Search

Background: Protein dynamics influence protein function and stability and modulate conformational changes. Such motions depend on the underlying networks of intramolecular interactions and communicating residues within the protein structure. Here, we provide the first characterization of the dynamic fingerprint of the dimeric alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 (VAP), which is among the APs with the highest known kcat at low temperatures., Methods: Multiple all-atom explicit solvent molecular dynamics simulations were employed in conjunction with different metrics to analyze the dynamical patterns and the paths of intra- and intermolecular communication., Results: Interactions and coupled motions at the interface between the two VAP subunits have been characterized, along with the networks of intramolecular interactions. It turns out a low number of intermolecular interactions and coupled motions, which result differently distributed in the two monomers. The paths of long-range communication mediated from the catalytic residues to distal sites were also characterized, pointing out a different information flow in the two subunits., Conclusions: A pattern of asymmetric flexibility is evident in the two identical subunits of the VAP dimer that is intimately linked to a different distribution of intra- and intermolecular interactions. The asymmetry was also evident in pairs of cross-correlated residues during the dynamics., General Significance: The results here discussed provide a structural rationale to the half-of-site mechanism previously proposed for VAP and other APs, as well as a general framework to characterize asymmetric dynamics in homomeric enzymes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Acylaminoacyl peptidase (AAP) subfamily belongs to the prolyl oligopeptidase (POP) family of serine-proteases. There is a great interest in the definition of molecular mechanisms related to the activity and substrate recognition of these complex multi-domain enzymes. The active site relies at the interface between the C-terminal catalytic domain and the β-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In AAP, the N-terminal extension is characterized by a structurally conserved α1-helix, which is known to affect thermal stability and thermal dependence of the catalytic activity. In the present contribution, results from hundreds nanosecond all-atom molecular dynamics simulations, along with analyses of the networks of cross-correlated motions of a member of the AAP subfamily are discussed. The MD investigation identifies a tunnel that from the surrounding of the N-terminal α1-helix bring to the catalytic site. This cavity seems to be regulated by conformational changes of the α1-helix itself during the dynamics. The evidence here provided can be a useful guide for a better understanding of the mechanistic aspects related to AAP activity, but also for drug design purposes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Protein dynamics and the underlying networks of intramolecular interactions and communicating residues within the three-dimensional (3D) structure are known to influence protein function and stability, as well as to modulate conformational changes and allostery. Acylaminoacyl peptidase (AAP) subfamily of enzymes belongs to a unique class of serine proteases, the prolyl oligopeptidase (POP) family, which has not been thoroughly investigated yet. POPs have a characteristic multidomain three-dimensional architecture with the active site at the interface of the C-terminal catalytic domain and a β-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In the present contribution, protein dynamics signatures of a hyperthermophilic acylaminoacyl peptidase (AAP) of the prolyl oligopeptidase (POP) family, as well as of a deletion variant and alanine mutants (I12A, V13A, V16A, L19A, I20A) are reported. In particular, we aimed at identifying crucial residues for long range communications to the catalytic site or promoting the conformational changes to switch from closed to open ApAAP conformations. Our investigation shows that the N-terminal α1-helix mediates structural intramolecular communication to the catalytic site, concurring to the maintenance of a proper functional architecture of the catalytic triad. Main determinants of the effects induced by α1-helix are a subset of hydrophobic residues (V16, L19 and I20). Moreover, a subset of residues characterized by relevant interaction networks or coupled motions have been identified, which are likely to modulate the conformational properties at the interdomain interface.

The well-known relationship between low birth weight and allergies prompted us to investigate a possible pleiotropic effect of ACP1 on these conditions. ACP1 is a polymorphic enzyme that affects signal transduction of insulin and other growth factors, T-cell receptor signaling, and the regulation of flavoenzyme activity. Our aim was to compare the relationship between ACP1 and allergy with the relationship between ACP1 and birth weight. We studied 299 subjects from the Caucasian population of England, 124 subjects from the Caucasian population of central Italy, and 302 healthy puerperae and their newborn babies from the same Caucasian populations. ACP1 phenotype was determined by starch gel electrophoresis on RBC hemolysate and by DNA analysis. Subjects with high ACP1 activity (ACP1 C,B phenotype) show a lower level of IgE compared to subjects with low ACP1 activity (p = 0.01). The proportion of infants with a birth weight below the first quartile is lower among infants born to mothers with high ACP1 activity than among infants born to mothers with medium-low activity (p = 0.01). The data suggest a protective effect of high-activity ACP1 C,B phenotype from low birth weight and from allergic manifestations after birth.

