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Biomedical subjects

J L Arrondo

Publications and source records attributed to J L Arrondo.

At least 19 recordsLinked to original sources
PubMed2004

Specific aspects of erectile function in urology/andrology.

The urologist/andrologist is the specialist responsible for diagnosis and treatment of health problems related to the genitourinary tract, and his or her participation in comprehensive care for a patient with erectile dysfunction (ED) is fundamental and often indispensable. The urologists/andrologists should characterize the origin of ED because of their knowledge and familiarity of all diagnostic tests and second- and third-line therapy. The origin of ED is important to determine for various reasons, such as young people suitable for etiologic treatment, medicolegal reasons, or patients' wishes for a better understanding of their condition. A review of the diagnostic tests available as well as indications for second- and third-line therapy is presented. The close relationship between ED and urological disorders, such as benign prostatic hyperplasia, prostate cancer and their treatments, and renal failure, in association with penile conditions like Peyronie's disease, priapism, and possible androgen deficiency in men older than 50 years, places the urologist at the center of integrated treatment of male ED.

Attitude of Health Personnel↗
PubMed2001

DNA-induced alpha-helical structure in the NH2-terminal domain of histone H1.

It is important to establish the structural properties of linker histones to understand the role they play in chromatin higher order structure and gene regulation. Here, we use CD, NMR, and IR spectroscopy to study the conformation of the amino-terminal domain of histone H1 degrees, free in solution and bound to the DNA. The NH(2)-terminal domain has little structure in aqueous solution, but it acquires a substantial amount of alpha-helical structure in the presence of trifluoroethanol (TFE). As in other H1 subtypes, the basic residues of the NH(2)-terminal domain of histone H1 degrees are clustered in its COOH-terminal half. According to the NMR results, the helical region comprises the basic cluster (Lys(11)-Lys(20)) and extends until Asp(23). The fractional helicity of this region in 90% TFE is about 50%. His(24) together with Pro(25) constitute the joint between the NH(2)-terminal helix and helix I of the globular domain. Infrared spectroscopy shows that interaction with the DNA induces an amount of alpha-helical structure equivalent to that observed in TFE. As coulombic interactions are involved in complex formation, it is highly likely in the complexes with DNA that the minimal region with alpha-helical structure is that containing the basic cluster. In chromatin, the high positive charge density of the inducible NH(2)-terminal helical element may contribute to the binding stability of the globular domain.

Amino Acid Sequence↗
PubMed2001

Induction of secondary structure in a COOH-terminal peptide of histone H1 by interaction with the DNA: an infrared spectroscopy study.

We have studied the conformation of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), free in solution and bound to the DNA, by Fourier-transform infrared spectroscopy. The peptide belongs to the COOH-terminal domain of histone H1(0) (residues 99-121) and is adjacent to the central globular domain of the protein. In aqueous (D(2)O) solution the amide I' is dominated by component bands at 1643 cm(-1) and 1662 cm(-1), which have been assigned to random coil conformations and turns, respectively. In accordance with previous NMR results, the latter component has been interpreted as arising in turn-like conformations in rapid equilibrium with unfolded states. The peptide becomes fully structured either in 90% trifluoroethanol (TFE) solution or upon interaction with the DNA. In these conditions, the contributions of turn (1662 cm(-1)) and random coil components virtually disappear. In TFE, the spectrum is dominated by the alpha-helical component (1654 cm(-1)). The band at 1662 cm(-1) shifts to 1670 cm(-1), and has been assigned to the COOH-terminal TPKK motif in a more stable turn conformation. A band at 1637 cm(-1), also present in TFE, has been assigned to 3(10) helical structure. The amide I' band of the complexes with the DNA retains the components that were attributed to 3(10) helix and the TPKK turn. In the complexes with the DNA, the alpha-helical component observed in TFE splits into two components at 1657 cm(-1) and 1647 cm(-1). Both components are inside the spectral region of alpha-helical structures. Our results support the presence of inducible helical and turn elements, both sharing the character of DNA-binding motifs.

