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A Aliverti

Publications and source records attributed to A Aliverti.

At least 37 records · Page 2Linked to original sources
PubMed2001

[Optoelectronic plethysmography: a new tool in respiratory medicine].

Opto-electronic plethysmography (OEP) is a new noninvasive technique, recently introduced, which is highly accurate in the measurement of total chest wall volume variations, allowing partitioning of the complex shape of the chest wall into different functional compartments. It can measure breathing patterns and, if combined with pressure measurements, can be used to study statics, dynamics and energetics of the respiratory system. OEP does not require connection to the patient and can be used without a subject-specific calibration. All these features and the results obtained in several studies make the OEP not only a reliable system for basic physiological and pathophysiological research studies, but also an attractive tool to be used in clinics.

Electronics, Medical↗
PubMed2000

The CrIIL reduction of [2Fe-2S] ferredoxins and site of attachment of CrIII using 1H NMR and site-directed mutagenesis.

The recently reported NMR solution structure of FeIIIFeIII parsley FdI has made possible 2D NOESY NMR studies to determine the point of attachment of CrIIIL in FeIIIFeIII...CrIIIL. The latter Cr-modified product was obtained by reduction of FeIIIFeIII parsley and spinach FdI forms with [Cr(15-aneN4) (H2O)2]2+ (15-aneN4 = 1,4,8,12-tetraazacyclopentadecane), referred to here as CrIIL, followed by air oxidation and chromatographic purification. From a comparison of NMR cross-peak intensities of native and Cr-modified proteins, two surface sites designated A and B, giving large paramagnetic CrIIIL broadening of a number of amino acid peaks, have been identified. The effects at site A (residues 19-22, 27, and 30) are greater than those at site B (residues 92-94 and 96), which is on the opposite side of the protein. From metal (ICP-AES) and electrospray ionization mass spectrometry (EIMS) analyses on the Cr-modified protein, attachment of a single CrIIIL only is confirmed for both parsley and spinach FdI and FdII proteins. Electrostatic interaction of the 3+ CrIIIL center covalently attached to one protein molecule (charge approximately -18) with a second (like) molecule provides an explanation for the involvement of two regions. Thus for 3-4 mM FeIIIFeIII...CrIIIL solutions used in NMR studies (CrIIIL attached at A), broadening effects due to electrostatic interactions at B on a second molecule are observed. Experiments with the Cys18Ala spinach FdI variant have confirmed that the previously suggested Cys-18 at site A is not the site of CrIIIL attachment. Line broadening at Val-22 of A gives the largest effect, and CrIIIL attachment at one or more adjacent (conserved) acidic residues in this region is indicated. The ability of CrIIL to bind in some (parsley and spinach) but not all cases (Anabaena variabilis) suggests that intramolecular H-bonding of acidic residues at A is relevant. The parsley and spinach FeIIFeIII...CrIIIL products undergo a second stage of reduction with the formation of FeIIFeII...CrIIIL. However, the spinach Glu92Ala (site B) variant undergoes only the first stage of reduction, and it appears that Glu-92 is required for the second stage of reduction to occur. A sample of CrIIIL-modified parsley FeIIIFeIII Fd is fully active as an electron carrier in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase.

Amino Acid Sequence↗
PubMed2000

Competition between C-terminal tyrosine and nicotinamide modulates pyridine nucleotide affinity and specificity in plant ferredoxin-NADP(+) reductase.

Chloroplast ferredoxin-NADP(+) reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP(+) photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2'-phosphoryl group of NADP(+), replacement of the C-terminal tyrosine (Tyr(308) in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k(cat) value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD(+) complexes revealed that the nicotinamide ring of NAD(+) binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k(cat) values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2'-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP(+) reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.

Amino Acid Sequence↗
PubMed2000

Optoelectronic plethysmography in intensive care patients.

