 A2) falling close to the range of generally observed recognition interfaces, with a distance of 10.0 A between the redox centers.

Amino Acid Sequence↗
PubMed1999

The hemes of Escherichia coli nitrate reductase A (NarGHI): potentiometric effects of inhibitor binding to narI.

We have potentiometrically characterized the two hemes of Escherichia coli nitrate reductase A (NarGHI) using EPR and optical spectroscopy. NarGHI contains two hemes, a low-potential heme b(L) (E(m,7) = 20 mV; g(z)() = 3.36) and a high-potential heme b(H) (E(m, 7) = 120 mV; g(z)() = 3.76). Potentiometric analyses of the g(z)() features of the heme EPR spectra indicate that the E(m,7) values of both hemes are sensitive to the menaquinol analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO). This inhibitor causes a potential-inversion of the two hemes (for heme b(L), E(m,7) = 120 mV; for heme b(H), E(m,7) = 60 mV). This effect is corroborated by optical spectroscopy of a heme b(H)-deficient mutant (NarGHI(H56R)) in which the heme b(L) undergoes a DeltaE(m,7) of 70 mV in the presence of HOQNO. Another potent inhibitor of NarGHI, stigmatellin, elicits a moderate heme b(L) DeltaE(m,7) of 30 mV, but has no detectable effect on heme b(H). No effect is elicited by either inhibitor on the line shape or the E(m,7) values of the [3Fe-4S] cluster coordinated by NarH. When NarI is expressed in the absence of NarGH [NarI(DeltaGH)], two hemes are detected in potentiometric titrations with E(m,7) values of 37 mV (heme b(L); g(z)() = 3.15) and -178 mV (heme b(H); g(z)() = 2.92), suggesting that heme b(H) may be exposed to the aqueous milieu in the absence of NarGH. The identity of these hemes was confirmed by recording EPR spectra of NarI(DeltaGH)(H56R). HOQNO binding titrations followed by fluorescence spectroscopy suggest that in both NarGHI and NarI(DeltaGH), this inhibitor binds to a single high-affinity site with a K(d) of approximately 0.2 microM. These data support a functional model for NarGHI in which a single dissociable quinol binding site is associated with heme b(L) and is located toward the periplasmic side of NarI.

Binding Sites↗
PubMed1998

Molybdenum cofactor properties and [Fe-S] cluster coordination in Escherichia coli nitrate reductase A: investigation by site-directed mutagenesis of the conserved his-50 residue in the NarG subunit.

Most of the molybdoenzymes contain, in the amino-terminal region of their catalytic subunits, a conserved Cys group that in some cases binds an [Fe-S] cluster. In dissimilatory nitrate reductases, the first Cys residue of this motif is replaced by a conserved His residue. Site-directed mutagenesis of this residue (His-50) was performed on the NarG subunit from Escherichia coli nitrate reductase A. The results obtained by EPR spectroscopy enable us to exclude the implication of this residue in [Fe-S] binding. Additionally, we showed that the His-50 residue does not coordinate the molybdenum atom, but its substitution by Cys or Ser introduces a perturbation of the hydrogen bonding network around the molybdenum cofactor. From potentiometric studies, it is proposed that the high-pH and the low-pH forms of the Mo(V) are both involved during the redox turnover of the enzyme. Perturbation of the Mo(V) pKV value might be responsible for the low activity reported in the His-50-Cys mutant enzyme. A catalytic model is proposed in which the protonation/deprotonation of the Mo(V) species is an essential step. Thus, one of the two protons involved in the catalytic cycle could be the one coupled to the molybdenum atom in the dissimilatory nitrate reductase of E. coli.

Cell Membrane↗
PubMed1998

Inhibitor binding within the NarI subunit (cytochrome bnr) of Escherichia coli nitrate reductase A.

We have used inhibitors and site-directed mutants to investigate quinol binding to the cytochrome bnr (NarI) of Escherichia coli nitrate reductase (NarGHI). Both stigmatellin and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) inhibit menadiol:nitrate oxidoreductase activity with I50 values of 0.25 and 6 microM, respectively, and prevent the generation of a NarGHI-dependent proton electrochemical potential across the cytoplasmic membrane. These inhibitors have little effect on the rate of reduction of the two hemes of NarI (bL and bH), but have an inhibitory effect on the extent of nitrate-dependent heme reoxidation. No quinol-dependent heme bH reduction is detected in a mutant lacking heme bL (NarI-H66Y), whereas a slow but complete heme bL reduction is detected in a mutant lacking heme bH (NarI-H56R). This is consistent with physiological quinol binding and oxidation occurring at a site (QP) associated with heme bL which is located toward the periplasmic side of NarI. Optical and EPR spectroscopies performed in the presence of stigmatellin or HOQNO provide further evidence that these inhibitors bind at a heme bL-associated QP site. These results suggest a model for electron transfer through NarGHI that involves quinol binding and oxidation in the vicinity of heme bL and electron transfer through heme bH to the cytoplasmically localized membrane-extrinsic catalytic NarGH dimer.

Electron Spin Resonance Spectroscopy↗
PubMed1998

The molybdenum cofactor of Escherichia coli nitrate reductase A (NarGHI). Effect of a mobAB mutation and interactions with [Fe-S] clusters.

We have studied the effect of a mobAB mutation and tungstate on molybdo-molybdopterin-guanine dinucleotide (Mo-MGD) insertion into Escherichia coli nitrate reductase (NarGHI). Preparation of fluorescent oxidized derivatives of MGD (Form A and Form B) indicates that in a mobAB mutant there is essentially no detectable cofactor present in either the membrane-bound (NarGHI) or purified soluble (NarGH) forms of the enzyme. Electron paramagnetic resonance characterization of membrane-bound cofactor-deficient NarGHI suggests that it has altered electrochemistry with respect to the dithionite reducibility of the [Fe-S] clusters of NarH. Potentiometric titrations of membrane-bound NarGHI indicate that the NarH [Fe-S] clusters have midpoint potentials at pH 8.0 (Em,8.0 values) of +180 mV ([3Fe-4S] cluster), +130, -55, and -420 mV ([4Fe-4S] clusters) in a wild-type background and +180, +80, -35, and -420 mV in a mobAB mutant background. These data support the following conclusions: (i) a model for Mo-MGD biosynthesis and assembly into NarGHI in which both metal chelation and nucleotide addition to molybdopterin precede cofactor insertion; and (ii) the absence of Mo-MGD significantly affects Em,8.0 of the highest potential [4Fe-4S] cluster.

Binding Sites↗