Acylated and unacylated ghrelin levels in normal weight and obese children: influence of puberty and relationship with insulin, leptin and adiponectin levels.

BACKGROUND: Ghrelin circulates in blood as acylated (AG) and unacylated (UAG) ghrelin. The physiological role of the two forms is poorly understood, in particular in childhood. Aim of the study was to evaluate the AG and UAG levels in obese and normal weight (NW) children, pre-pubertal and pubertal, and their relationship with insulin, leptin and adiponectin levels. SUBJECTS AND METHODS: A population based study in which AG, UAG, leptin, adiponectin, glucose, insulin, testosterone or estradiol levels, insulinemic indexes were evaluated in 82 NW and 58 obese (OB) children. RESULTS: Both ghrelin forms in NW were higher (AG, p<0.02; UAG, p<0.0001) than in OB subjects, with similar ratio AG/UAG . While no differences were observed for gender, puberty AG (p<0.01) and UAG (p<0.0001) levels were higher in pre-pubertal than pubertal NW and OB subjects. Adiponectin levels in NW subjects were higher (p<0.001), while leptin and insulin levels were lower (p<0.0001) than in OB subjects. NW children showed homeostasis model assessment (HOMA) and HOMAß indices lower than OB children (p<0.0001) with a higher a quantitative insulin sensitivity check index (p<0.0001). AG and UAG levels correlated to each other (p<0.0001), each showing a negative correlation to age, height, weight and body mass index. Both forms, but more strongly UAG, correlated with adiponectin, leptin, and insulin. CONCLUSIONS: OB children show lower levels of both AG and UAG when compared to NW subjects, with lower levels during puberty. These results demonstrate a peculiar strong relationship between UAG levels and metabolic parameters in the pediatric population, suggesting a role for UAG in metabolic functions.

Etiopathogenetic advances and management of holoprosencephaly: from bench to bedside.

Holoprosencephaly (HPE) is a complex brain malformation caused by impaired or incomplete midline division of the prosencephalon. It's characterized by cerebral and facial anomalies of different levels of severity. Both genetic and environmental factors are known to cause HPE, but they cover only few cases. Genetic causes are responsible for about 20% of cases: they are chromosomal abnormalities and gene mutations: up to date, nine genes (SHH, ZIC2, SIX3, TGIF, PATCHED1, TDGF1/CRIPTO, FAST1, GLI2 and DHCR) are definitely associated with HPE, but many others candidate gene are under investigation. The diagnosis of HPE is usually prenatal and is based on systematic ultrasound scan (US) and magnetic resonance imaging (MRI). Children with HPE have many medical problems in agreement with the severity of the brain malformation: craniofacial abnormalities, neurological signs, endocrine disorders, oromotor and dysautonomic dysfunction, thus requiring a multidisciplinary team for symptomatic treatment of manifestations, prevention of complications and parental support. Genetic counselling is an important step, often made difficult by extreme phenotypic variability, genetic heterogeneity, and a high risk of recurrence in apparently sporadic cases. In conclusion it can be concluded that we are far from a complete explanation of the etiopathogenesis. Future researches on genomic rearrangements all over the genome with techniques like the CGH array should lead to the identification of other causal genes and could improve diagnosis and prognosis. A skill multidisciplinary approach is mandatory to offer the better clinical assistance to patients and their parents.

Retention of nativelike conformation by proteins embedded in high external electric fields.

In this Communication, we show that proteins embedded in high external electric fields are capable of retaining a nativelike fold pattern. We have tested the metalloprotein azurin, immobilized onto SiO2 substrates in air with proper electrode configuration, by applying static fields up to 10(6)-10(7) Vm. The effects on the conformational properties of protein molecules have been determined by means of intrinsic fluorescence measurements. Experimental results indicate that no significant field-induced conformational alteration occurs. Such results are also discussed and supported by theoretical predictions of the inner protein fields.

Computational approaches to structural and functional analysis of plastocyanin and other blue copper proteins.

Computational techniques are becoming increasingly important in structural and functional biology, in particular as tools to aid the interpretation of experimental results and the design of new systems. This review reports on recent studies employing a variety of computational approaches to unravel the microscopic details of the structure-function relationships in plastocyanin and other proteins belonging to the blue copper superfamily. Aspects covered include protein recognition, electron transfer and protein-solvent interaction properties of the blue copper protein family. The relevance of integrating diverse computational approaches to address the analysis of a complex protein system, such as a cupredoxin metalloprotein, is emphasized.

Theoretical descriptors for the quantitative rationalisation of plastocyanin mutant functional propertiess.

A quantitative rationalisation of the effect of specific amino acids on the recognition process and redox characteristics of plastocyanin towards cytochrome f, as determined by point mutation experiments, has been attempted in this study. To achieve this goal we derived theoretical descriptors directly from the three-dimensional structure of the plastocyanin mutants, in the same manner as it is usually done for small drug-like molecules. The protein descriptors computed can be related to: (a) the electrostatic and dipole-dipole interactions, effective at long distance; (b) polar interactions whose features are encoded by charged partial surface area descriptors; (c) the propensity of the surface residues to form hydrogen bonding interactions; and (d) dispersion and repulsive interactions. Moreover, an estimation of mutation-dependent variation of redox potential observed has been obtained by electrostatic free energy calculations. The quantitative structure-activity relationship (QSAR) models offer structural interpretation of the point mutation experiment responses and can be of help in the design of new protein engineering experiments.