We used optoelectronic plethysmography to study 11 normal subjects during quiet and deep breathing, six sedated and paralyzed patients with acute lung injury and acute respiratory distress syndrome (ALI/ARDS) receiving continuous positive pressure ventilation (CPPV) (positive end-expiratory pressure [PEEP] = 10 cm H(2)O, tidal volume [VT] = 300, 600, 900 ml), and seven ALI/ARDS patients receiving pressure support ventilation (PSV) (PEEP 10 cm H(2)O, pressure support = 5, 10, 15, 25 cm H(2)O). The volumes measured using optoelectronic plethysmography were compared with measurements taken using spirometry and pneumotachography. The three methods were highly correlated. The discrepancies found were 1.7 +/- 5.9%, -1.6 +/- 5.4%, and 4.9 +/- 6.4% when comparing optoelectronic plethysmography with spirometry, optoelectronic plethysmography with pneumotachography, and spirometry with pneumotachography, respectively. Accuracy of the compartmentalization procedure (upper thorax, lower thorax, and abdomen) was assessed by calculating compartmental volume changes during isovolume maneuvers. The discrepancy from the ideal zero line was -2.1 +/- 48.3 ml. Abdominal contribution to inspired volume was greater for normal subjects than for PSV patients (63 +/- 11% versus 43 +/- 14%, p < 0.001). It decreased with VT for normal subjects (48.5 +/- 15%, p < 0.05), whereas it increased for CPPV patients (61 +/- 10%, p < 0.05). No significant distribution differences were found between 5 and 25 cm H(2)O PSV. We conclude that optoelectronic plethysmography is a feasible technique able to provide unique data on the distribution of chest wall volume changes in intensive care patients.

Adult↗
PubMed1999

Interaction of the soluble recombinant PsaD subunit of spinach photosystem I with ferredoxin I.

Photosystem I of higher plants functions in photosynthesis as a light-driven oxidoreductase producing reduced ferredoxin. Its peripheral subunit PsaD has been identified as the docking site for ferredoxin I. With the aim of elucidating the structure-function relationship and the role of this subunit, a recombinant form of the spinach protein was produced by heterologous expression in Escherichia coli. The PsaD protein was synthesized in soluble form and purified to homogeneity. The interaction of the PsaD subunit with ferredoxin I was investigated using three different approaches: chemical cross-linking between the two purified proteins in solution, affinity chromatography of the PsaD subunit on a ferredoxin-coupled resin, and titration with ferredoxin of the protein fluorescence of the subunit. All these studies indicated that the isolated PsaD in solution has a definite conformation and maintains the ability to bind ferredoxin I with high affinity and specificity. The Kd value of the complex of PsaD and ferredoxin is in the nanomolar range, which is consistent with reported Km values for ferredoxin I photoreduction by thylakoid membranes. The ionic strength dependence of the K(d) suggests that the protein-protein interaction is at least partially electrostatic in nature. Nevertheless, none of the glutamate residues of the acidic cluster of residues 92-94 of ferredoxin I, which have been reported to be involved in the interaction with the subunit, seems to be essential for PsaD binding, as borne out by experiments using ferredoxin I mutants in positions 92-94.

Cross-Linking Reagents↗
PubMed1999

A productive NADP+ binding mode of ferredoxin-NADP + reductase revealed by protein engineering and crystallographic studies.

The flavoenzyme ferredoxin-NADP+ reductase (FNR) catalyzes the production of NADPH during photosynthesis. Whereas the structures of FNRs from spinach leaf and a cyanobacterium as well as many of their homologs have been solved, none of these studies has yielded a productive geometry of the flavin-nicotinamide interaction. Here, we show that this failure occurs because nicotinamide binding to wild type FNR involves the energetically unfavorable displacement of the C-terminal Tyr side chain. We used mutants of this residue (Tyr 308) of pea FNR to obtain the structures of productive NADP+ and NADPH complexes. These structures reveal a unique NADP+ binding mode in which the nicotinamide ring is not parallel to the flavin isoalloxazine ring, but lies against it at an angle of approximately 30 degrees, with the C4 atom 3 A from the flavin N5 atom.

Amino Acid Sequence↗
PubMed1998

Probing the function of the invariant glutamyl residue 312 in spinach ferredoxin-NADP+ reductase.

Ferredoxin-NADP+ reductase, the prototype of a large family of structurally related flavoenzymes, pairs single electrons carried by ferredoxin I and transfers them as a hydride to NADP+. Four mutants of the enzyme, in which Glu-312 was replaced with Asp, Gln, Leu, and Ala to probe the role of the residue charge, size, and polarity in the enzyme activity, have been heterologously expressed, purified, and characterized through steady-state, rapid kinetic studies, ligand-binding experiments, and three-dimensional structure determination by x-ray crystallography. The E312L mutant was the only one that was almost inactive (approximately 1%), whereas unexpectedly the E312A reductase was 10-100% active with the various acceptors tested. Rapid kinetic absorption spectroscopy studies demonstrated that flavin reduction by NADPH was impaired in the mutants. Furthermore, NADP(H) binding was partially perturbed. These functional and structural studies lead us to conclude that Glu-312 does not fulfil the role of proton donor during catalysis, but it is required for proper binding of the nicotinamide ring of NADP(H). In addition, its charge modulates the two one-electron redox potentials of the flavin to stabilize the semiquinone form.

