Detection of blood pressure increments in active elderly individuals: the role of ambulatory blood pressure monitoring.

BACKGROUND AND AIMS: Physiological aging can lead to an increase in blood pressure (BP) over time even in regularly exercising elders. Office BP measurements (OBPM) might be unable to detect these BP variations. The aim of this study was to analyze BP changes over 3.5 years in active elders using ABPM. METHODS AND RESULTS: The study involved 80 active subjects ≥65 years old who exercised regularly. At baseline and again 3.5 years later, all subjects had lab tests, weight, body mass index (BMI), body composition, resting energy expenditure (REE) recorded; they underwent OBPM, ABPM and physical activity assessment. Over 3.5 years, our sample's mean weight, BMI, body composition, REE, albumin, and physical activity levels, did not change significantly. The prevalence of hypertension detected by OBPM dropped from 68.8% to 61.3%. ABPM revealed an increase in mean 24-h BP (Δsystolic: 5.3 ± 13.6 mmHg; p = 0.001; Δdiastolic: 1.8 ± 6.7 mmHg; p = 0.018) and mean daytime BP (Δsystolic: 5.8 ± 13.5 mmHg; p = 0.001; Δdiastolic: 1.9 ± 7.1 mmHg; p = 0.022); the prevalence of hypertension detected by ABPM increased from 50% to 65%, also due to an increase (from 8.8% to 16.3%) in masked hypertension. There was no correlation between BP changes and changes in body composition and REE. CONCLUSION: BP tends to increase over time in active elders, regardless of changes in body composition or level of physical activity. ABPM is an appropriate method for detecting these BP variations in active elders and to reveal cases of masked hypertension that might otherwise escape detection by OBPM.

Impact of antisecretory treatment on respiratory symptoms of gastroesophageal reflux disease in children.

The effect of antisecretory treatment on extraesophageal symptoms of gastroesophageal reflux disease was evaluated. Seventy-eight children presenting with typical and extraesophageal symptoms of gastroesophageal reflux disease underwent a multichannel intraluminal impedance and pH monitoring (MII/pH). Children with a positive MII/pH were randomly treated with proton pump inhibitors (PPIs) or histamine H(2) -receptor antagonists (H(2) RAs) during 3 months. At the end of the treatment period, all patients were recalled. A second treatment period of 3 months was given to those patients who were not symptom-free after 3 months. Thirty-five of the forty-one (85.4%) children with a pathologic MII/pH presented with extraesophageal symptoms and were treated with PPIs (omeprazole; n:19) or H(2) RAs (ranitidine; n:16) for 12 weeks. After 3 months, 11/19 (57.9%) PPI-treated patients had a complete resolution of symptoms; 6/8 nonresponders were treated with PPI for another 3 months and became all symptom-free. The other two underwent a Nissen fundoplication. Only 5/16 (31.2 %) patients treated with H(2) RAs had a complete resolution of symptoms after 3 months; 1/11 was treated again with H(2) RAs during 3 months, and 10/11 were changed to PPIs. In 3/10, a partial resolution of symptoms was achieved, while in 7/10, a complete remission was obtained (P < 0.05). Antisecretory reflux treatment improves extraesophageal reflux symptoms. The efficacy of PPIs is superior to that of H(2) RAs in these children.

Circulating endothelial microparticles in diabetes mellitus.

BACKGROUND: Endothelial Microparticles (EMPs) are small vesicles shed from activated or apoptotic endothelial cells and involved in cellular cross-talk. Whether EMP immunophenotypes vary according to stimulus in Diabetes Mellitus (DM) is not known. We studied the cellular adhesion molecule (CAM) profile of circulating EMPs in patients with and without Diabetes Mellitus type 2, who were undergoing elective cardiac catheterization. METHODS AND RESULTS: EMPs were analyzed by flow cytometry. The absolute median number of EMPs (EMPs/microL) specific for CD31, CD105, and CD106 was significantly increased in the DM population. The ratio of CD62E/CD31 EMP populations reflected an apoptotic process. CONCLUSION: Circulating CD31+, CD105+, and CD106+ EMPs were significantly elevated in patients with DM. EMPs were the only independent predictors of DM in our study cohort. In addition, the EMP immunophenotype reflected an apoptotic process. Circulating EMPs may provide new options for risk assessment.