Amino Acid Sequence↗
PubMed2001

Interaction of cholesterol with sphingomyelin in mixed membranes containing phosphatidylcholine, studied by spin-label ESR and IR spectroscopies. A possible stabilization of gel-phase sphingolipid domains by cholesterol.

The ESR spectra from different positional isomers of sphingomyelin and phosphatidylcholine spin-labeled in their acyl chain have been studied in sphingomyelin(cerebroside)-phosphatidylcholine mixed membranes that contain cholesterol. The aim was to investigate mechanisms by which cholesterol could stabilize possible domain formation in sphingolipid-glycerolipid membranes. The outer hyperfine splittings in the ESR spectra of sphingomyelin and phosphatidylcholine spin-labeled on the 5 C atom of the acyl chain were consistent with mixing of the components, but the perturbations on adding cholesterol were greater in the membranes containing sphingomyelin than in those containing phosphatidylcholine. Infrared spectra of the amide I band of egg sphingomyelin were shifted and broadened in the presence of cholesterol to a greater extent than the carbonyl band of phosphatidylcholine, which was affected very little by cholesterol. Two-component ESR spectra were observed from lipids spin-labeled on the 14 C atom of the acyl chain in cholesterol-containing membranes composed of sphingolipids, with or without glycerolipids (sphingomyelin/cerebroside and sphingomyelin/cerebroside/phosphatidylcholine mixtures). These results indicate the existence of gel-phase domains in otherwise liquid-ordered membranes that contain cholesterol. In the gel phase of egg sphingomyelin, the outer hyperfine splittings of sphingomyelin spin-labeled on the 14-C atom of the acyl chain are smaller than those for the corresponding spin-labeled phosphatidylcholine. In the presence of cholesterol, this situation is reversed; the outer splitting of 14-C spin-labeled sphingomyelin is then greater than that of 14-C spin-labeled phosphatidylcholine. This result provides some support for the suggestion that transbilayer interdigitation induced by cholesterol stabilizes the coexistence of gel-phase and "liquid-ordered" domains in membranes containing sphingolipids.

Brain Chemistry↗
PubMed2001

Differential interaction of equinatoxin II with model membranes in response to lipid composition.

Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (approximately 26 mN m(-1)) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (approximately 33 mN m(-1)), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101-120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Early stages of LDL oxidation: apolipoprotein B structural changes monitored by infrared spectroscopy.

Changes in the conformation of apoliprotein B-100 in the early stages of copper-mediated low density lipoprotein oxidation have been monitored by infrared spectroscopy. During the lag phase no variation in structure is observed, indicating that copper binding to the protein does not significantly affect its structure. In the propagation phase, while hydroperoxides are formed but the protein is not modified, no changes in secondary structure are observed, but the thermal profile of the band corresponding to alpha-helix is displaced in frequency, indicating changes in tertiary structure associated with this conformation but not with beta-sheet components. When aldehyde formation starts, a decrease of approximately 3% in the area of bands corresponding to alpha-helix and beta-sheet is produced, concomitantly with an increase in beta-turns and unordered structure. The two bands corresponding to beta-turns vary as well under these conditions, indicating changes in these structures. Also at this stage the thermal profile shows variations in frequency for the bands corresponding to both alpha-helix and beta-sheet. The results are consistent with the hypothesis that as soon as the polyunsaturated fatty acids from the particle core are modified, this change is reflected at the surface, in the alpha-helical components contacting the monolayer.

Apolipoproteins B↗
PubMed2000

Do sequence repeats play an equivalent role in the choline-binding module of pneumococcal LytA amidase?