Amino Acid Sequence↗
PubMed1998

Structure of the mutant E92K of [2Fe-2S] ferredoxin I from Spinacia oleracea at 1.7 A resolution.

Ferredoxin I (Fd I) from Spinacia oleracea is composed of 97 amino-acid residues and a [2Fe-2S] cluster. The crystal structure of the E92K mutant of Fd I was solved by molecular replacement and refined to an R factor of 19.6% for 11755 reflections at 1.7 A resolution. The overall structure and the active centre of spinach Fd is highly conserved with respect to ferredoxins of known structure. The E92K mutation appears to disturb a hydrogen-bond network which stabilizes the loop bearing the [2Fe-2S] cluster. This observation provides a rationale for the reduced electron-transfer efficiency displayed by the E92K mutant. Inspection of the crystal packing reveals that the side chain of Lys92 is engaged in an intermolecular interaction with Asp26 of a symmetry-related molecule. This feature may explain why only the mutant E92K and not wild-type Fd I could be successfully crystallized.

Amino Acid Sequence↗
PubMed1997

A three-domain iron-sulfur flavoprotein obtained through gene fusion of ferredoxin and ferredoxin-NADP+ reductase from spinach leaves.

Ferredoxin and ferredoxin-NADP+ reductase are the two last partners of the photosynthetic electron-transfer chain, responsible for the final reduction of NADP+ to NADPH. Herein, we report the engineering and characterization of a novel protein molecule in which the electron-carrier protein (ferredoxin I) and the reductase (a flavoprotein) were covalently linked in a single polypeptide chain by gene fusion. The gene was obtained by joining the cDNAs encoding the respective proteins and subsequently by deleting the intervening sequence between them by site-directed mutagenesis. No extra amino acid residues were introduced between the C-terminus of ferredoxin I and the N-terminus of the flavoenzyme. The chimera was purified to homogeneity and characterized. The M(r) of the chimera apoprotein was 45,800 as determined by mass spectrometry, in agreement with the expected value of 45,846. Both flavin and iron-sulfur cluster were in 1:1 ratio with respect to the apoprotein. The chimera was found active as a diaphorase and, more interestingly, highly efficient as a cytochrome c reductase, without need for free ferredoxin addition in the assay medium. Several lines of evidence indicate that the ferredoxin and the reductase in the chimera assume a configuration quite similar to that in the dissociable physiological complex. Thus, the fusion protein could be a useful tool for studying the mechanism of protein-protein recognition and electron transfer in the ferredoxin-ferredoxin-NADP+ reductase system.

Amino Acid Sequence↗
PubMed1997

On the role of the acidic cluster Glu 92-94 of spinach ferredoxin I.

The role of the acidic cluster Glu 92-94 of spinach ferredoxin I in the interaction both with the photosystem I multisubunit complex and the ferredoxin-NADP+ reductase, either membrane-bound or purified, was studied by kinetic characterization of site-directed mutants. Three mutants of ferredoxin have been produced to evaluate the effects of elimination of one or two negative charges in the three specific positions of the acidic cluster. Kinetic characterization of the ferredoxin mutants E92A/E93A, E93A and E93A/E94A as electron carriers in the photosynthetic electron transport chain, allowed to establish that the two latter mutants were nearly indistinguishable from the wild-type protein in their ability to be photoreduced by photosystem I and as electron donor to the reductase in the NADP+ photoreduction with thylakoid membranes. The E92A/E93A ferredoxin mutant behaved very similarly to E92 mutants previously characterized. Thus, the elimination of the carboxyl groups adjacent to residue 92 did not further impaired ferredoxin I main function, i.e., as an electron carrier in NADP+ photoreduction. The two double mutants showed a reduced rate in the cross-linking of ferredoxin to the reductase promoted by a soluble carbodiimide, indicating an involvement of the acidic cluster in the formation of the active covalent complex between the two proteins.

Amino Acid Sequence↗
PubMed1997

Rib cage mechanics during quiet breathing and exercise in humans.