Catalytic antibodies.

Monoclonal antibodies elicited to haptens that are analogs of the transition state for hydrolysis of carboxylic esters behaved as enzymic catalysts with the appropriate substrates. These substrates are distinguished by the structural congruence of both hydrolysis products with haptenic fragments. The haptens were potent inhibitors of this esterolytic activity, in agreement with their classification as transition state analogs. Mechanisms are proposed to account for the different chemical behavior of these antibodies with two types of ester substrates. The generation of an artificial enzyme through transition state stabilization by antibodies was thus demonstrated. These studies indicate a potentially general approach to catalyst design.

A stereospecific cyclization catalyzed by an antibody.

A monoclonal antibody elicited by a transition-state analog that is representative of an intramolecular six-membered ring cyclization reaction acted as a stereospecific, enzyme-like catalyst for the appropriate substrate. Formation of a single enantiomer of a delta-lactone from the corresponding racemic delta-hydroxyester was accelerated by the antibody by about a factor of 170, which permitted isolation of the lactone in an enantiomeric excess of about 94 percent. This finding demonstrates the feasibility of catalytic-antibody generation for chemical transformations that require stereochemical control.

Advances and pitfalls in protein structure prediction.

Three dimensional protein structures are crucial for understanding biology at both molecular and system level. Despite the advances in experimental structural biology, the pace of sequence deposition into databanks considerably exceeds that of structure determination. Inevitably the functional annotation of genes and genomes requires the exploitation of bioinformatics methods for protein sequence comparison and structure prediction. Hence monitoring objectively the state of art of the field is of paramount importance, in order to make best use of computational protein models to address biological questions. This review describes some relevant issues in the field of structural bioinformatics, emphasizig both open basic questions and the progress being continuously achieved. It is reasonably expected that these bioinformatics methods will increasingly contribute to the biomedical, pharmaceutical and biotechnological research.

Balancing urgency, age and quality of life in organ allocation decisions--what would you do? A survey.

PURPOSE: Explore public attitudes towards the trade-offs between justice and medical outcome inherent in organ allocation decisions. BACKGROUND: The US Task Force on Organ Transplantation recommended that considerations of justice, autonomy and medical outcome be part of all organ allocation decisions. Justice in this context may be modeled as a function of three types of need, related to age, clinical urgency, and quality of life. METHODS: A web-based survey was conducted in which respondents were asked to choose between two hypothetical patients who differed in clinical urgency (time to death <1 year), age, pretransplant and post-transplant quality of life, and life expectancy. RESULTS: A pool of 1600 people were notified via email about the survey; 623 (39%) responded. Respondents preferred giving organs to younger people up to an age difference of <15.4 years (SD 18) and more clinically urgent people up to a difference in urgency of <2.54 months (SD 3). Priority varied with the quality of life of the worst-off patient and the relative status of the patients. If both had worse than average quality of life, respondents preferred the better-off patient. When both had better than average quality of life, they preferred the worse-off patient. In analysis according to age versus clinical urgency, the older the patient, the more urgency needed to receive priority. In quality of life versus clinical urgency, the better the control's quality of life, the more urgency the competing patient required. The worse the patient's post-transplant outcome, the more urgency needed to receive priority. CONCLUSIONS: It appears that clinical urgency is only one of many factors influencing attitudes about allocation decisions and that respondents may invoke different principles of fairness depending the relative clinical status of patients.

The mepsMAP server. Mapping epitopes on protein surface: mining annotated proteins.