LytA amidase breaks down the N-acetylmuramoyl-l-alanine bonds in the peptidoglycan backbone of Streptococcus pneumoniae. Its polypeptide chain has two modules: the NH(2)-terminal module, responsible for the catalytic activity, and the COOH-terminal module, constructed by six tandem repeats of 20 or 21 amino acids (p1-p6) and a short COOH-terminal tail. The polypeptide chain must contain at least four repeats to efficiently anchor the autolysin to the choline residues of the cell wall. Nevertheless, the catalytic efficiency decreases by 90% upon deletion of the final tail. The structural implications of deleting step by step the two last (p5 and p6) repeats and the final COOH-tail and their effects on choline-amidase interactions have been examined by comparing four truncated mutants with LytA amidase by means of different techniques. Removal of this region has minor effects on secondary structure content but significantly affects the stability of native conformations. The last 11 amino acids and the p5 repeat stabilize the COOH-terminal module; each increases the module transition temperature by about 6 degrees C. Moreover, the p5 motif also seems to participate, in a choline-dependent way, in the stabilization of the NH(2)-terminal module. The effects of choline binding on the thermal stability profile of the mutant lacking the p5 repeat might reflect a cooperative pathway providing molecular communication between the choline-binding module and the NH(2)-terminal region. The three sequence motives favor the choline-amidase interaction, but the tail is an essential factor in the monomer <--> dimer self-association equilibrium of LytA and its regulation by choline. The final tail is required for preferential interaction of choline with LytA dimers and for the existence of different sets of choline-binding sites. The p6 repeat scarcely affects the amidase stability but could provide the proper three-dimensional orientation of the final tail.

Choline↗
PubMed1999

Thermal and pH-induced conformational changes of a beta-sheet protein monitored by infrared spectroscopy.

The stability of a lentil lectin, an all-beta protein, has been perturbed by changes in pH and temperature. In the pH interval 5.0 --> 10.0, the overall secondary structure does not undergo significant changes. However, if the individual components of the infrared amide I band are considered, changes in band components attributed to variations in beta-sheet and beta-turns cross-interactions are detected. The combined effects of pH and temperature reveal that the protein is more compact at pH 7.5 with lower denaturation temperatures at pH 5.0 or 10.0, indicating a less stable protein under those conditions. According to our results, the structural stability of the beta-sheet would depend not only on the intermolecular interactions among the strands but also on the conformation of the segments connecting these strands. The protein infrared band assignment has also been examined since the three-dimensional structure of the lentil lectin protein is known from X-ray diffraction studies. Two of the bands observed are attributed to beta-sheet. The one at 1620 cm-1, not affected if the medium is deuterated, is assigned to hairpins composed by two strands connected by a rigid turn whereas that located at 1633 cm-1 corresponds to strands associated by more flexible segments. The band appearing at 1645 cm-1 in H2O corresponds to the open, flexible loops that are connecting the beta-strands. The simplest assumption of the various secondary structure components having identical IR extinction coefficients is enough to provide IR-derived data that are in good agreement with the structure solved by X-ray diffraction.

Amides↗
PubMed1999

pH-dependent thermal transitions of lentil lectin.

The thermal stability of lentil lectin in the 5.0-10.0 pH range was studied by high-sensitivity differential scanning calorimetry and infrared spectroscopy. The thermally induced transitions for protein were irreversible and strongly dependent upon the scan rate at all pH values, suggesting that the denaturation is under kinetic control. It is shown that process of lentil lectin denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, N-->D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation, N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Calorimetry, Differential Scanning↗
PubMed1999

Ceramides in phospholipid membranes: effects on bilayer stability and transition to nonlamellar phases.

The effects of ceramides of natural origin on the gel-fluid and lamellar-inverted hexagonal phase transitions of phospholipids (mainly dielaidoylphosphatidylethanolamine) have been studied by differential scanning calorimetry, with additional support from infrared and 31P nuclear magnetic resonance (NMR) spectroscopy. In the lamellar phase, ceramides do not mix ideally with phospholipids, giving rise to the coexistence of domains that undergo the gel-fluid transition at different temperatures. The combination of differential scanning calorimetry and infrared spectroscopy, together with the use of deuterated lipids, allows the demonstration of independent melting temperatures for phospholipid and ceramide in the mixtures. In the lamellar-hexagonal phase transitions, ceramides (up to 15 mol %) decrease the transition temperature, without significantly modifying the transition enthalpy, thus facilitating the inverted hexagonal phase formation. 31P-NMR indicates the coexistence, within a certain range of temperatures, of lamellar and hexagonal phases, or hexagonal phase precursors. Ceramides from egg or from bovine brain are very similar in their effects on the lamellar-hexagonal transition. They are also comparable to diacylglycerides in this respect, although ceramides are less potent. These results are relevant in the interpretation of certain forms of interfacial enzyme activation and in the regulation and dynamics of the bilayer structure of cell membranes.