During exercise, large pleural, abdominal, and transdiaphragmatic pressure swings might produce substantial rib cage (RC) distortions. We used a three-compartment chest wall model (J. Appl. Physiol. 72: 1338-1347, 1992) to measure distortions of lung- and diaphragm-apposed RC compartments (RCp and RCa) along with pleural and abdominal pressures in five normal men. RCp and RCa volumes were calculated from three-dimensional locations of 86 markers on the chest wall, and the undistorted (relaxation) RC configuration was measured. Compliances of RCp and RCa measured during phrenic stimulation against a closed airway were 20 and 0%, respectively, of their values during relaxation. There was marked RC distortion. Thus nonuniform distribution of pressures distorts the RC and markedly stiffens it. However, during steady-state ergometer exercise at 0, 30, 50, and 70% of maximum workload, RC distortions were small because of a coordinated action of respiratory muscles, so that net pressures acting on RCp and RCa were nearly the same throughout the respiratory cycle. This maximizes RC compliance and minimizes the work of RC displacement. During quiet breathing, plots of RCa volume vs. abdominal pressure were to the right of the relaxation curve, indicating an expiratory action on RCa. We attribute this to passive stretching of abdominal muscles, which more than counterbalances the insertional component of transdiaphragmatic pressure.

Abdomen↗
PubMed1997

Human respiratory muscle actions and control during exercise.

We measured pressures and power of diaphragm, rib cage, and abdominal muscles during quiet breathing (QB) and exercise at 0, 30, 50, and 70% maximum workload (Wmax) in five men. By three-dimensional tracking of 86 chest wall markers, we calculated the volumes of lung- and diaphragm-apposed rib cage compartments (Vrc,p and Vrc,a, respectively) and the abdomen (Vab). End-inspiratory lung volume increased with percentage of Wmax as a result of an increase in Vrc,p and Vrc,a. End-expiratory lung volume decreased as a result of a decrease in Vab. DeltaVrc,a/DeltaVab was constant and independent of Wmax. Thus we used DeltaVab/time as an index of diaphragm velocity of shortening. From QB to 70% Wmax, diaphragmatic pressure (Pdi) increased approximately 2-fold, diaphragm velocity of shortening 6.5-fold, and diaphragm workload 13-fold. Abdominal muscle pressure was approximately 0 during QB but was equal to and 180 degrees out of phase with rib cage muscle pressure at all percent Wmax. Rib cage muscle pressure and abdominal muscle pressure were greater than Pdi, but the ratios of these pressures were constant. There was a gradual inspiratory relaxation of abdominal muscles, causing abdominal pressure to fall, which minimized Pdi and decreased the expiratory action of the abdominal muscles on Vrc,a gradually, minimizing rib cage distortions. We conclude that from QB to 0% Wmax there is a switch in respiratory muscle control, with immediate recruitment of rib cage and abdominal muscles. Thereafter, a simple mechanism that increases drive equally to all three muscle groups, with drive to abdominal and rib cage muscles 180 degrees out of phase, allows the diaphragm to contract quasi-isotonically and act as a flow generator, while rib cage and abdominal muscles develop the pressures to displace the rib cage and abdomen, respectively. This acts to equalize the pressures acting on both rib cage compartments, minimizing rib cage distortion.

Abdominal Muscles↗
PubMed1996

Analysis of the oxidation-reduction potentials of recombinant ferredoxin-NADP+ reductase from spinach chloroplasts.

Midpoint oxidation-reduction potentials for the two-electron reduction of the bound FAD in spinach ferredoxin-NADP+ reductase were measured by potentiometry (Em = -342 +/- 1 mV at pH 7 and 10 degrees C). They were used with the semiquinone formation constant, obtained by spectroscopic measurement of the semiquinone concentration, to calculate values for the redox potentials of the two one-electron steps in the reduction. The redox potential for the oxidized enzyme/enzyme semiquinone couple (EOX/SQ) at pH 7 is -350 +/- 2 mV (10 degrees C) while the value for the enzyme semiquinone/enzyme hydroquinone couple (ESQ/HQ) under the same conditions is -335 +/- 1 mV. These values correspond to a semiquinone formation constant of 0.55. Measurement of the effects of pH on the potentials showed that EOX/SQ varies linearly with pH (slope -46 +/- 4 mV), while ESQ/HQ is independent of pH at high pH values, but below about pH 7.5 the potential becomes less negative with decreasing pH. indicating that there is a redox-linked protonation of the fully reduced enzyme (pKa = 7.2, 10 degrees C). The absorption spectrum of the fully reduced enzyme was found to depend on pH with the changes giving a calculated pKa of 7.5 (at 15 degrees C). The spectrum at high pH is similar to that of the anionic form of free flavin hydroquinone. The observations suggest that at physiological pH, the enzyme FAD cycles between the three redox states: oxidized, neutral semiquinone and hydroquinone anion.

Anions↗
PubMed1996

Mutations of Glu92 in ferredoxin I from spinach leaves produce proteins fully functional in electron transfer but less efficient in supporting NADP+ photoreduction.