Electrostatic analysis and Brownian dynamics simulation of the association of plastocyanin and cytochrome f.

The oxidation of cytochrome f by the soluble cupredoxin plastocyanin is a central reaction in the photosynthetic electron transfer chain of all oxygenic organisms. Here, two different computational approaches are used to gain new insights into the role of molecular recognition and protein-protein association processes in this redox reaction. First, a comparative analysis of the computed molecular electrostatic potentials of seven single and multiple point mutants of spinach plastocyanin (D42N, E43K, E43N, E43Q/D44N, E59K/E60Q, E59K/E60Q/E43N, Q88E) and the wt protein was carried out. The experimentally determined relative rates (k(2)) for the set of plastocyanin mutants are found to correlate well (r(2) = 0.90 - 0.97) with the computed measure of the similarity of the plastocyanin electrostatic potentials. Second, the effects on the plastocyanin/cytochrome f association rate of these mutations in the plastocyanin "eastern site" were evaluated by simulating the association of the wild type and mutant plastocyanins with cytochrome f by Brownian dynamics. Good agreement between the computed and experimental relative rates (k(2)) (r(2) = 0.89 - 0.92) was achieved for the plastocyanin mutants. The results obtained by applying both computational techniques provide support for the fundamental role of the acidic residues at the plastocyanin eastern site in the association with cytochrome f and in the overall electron-transfer process.

Control of metalloprotein reduction potential: the role of electrostatic and solvation effects probed on plastocyanin mutants.

The changes in the thermodynamics of Cu(II) reduction for spinach plastocyanin induced by point mutations altering the electrostatic potential in proximity of the copper center were determined through variable temperature direct electrochemistry experiments. In particular, the functionally important surface residues Leu12 and Gln88 were replaced with charged and polar residues, and Asn38 was substituted with Asp. The mutational variations of the reduction enthalpy and entropy were analyzed with a QSPR (quantitative structure-property relationships) approach, employing global and local theoretical descriptors defined and computed on the three-dimensional protein structure. The correlations found are informative on how electrostatic and solvation effects control the E degrees ' values in this species through the combined effects on the reduction enthalpy and entropy. The changes in reduction enthalpy can be justified with electrostatic considerations. Most notably, enthalpy-entropy compensation phenomena play a significant role: the entropic effects due to the insertion of charged residues determine E degrees ' changes that are invariably opposite to those induced by the concomitant enthalpic effects. Therefore, the resulting E degrees ' changes are small or even opposite to those expected on simple electrostatic grounds. The mutational variation in the reduction entropy appears to be linked to the hydrogen bonding donor/acceptor character of the northern part of the protein, above the metal site, and to the electrostatic potential distribution around the copper site. Both properties influence the reduction-induced reorganization of the water molecules on the protein surface in the same region.

Blue copper proteins: a comparative analysis of their molecular interaction properties.

Blue copper proteins are type-I copper-containing redox proteins whose role is to shuttle electrons from an electron donor to an electron acceptor in bacteria and plants. A large amount of experimental data is available on blue copper proteins; however, their functional characterization is hindered by the complexity of redox processes in biological systems. We describe here the application of a semiquantitative method based on a comparative analysis of molecular interaction fields to gain insights into the recognition properties of blue copper proteins. Molecular electrostatic and hydrophobic potentials were computed and compared for a set of 33 experimentally-determined structures of proteins from seven blue copper subfamilies, and the results were quantified by means of similarity indices. The analysis provides a classification of the blue copper proteins and shows that (I) comparison of the molecular electrostatic potentials provides useful information complementary to that highlighted by sequence analysis; (2) similarities in recognition properties can be detected for proteins belonging to different subfamilies, such as amicyanins and pseudoazurins, that may be isofunctional proteins; (3) dissimilarities in interaction properties, consistent with experimentally different binding specificities, may be observed between proteins belonging to the same subfamily, such as cyanobacterial and eukaryotic plastocyanins; (4) proteins with low sequence identity, such as azurins and pseudoazurins, can have sufficient similarity to bind to similar electron donors and acceptors while having different binding specificity profiles.

Theoretical investigation of substrate specificity for cytochromes P450 IA2, P450 IID6 and P450 IIIA4.

Three-dimensional models of the cytochromes P450 IA2, P450 IID6 and P450 IIIA4 were built by means of comparative modeling using the X-ray crystallographic structures of P450 CAM, P450 BM-3, P450 TERP and P450 ERYF as templates. The three cytochromes were analyzed both in their intrinsic structural features and in their interaction properties with fifty specific and non-specific substrates. Substrate/enzyme complexes were obtained by means of both automated rigid and flexible body docking. The comparative analysis of the three cytochromes and the selected substrates, in their free and bound forms, allowed for the building of semi-quantitative models of substrate specificity based on both molecular and intermolecular interaction descriptors. The results of this study provide new insights into the molecular determinants of substrate specificity for the three different eukaryotic P450 isozymes and constitute a useful tool for predicting the specificity of new compounds.