Animals↗
PubMed1999

Structure and dynamics of membrane proteins as studied by infrared spectroscopy.

Infrared (IR) spectroscopy is a useful technique in the study of protein conformation and dynamics. The possibilities of the technique become apparent specially when applied to large proteins in turbid suspensions, as is often the case with membrane proteins. The present review describes the applications of IR spectroscopy to the study of membrane proteins, with an emphasis on recent work and on spectra recorded in the transmission mode, rather than using reflectance techniques. Data treatment procedures are discussed, including band analysis and difference spectroscopy methods. A technique for the analysis of protein secondary and tertiary structures that combines band analysis by curve-fitting of original spectra with protein thermal denaturation is described in detail. The assignment of IR protein bands in H2O and in D2O, one of the more difficult points in protein IR spectroscopy, is also reviewed, including some cases of unclear assignments such as loops, beta-hairpins, or 3(10)-helices. The review includes monographic studies of some membrane proteins whose structure and function have been analysed in detail by IR spectroscopy. Special emphasis has been made on the role of subunit III in cytochrome c oxidase structure, and the proton pathways across this molecule, on the topology and functional cycle of sarcoplasmic reticulum Ca(2+)-ATPase, and on the role of lipids in determining the structure of the nicotinic acetylcholine receptor. In addition, shorter descriptions of retinal proteins and references to other membrane proteins that have been studied less extensively are also included.

Bacteriorhodopsins↗
PubMed1998

Domain structure and stability of human phenylalanine hydroxylase inferred from infrared spectroscopy.

We have studied the conformation and thermal stability of recombinant human phenylalanine hydroxylase (hPAH) and selected truncated forms, corresponding to distinct functional domains, by infrared spectroscopy. The secondary structure of wild-type hPAH was estimated to be 48% alpha-helix, 28% extended structures, 12% beta-turns and 12% non-structured conformations. The catalytic C-terminal domain (residues 112-452) holds most of the regular secondary structure elements, whereas the regulatory N-terminal domain (residues 2-110) adopts mainly an extended and disordered, flexible conformation. Thermal stability studies of the enzyme forms indicate the existence of interactions between the two domains. Our results also demonstrate that the conformational events involved in the activation of hPAH by its substrate (L-Phe) are mainly related to changes in the tertiary/quaternary structure. The activating effect of phosphorylation, however, affects the secondary structure of the N-terminal domain of the protein.

Binding Sites↗
PubMed1998

Topology of sarcoplasmic reticulum Ca2+-ATPase: an infrared study of thermal denaturation and limited proteolysis.

Sarcoplasmic reticulum Ca2+-ATPase structure and organization in the membrane has been studied by infrared spectroscopy by decomposition of the amide I band. Besides the component bands assignable to secondary structure elements such as alpha-helix, beta-sheet, etc...., two unusual bands, one at 1,645 cm(-1) in H2O buffer and the other at 1,625 cm(-1) in D2O buffer are present. By perturbing the protein using temperature and limited proteolysis, the band at 1,645 cm(-1) is tentatively assigned to alpha-helical segments located in the cytoplasmic domain and coupled to beta-sheet structure, whereas the band at 1,625 cm(-1) arises probably from monomer-monomer contacts in the native oligomeric protein. The secondary structure obtained is 33% alpha-helical segments in the transmembrane plus stalk domain; 20% alpha-helix and 22% beta-sheet in the cytoplasmic domain plus 19% turns and 6% unordered structure. Thermal unfolding of Ca2+-ATPase is a complex process that cannot be described as a two-state denaturation. The results obtained are compatible with the idea that the protein is an oligomer at room temperature. The loss of the 1,625 cm(-1) band upon heating would be consistent with a disruption of the oligomers in a process that later gives rise to aggregates (appearance of the 1,618 cm(-1) band). This picture would also be compatible with early results suggesting that processes governing Ca2+ accumulation and ATPase activity are uncoupled at temperatures above 37 degrees C, so that while ATPase activity proceeds at high rates, Ca2+ accumulation is inhibited.