Ferredoxin I in spinach chloroplasts fulfils the role of distributing electrons of low redox potential produced by photosystem I to several metabolic routes, NADP+ reduction being the major output. To investigate the role of Glu92, which is conserved in the chloroplast-type ferredoxins, mutations of this residue to either Gln, Ala or Lys were obtained through site-directed mutagenesis. A Glu93Ala mutant was also designed. The four mutants of ferredoxin I were overproduced in Escherichia coli, purified and characterised. The different migration in nondenaturing gel electrophoresis of wild-type and mutant proteins confirmed that the desired mutation was present in the expressed proteins. Spectral and physical properties of the mutants were similar to those of wild-type ferredoxin; electron-transfer properties were, however, quite different in the case of the mutants at position 92. Unexpectedly, these mutant ferredoxins were found to be twice as active as the wild-type protein in supporting the NADPH--cytochrome c reductase reaction catalysed by ferredoxin--NADP+ reductase. However, interactions of the mutant ferredoxins with the isolated thylakoid membranes deprived of endogenous ferredoxin showed that the mutants were less capable of supporting NADP+ photoreduction than the wild-type protein: both V and the apparent Km for reduced ferredoxin were influenced. On the other hand, the Kd values for the complex between oxidised ferredoxin and the reductase, measured at low ionic strength, were substantially changed only in the case of the Glu-->Lys mutation. With this mutant the rate of cross-linking between the two proteins induced by a carbodiimide was also decreased. It was found that the redox potentials of the iron-sulfur cluster of the mutants were more positive by 73-93 mV than that of ferredoxin I. Thus, the behavior of the ferredoxin mutants can be rationalised in terms of the effect of the side-chain replacement on the electrochemical properties of the [2Fe-2S] cluster and of an impairment in the interaction with the reductase under physiological conditions.

Base Sequence↗
PubMed1996

Chest wall and lung volume estimation by optical reflectance motion analysis.

Estimation of chest wall motion by surface measurements only allows one-dimensional measurements of the chest wall. We have assessed on optical reflectance system (OR), which tracks reflective markers in three dimensions (3-D) for respiratory use. We used 86 (6-mm-diameter) hemispherical reflective markers arranged circumferentially on the chest wall in seven rows between the sternal notch and the anterior superior iliac crest in two normal standing subjects. We calculated the volume of the entire chest wall and compared inspired and expired volumes with volumes obtained by spirometry. Marker positions were recorded by four TV cameras; two were 4 m in front of and two were 4 m behind the subject. The TV signals were sampled at 100 Hz and combined with grid calibration parameters on a personal computer to obtain the 3-D coordinates of the markers. Chest wall surfaces were reconstructed by triangulation through the point data, and chest wall volume was calculated. During tidal breathing and vital capacity maneuvers and during CO2-stimulated hyperpnea, there was a very close correlation of the lung volumes (VL) estimated by spirometry [VL(SP)] and OR [VL(OR)]. Regression equations of VL(OR) (y) vs. VL(SP) (x, BTPS in liters) for the two subjects were given by y = 1.01x-0.01 (r = 0.996) and y = 0.96x + 0.03 (r = 0.997), and by y = 1.04x + 0.25 (r = 0.97) and y = 0.98x + 0.14 (r = 0.95) for the two maneuvers, respectively. We conclude spirometric volumes can be estimated very accurately and directly from chest wall surface markers, and we speculate that OR may be usefully applied to calculations of chest wall shape, regional volumes, and motion analysis.

Humans↗
PubMed1996

A new method for 3D optical analysis of chest wall motion.

A method for kinematic analysis of chest wall motion is presented, based on a TV-image processor which allows a three-dimensional assessment of volume change of the trunk by automatically computing the co-ordinates of several passive markers placed on relevant landmarks of the thorax and abdomen. The parallel computation used for the image processing allows a real time recognition of the passive markers with the necessary accuracy. A geometric model also allows the on-line computation of the contribution to the chest volume by the different parts. For this purpose the model is based on 96 tetrahedrons which can be grouped into 16 compartments and into four sections representing: upper thorax (reflecting the action of neck and parasternal muscles and the effect of pleural pressure), lower thorax (reflecting the action of diaphragm and the effect of pleural and abdominal pressure), upper and lower abdomen (reflecting the actions of diaphragm and abdominal muscles). The volume can also be split into two vertical sections pointing out asymmetrics between the right and left side. The method is non-invasive, non-ionizing and leaves the subject maximum freedom of movement during the test, thus being suitable for routine clinical analysis. The monitoring of the subject can be prolonged in time and can be performed in different postures: standing, sitting, supine and lying on one side. The method was tested on eight healthy subjects showing good accuracy, reliability and reproducibility.

Biomechanical Phenomena↗