For a growing number of biologists DNA or protein data are typically retrieved and managed on the Web, and not in the laboratory. A large number of bioinformatics datasets from primary and (thousands of) secondary databases are scattered on the Web in various formats. A biologist end-user might need to access and use tens of databases and tools every day. For this reason, the bioinformatics community is developing more and more service-oriented architectures (SOAs): software architecture of loosely coupled software services that can be accessed without knowledge of, or control over, their internal architecture. Data-processing and analysis tasks can be automated by having free access to bioinformatics Web services (WSs) that are the building blocks of the SOAs. In this paper we introduce a new bioinformatics Web server, mepsMAP (mapping epitopes on protein surface: Mining Annotated Proteins), developed to identify the recognition sites between antibodies and their cognate antigens. In some cases, the recognition site is represented by a continuous segment of the antigen sequence, but much more often the epitope is "conformational," i.e., the antibody recognizes the location and type of exposed antigen side chains that are not necessarily contiguous in the antigen's sequence, but brought together by its three-dimensional structure. A facility on the server allows the user to search putative conformational epitopes on protein surface, querying the system for proteins with a given annotation. The mepsMAP server has been implemented as a SOA composed by a database and a set of four WSs. We present here the software architecture of the system with a detailed description of the WS dataflow that has been optimized to provide the best computing performance while maintaining the easiest end-user access to the system via a Web interface.

Structural model and ligand interactions of the Xanthomonas axonopodis pv. citri oligopeptide-binding protein.

The oligopeptide-binding protein, OppA, ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides by several bacterial species. In the present study, we report a structural model and an oligopeptide docking analysis of the OppA protein expressed by Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The X. citri OppA structural model showed a conserved three-dimensional structure, irrespective of the low amino acid identities with previously defined structures of Bacillus subtilis and Salmonella typhimurium orthologs. Oligopeptide docking analysis carried out with the proposed model indicated that the X. citri OppA preferentially binds tri- and tetrapeptides. The present study represents the first structural analysis of an OppA ortholog expressed by a phytopathogen and contributes to the understanding of the physiology and nutritional strategies of X. citri.

The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition.

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT.

Early treatment with ursodeoxycholic acid for cholestasis in children on parenteral nutrition because of primary intestinal failure.

BACKGROUND: There is conflicting evidence as to whether ursodeoxycholic acid (UDCA) reduces the incidence of parenteral nutrition-associated cholestasis. AIM: To investigate the efficacy of UDCA on parenteral nutrition-associated cholestasis in children with intestinal failure due to short bowel syndrome or to other causes. METHODS: Children with cholestasis received 30 mg/kg/day UDCA. Improvement or normalization of parenteral nutrition-associated cholestasis was evaluated at 6 months of therapy and at the last follow-up. In a subgroup of children, serum UDCA levels were measured while receiving UDCA and after 4 weeks withdrawal. RESULTS: Twelve children were treated with UDCA. Full remission or partial improvement of parenteral nutrition-associated cholestasis occurred in 11 of 12 children. In three of four children, withdrawal of UDCA was associated with a rebound rise of cholestasis. Only one of 12 treated children showed no improvement and in this patient, in contrast to four other patients, plasma levels of UDCA did not increase during treatment. CONCLUSIONS: Ursodeoxycholic acid was effective in controlling parenteral nutrition-associated cholestasis. The efficacy of UDCA also in children with short bowel is related to intestinal absorption.

Homology-derived three-dimensional structure prediction of Candida cylindracea lipase.

We propose a structural model of Candida cylindracea lipase (CCL) based on the reported X-ray structure of the highly homologous Geotrichum candidum lipase (GCL). The network of interactions around the active site, the salt and disulfide bridge pattern is conserved in the proposed structure. Functional, structural and evolutionary aspects of the peculiar usage of CTG codons by C. cylindracea ATCC 14830 are discussed.

PUZZLE: a new method for automated protein docking based on surface shape complementarity.

We describe here a novel procedure for automated protein docking, based only on geometric criteria. In our algorithm we project protein surfaces into bi-dimensional matrices; the search for complementary regions is performed by detecting matching sub-matrices. An exhaustive sampling of the rotation space is made in order to analyse all the possible relative orientations of the two proteins, but nevertheless this procedure requires a relatively short processing time (3 h to 24 h cpu time on a SG4D320, depending on the complexity of the input information). When tested with co-crystallized, free components and models of components of known protein-protein complexes, the method gave very satisfactory results. The procedure selects no more than four relative orientations of the molecular components, but the correct orientation is always present among them, ranking either first or second. In more than half the cases the "wrong" solutions nevertheless correctly identify most of the residues involved in the interaction. This is remarkable also in view of the fact that the chosen test complexes (trypsin-trypsin inhibitor and antibody-lysozyme) have a very different geometry of surface complementarity: trypsin inhibitor inserts a long side-chain into the deep specificity pocket of the protease, while the interface between antibody and lysozyme is rather flat and contains buried water molecules (not included in the calculation). In order to simulate a more realistic protein docking problem, we also used a trypsin inhibitor and an anti-lysozyme antibody model in our simulations, again with satisfying results.