Aim: This is the first systematic study using quantitative real-time PCR to analyze and compare the expression profiles for critical members of the epidermal growth factor (EGF), transforming growth factor beta (TGFbeta), and vascular endothelial growth factor (VEGF) families in developing rat lungs., Methods: mRNA expression was quantified at embryonic (E) day 15, 17, 19, 21, and postnatal age 1 day, 2 weeks, 12 weeks., Results: EGF and EGFR increased during gestation and development, then decreased in adulthood, whereas TGFalpha was highest at birth and remained unchanged afterwards. All TGFbeta isoforms increased slightly during pregnancy, reached highest expression during development, and returned to neonatal levels in adulthood. TGFbetaRI and TGFbetaRII patterns were similar to TGFbeta2 and TGFbeta1 respectively, whereas TGFbetaRIII expression was lowest at the postnatal time points. VEGF(164) and VEGF(120) showed a steady increase up to 2 weeks and declined at 12 weeks, whereas highest VEGF(188) expression occurred at 12 weeks. VEGF-A receptors expression paralleled the summation of all three isoforms, increasing steadily with age., Conclusion: Expression of growth factors in the developing lung is characterized by highly regulated distinctive patterns that may be critical to understand the early origin and progression of pulmonary diseases in childhood as well as in adulthood. Quantitative real-time PCR analysis revealed several differences compared to previously reported expression patterns defined with older methodologies.

We offer further biological characterization of the XK atelen/aprosencephaly syndrome in two infants, one with prolonged survival, the other presenting prenatally with apparent hydranencephaly and an orbital tumor (OS). Familial occurrence in the former born to presumably nonconsanguineous Lybian parents may represent parental germinal mosaicism or autosomal recessive inheritance. Both had apparently normal chromosomes; however, the Lybian infant had slightly increased induced chromosome breakage suggesting that this rare multiple congenital anomalies syndrome may involve a DNA repair defect. Virtual absence of atelen/aprosencephalic structures may lead to an arthrogryposis-like prenatal movement disorder. The orbital tumor in the Utah infant consisted of dystopic neural tissue compressing a rudimentary globe and was connected by a thin bridge of neural tissue to the small mass of disorganized brain tissue usually found in atelen/aprosencephalic infants and fetuses. No evidence of an encephaloclastic process was found in the autopsied Utah infant., (Copyright 2005 Wiley-Liss, Inc)

Respiratory syncytial virus (RSV) infection is the most common cause of bronchiolitis in infants and an important risk factor for the development of recurrent wheezing and asthma. Cysteinyl leukotrienes were implicated in the pathophysiology of these diseases, and are being targeted for their diagnosis and therapy. We measured urinary leukotriene E4 (LTE4) in infants with RSV bronchiolitis in comparison with controls without respiratory infection, and investigated whether medical and family history, age, and passive exposure to tobacco smoke are related to urinary leukotriene excretion. We studied 33 infants with bronchiolitis and 25 controls, 1-12 months of age. Demographic and historical data were obtained from informed-consent forms and questionnaires completed by the parents. RSV was detected in nasal secretions by enzyme-linked immunoassay. Urine samples were collected on day of admission and were analyzed for LTE4 with an enzyme-linked immunoassay. Urinary LTE4 was 8-fold higher in infants with bronchiolitis than in controls. Leukotriene excretion was significantly higher in infected infants <6 months of age with a medical history of eczema or dry cough and/or family history of asthma. Multivariate analysis revealed that eczema and dry cough are independently associated with high LTE4 excretion during bronchiolitis. Exposure to tobacco smoke did not affect urinary LTE4. Our study shows that leukotriene synthesis during bronchiolitis is particularly elevated in younger infants with an atopic/asthmatic background. Urinary LTE4 may become a valuable, noninvasive marker for the identification of patients who will benefit most from therapy with leukotriene modifiers for management of bronchiolitis., (2005 Wiley-Liss, Inc.)

Cholangiography carried out in laparoscopic surgery is a diagnostic exam considered by many authors to be very important for the anatomy and the research pre-existent pathologies of the biliary tree. Such are the effective advantages that many Schools suggest it be carried out any case as the risks, the costs and the results do not influence or alter the result of surgical treatment and moreover, its continuous use improves the positive results. The simple execution of cholangiography, allowed also by the presence of suitable forceps, makes this exam practicable at any moment and easily coupled to other diagnostic and therapeutic methods executable during the proper times of laparoscopic surgery.

To evaluate acceptability, safety, and efficacy of a Y set with two short branches (TAS) filled with electrolytic chloroxidizer solution during the dwell time, 60 patients were randomly allocated to be treated with the traditional Y set (TCS) or with the TAS. Twenty-three were new patients whereas the remaining 37 were patients already on continuous ambulatory peritoneal dialysis (CAPD) with the TCS. The follow-up was 416.5 months in the control group and 387.4 months in the test group. During the study period there were 6 peritonitis episodes in each group with an incidence of 1 episode every 69.4 patient-months in the control group and 1 episode every 64.6 patient-months in the test group. Twenty-four patients (80%) in the control group and 27 (90%) in the test group were free from peritonitis. The probability to remain free from peritonitis was respectively 87% and 83% in the test group and in control group after 12 months, 70% and 78% after 21 months. Seventy-nine percent of the patients who used both systems preferred the TAS for better handling, lower encumbrance, and major safety. One patient preferred the TCS, three patients did not find any differences between the two devices.