Animals↗
PubMed1998

Infrared studies of protein-induced perturbation of lipids in lipoproteins and membranes.

The paper reviews the main recent publications concerning infrared (IR) spectroscopy as applied to the study of lipid-protein interactions in model and cell membranes, lipoproteins, and related systems (e.g. lung surfactant). The review focuses mainly on transmission IR. Based on the available data, a number of general conclusions are presented on the perturbations caused by proteins on either the hydrocarbon chains, the polar headgroups or the interface region. Lipid-protein interactions in native cell membranes do not reveal significant differences from what is observed in semisynthetic model systems.

Animals↗
PubMed1997

Interaction of biotin with streptavidin. Thermostability and conformational changes upon binding.

The effect of biotin binding on streptavidin (STV) structure and stability was studied using differential scanning calorimetry, Fourier transform infrared spectroscopy (FT-IR), and fluorescence spectroscopy. Biotin increases the midpoint temperature Tm, of thermally induced denaturation of STV from 75 degrees C in unliganded protein to 112 degrees C at full ligand saturation. The cooperativity of thermally induced unfolding of STV changes substantially in presence of biotin. Unliganded STV monomer has at least one domain that unfolds independently. The dimer bound to biotin undergoes a single coupled denaturation process. Simulations of thermograms of STV denaturation that take into account only the thermodynamic effects of the ligand with a Ka approximately 10(15) reproduce the behavior observed, but the estimated values of Tm are 15-20 degrees C lower than those experimentally determined. This increased stability is attributed to an enhanced cooperativity of the thermal unfolding of STV. The increment in the cooperativity is as consequence of a stronger intersubunit association and an increased structural order upon binding. FT-IR and fluorescence spectroscopy data reveal that unordered structure found in unliganded STV disappears under fully saturating conditions. The data provide a rationale for previous suggestions that biotin binding induces an increase in protein tightness (structural cooperativity) leading, in turn, to a higher thermostability.

Bacterial Proteins↗
PubMed1996

Conformational properties and stability of tyrosine hydroxylase studied by infrared spectroscopy. Effect of iron/catecholamine binding and phosphorylation.

The conformation and stability of recombinant tetrameric human tyrosine hydroxylase isoenzyme 1 (hTH1) was studied by infrared spectroscopy and by limited tryptic proteolysis. Its secondary structure was estimated to be 42% alpha-helix, 35% beta-extended structures (including beta-sheet), 14% beta-turns, and 10% nonstructured conformations. Addition of Fe(II) or Fe(II) plus dopamine to the apoenzyme did not significantly modify its secondary structure. However, an increased thermal stability and resistance to proteolysis, as well as a decreased cooperativity in the thermal denaturation transition, was observed for the ligand-bound forms. Thus, as compared with the apoenzyme, the ligand-bound subunits of hTH1 showed a more compact tertiary structure but weaker intersubunit contacts within the protein tetramer. Phosphorylation of the apoenzyme by cyclic AMP-dependent protein kinase did not change its overall conformation but allowed on iron binding a conformational change characterized by an increase (about 10%) in alpha-helix and protein stability. Our results suggest that the conformational events involved in TH inhibition by catecholamines are mainly related to modifications of tertiary and quaternary structural features. However, the combined effect of iron binding and phosphorylation, which activates the enzyme, also involves modifications of the protein secondary structure.

Catecholamines↗
PubMed1996

Infrared evidence of a beta-hairpin peptide structure in solution.

The IR spectrum of an 16-amino acid peptide corresponding, according to NMR studies, to a beta-hairpin has been analysed. Two characteristic features distinguish its spectrum from that of an antiparallel beta-sheet: the low-frequency band that in a beta-sheet structure is located at approximately 1632 cm-1 appears here at approximately 1620 cm-1, and the high-frequency component does not undergo the isotopic shift typical of beta-sheet from 1690 to 1675 cm-1 when transferred to D2O. The infrared characteristics associated with beta-hairpins have been described so far in two proteins, in one of which, whose three-dimensional structure is known from X-ray diffraction, a beta-hairpin has actually been detected.

Bacterial Proteins↗