Framework residue 71 is a major determinant of the position and conformation of the second hypervariable region in the VH domains of immunoglobulins.

Analysis of the immunoglobulins of known structure reveals systematic differences in the position and main-chain conformation of the second hypervariable region of the VH domain (H2). We show that the major determinant of the position of H2 is the size of the residue at site 71, a site that is in the conserved framework of the VH domain. It is likely that for about two thirds of the known VH sequences the size of the residue at this site is also a major determinant of the conformation of H2. This effect can override the predisposition of the sequence, as in the case of the H2 loop of J539, which is an exception to the rules relating sequence and conformation of short hairpin loops. Understanding the relationship between the residue at position 71 and the position and conformation of H2 has applications to the prediction and engineering of antigen-binding sites of immunoglobulins.

High level expression and rational mutagenesis of a designed protein, the minibody. From an insoluble to a soluble molecule.

We recently described the design and chemical synthesis of the minibody, a 61-residue metal binding beta-protein with a novel fold. Characterization of the polypeptide by circular dichroism spectroscopy, size exclusion chromatography, and metal binding studies showed the molecule to be folded, monomeric, globular and able to bind metals. The main obstacle which prevented a more detailed characterization was the very low solubility of the protein in water (about 10 microM). To address this problem, we used two independent approaches: (1) mutagenesis of the beta-sheet framework residues and (2) addition of a solubilizing motif, made of three lysine residues, at the N or C termini. Engineering and production of mutants was facilitated by the achievement of high level expression of the protein in Escherichia coli. Both approaches led to minibody variants with a solubility ranging from tenfold higher up to millimolar levels. For the best-characterized variant obtained so far, the thermodynamic stability calculated from denaturant-induced transition is identical to that of the parent, poorly soluble, molecule.

Conformations of the third hypervariable region in the VH domain of immunoglobulins.

Antigen-combining sites of antibodies are constructed from six loops from VL and VH domains. The third hypervariable region of the heavy chain is far more variable than the others in length, sequence and structure, and was not included in the canonical-structure description of the conformational repertoire of the three hypervariable regions of V kappa chains and the first two of VH chains. Here we present an analysis of the conformations of the third hypervariable region of VH domains (the H3 regions) in antibodies of known structure. We define the H3 region as comprising the residues between 92Cys and 104Gly. We divide it into a torso comprising residues proximal to the framework, four residues from the N terminus and six residues from the C terminus, and a head. There are two major classes of H3 structures that have more than ten residues between 92Cys and 104Gly: (1) the conformation of the torso has a beta-bulge at residue 101, and (2) the torso does not contain a bulge, but continues the regular hydrogen-bonding pattern of the beta-sheet hairpin. The choice of bulged versus non-bulged torso conformation is dictated primarily by the sequence, through the formation of a salt bridge between the side-chains of an Arg or Lys at position 94 and an Asp at position 101. Thus the torso region appears to have a limited repertoire of conformations, as in the canonical structure model of other antigen-binding loops. The heads or apices of the loops have a very wide variety of conformations. In shorter H3 regions, and in those containing the non-bulged torso conformation, the heads follow the rules relating sequence to structure in short hairpins. We surveyed the heads of longer H3 regions, finding that those with bulged torsos present many very different conformations of the head. We recognize that H3, unlike the other five antigen-binding loops, has a conformation that depends strongly on the environment, and we have analysed the interactions of H3 with residues elsewhere in the VH domain, in the VL domain, and with ligands, and their effects on the conformation of H3. We tested these results by attempts to predict the conformations of H3 regions in antibody structures solved after the results were derived. The general conclusion of this work is that the conformation of H3 shows some regularities, from which rules relating sequence to conformation can be stated, but to a less complete degree than for the other five antigen-binding loops. Accurate prediction of the torso conformation is possible in most cases; predictions of the conformation of the head is possible in some cases. However, our understanding of the sequence-structure relationships has reduced the uncertainty to no more than a few residues at the apex of the H3 region.

Bacteriophage lambda display of complex cDNA libraries: a new approach to functional genomics.

We describe the construction and characterization of two lambda surface displayed cDNA expression libraries derived from human brain and mouse embryo. cDNA inserts were obtained by tagged random-priming elongation of commercially available cDNA libraries and cloned into a novel lambda vector at the 3' end of the D capsid protein gene, which produced highly complex repertoires (1x10(8) and 2x10(7) phage). These libraries were affinity selected with a monoclonal antibody against the neural specific factor GAP-43 and with polyclonal antibodies that recognize the EMX1 and EMX2 homeoproteins. In both cases rapid identification of specific clones was achieved, which demonstrates the great potential of the lambda display system for generating affinity selectable cDNA libraries from complex genomes.

Mutations of Gly to Ala in human glutathione transferase P1-1 affect helix 2 (G-site) and induce positive cooperativity in the binding of glutathione.

Previous kinetic studies on human glutathione transferase P1-1 have indicated that the motions of an irregular alpha-helix (helix 2) lining the glutathione (GSH) binding site are viscosity dependent and may modulate the affinity of GSH binding. The effect of single amino acid residue substitutions (Gly to Ala) in this region is investigated here by site-directed mutagenesis. Three mutants (Gly41Ala, Gly50Ala and Gly41Ala/Gly50Ala) were overexpressed in Escherichia coli, purified, and characterized by kinetic, structural, and spectroscopic studies. All these mutant enzymes show kcat values similar to that of the wild-type enzyme, while the [S]0.5 for GSH increases about eight-fold in the Gly41Ala mutant and more than 100-fold in the Gly41Ala/Gly50Ala double mutant. This change in affinity towards GSH is accompanied by an induced positive cooperativity as reflected by Hill coefficients of 1.4 (Gly41Ala) and 1.7 (Gly41Ala/Gly50Ala) upon substrate binding. Taken together, these data suggest that the region around helix 2 is markedly altered leading to the observed intersubunit communication. Molecular modeling of the Gly41Ala/Gly50Ala mutant and of the inactive oxidized form of the native enzyme provides a structural explanation of our results.

Identification of biologically active peptides using random libraries displayed on phage.

The construction of new and increasingly diverse libraries, as well as the implementation of more powerful selection schemes, has led to the identification of linear peptides that mimic complex epitopes. Phage display techniques are allowing the selection of disease-related peptides, which reproduce the antigenic and immunogenic properties of natural antigens, using whole sera from patients. The range of applications of phage technology has been extended to include the search for peptides binding to molecules other than antibodies, such as cell receptors and enzymes.

Modelling antibody-antigen interactions: ferritin as a case study.

In this work, we propose a model for the structure of the antigen-antibody complex formed by human H-ferritin and an antibody that specifically recognizes it. We cloned and sequenced the antibody gene, predicted the antibody three-dimensional structure, and reconstructed the H-ferritin-antibody complex using an automated docking procedure previously validated on known complexes. This procedure allowed us to identify one putative complex which we carefully analysed, in order both to evaluate its likelihood, in light of a set of experimental results described in the literature, and to predict precisely which are the sites of interaction between the two molecules. Our model is compatible with the experimentally determined characteristics of the complex. Some of the residues that form the predicted antigenic site of ferritin can be found in the amino acid sequence of peptides selected from a random peptide library because of their affinity for the ferritin monoclonal antibody. Furthermore, the structural difference between the antigenic site in human H-ferritin and the corresponding region in other species permits us to rationalize the inability of the antibody to recognize human L-ferritin and rat, chicken and mouse H-ferritin. Through the analysis of our model complex, we identify a number of other residues putatively involved in the interaction. This multidisciplinary approach shows that synergy between computational and experimental methods may bring further insight into the understanding of antibody-antigen recognition rules.