Molecular and Supramolecular Architecture of theSalmo gairdneriProteinaceous Eggshell during Development

A detailed developmental study of eggshell architecture of the fishSalmo gairdneri(rainbow trout) was performed using transmission and scanning electron microscopy. Thioglycollic acid treatment and freeze-fracturing reveal that fibrils ca. 5–10 nm in diameter constitute each lamella of the helicoidal eggshell. Freeze-fracturing also permits a direct visualization of the helicoidal architecture. Laser-Raman studies of the eggshell indicate abundant antiparallel β-pleated-sheet conformation in the eggshell proteins ofS. gairdneriduring all developmental stages. Apparently, this conformation dictates formation of the helicoidal structure. Disulfide bonds, together with isopeptide bonds, cross-linkS. gairdnerieggshell proteins throughout development.     

Mutation analysis of ten exons of the CFTR gene in Greek cystic fibrosis patients: characterization of 74.5% of CF alleles including one novel mutation

To initiate the complete characterization of mutations in the CFTR gene in Greek cystic fibrosis (CF) patients, we screened 184 patients for six relatively common mutations (AF 508, G542X, G551D, 621+1 GT, N1303K, W1282X) using allele-specific hybridization and, in addition, analyzed exons 4, 5, 7, 8, 10, 11, 17b, 19, 20 and 21 using the method of denaturing gradient gel electrophoresis (DGGE). Six mutations accounted for 65.9% of the CF alleles in Greek patients, of which the F 508 mutation had a frequency of 52.7%. A further 15 previously described mutations accounted for another 8.3% CF alleles and one previously undescribed mutation (3272-4AG) was found in one chromosome. The W1282X mutation was not detected at all. Thus, so far, we have identified 21 mutations in the CFTR gene in Greek CF patients, accounting for 74.5% of the CF alleles.     

Thermal and circulatory responses during exercise: effects of hypohydration, dehydration, and water intake

Armstrong, Lawrence E., Carl M. Maresh, Catherine V. Gabaree, Jay R. Hoffman, Stavros A. Kavouras, Robert W. Kenefick, JohnW. Castellani, and Lynn E. Ahlquist. Thermal and circulatory responses during exercise: effects of hypohydration, dehydration, andwater intake. J. Appl. Physiol. 82(6):2028-2035, 1997.This investigation examined the distinct andinteractive effects of initial hydration state, exercise-induceddehydration, and water rehydration in a hot environment. On fouroccasions, 10 men performed a 90-min heat stress test (treadmillwalking at 5.6 km/h, 5% grade, 33°C, 56% relative humidity).These heat stress tests differed in pretest hydration [2euhydrated (EU) and 2 hypohydrated (HY) trials] and water intakeduring exercise [2 water ad libitum (W) and 2 no water (NW)trials]. HY + NW indicated greater physiological strain than allother trials (P < 0.05-0.001)in heart rate, plasma osmolality(P_osm), sweat sensitivity(g / °C · min), and rectal temperature.Unexpectedly, final HY + W and EU + W responses for rectal temperature,heart rate, and P_osm were similar,despite the initial 3.9 ± 0.2% hypohydration in HY + W. Weconcluded that differences in pretestP_osm (295 ± 7 and 287 ± 5 mosmol/kg for HY + W and EU + W, respectively) resulted in greaterwater consumption (1.65 and 0.31 liter for HY + W and EU + W,respectively), no voluntary dehydration (0.9% body mass increase), andattenuated thermal and circulatory strain during HY + W.

     

Relevance of Divalent Cations to ATP-Driven Proton Pumping in Beef Heart Mitochondrial F0F1-ATPase

The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F₀F₁-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me–ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the K _m of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the K _m value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F₀F₁-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely.     

The discovery and structure-activity relationships of nonpeptide, low molecular weight antagonists selective for the endothelin ETB receptor

The systematic modification of the ET_A selective N-(5-isoxazolyl)benzene-sulfonamide endothelin antagonists to give ET_B selective antagonists is reported. The reversal in selectivity was brought about by substitution of the 4-position with aryl and substituted aryl groups. Of all the aromatic substituents studied, the para-tolyl group gave rise to the most active and selective ET_B antagonist. Larger substituents caused a decrease in both ET_B activity and selectivity. A similar trend was observed by substitution at the 5-position of the N-(5-isoxazolyl)-2-thiophenesulfonamide ET_A receptor antagonists. The para-tolyl group was again found to be optimal for the ET_B activity and selectivity. The structural features that were found to be favorable for binding to the ET_B receptor, that is, the presence of a linear, conjugated π-system of definite shape and size, have been successfully incorporated into the design of ET_B selective polycyclic aromatic sulfonamides antagonists.     

Conformational analysis of peptide analogues of silkmoth chorion protein segments using CD, NMR and molecular modelling.

Silkmoth proteins secreted from the follicular cells that surround the oocyte form a large extracellular assembly which is important for protecting and sustaining the structure of the oocyte and the developing embryo. These proteins have been classified into two major families (A and B). Sequence analysis showed conservation of a central domain containing long stretches of six amino acid residue repeats in both families, which have been suggested to be organized in beta-sheet structures. In this work NMR and CD spectra, as well as molecular calculations, have been used to investigate the conformational properties of two synthetic peptides (A and B), analogues of parts of the central domain of silkmoth chorion proteins of the A and B families, respectively. These peptides consist of three tandem repeats of the six-residue basic motif. Analysis of CD spectra of the two peptides in aqueous solutions and mixtures with organic solvents revealed beta-sheet and turn structural elements with a percentage higher than 40%. NOESY spectra at low temperatures (263-273 K) show sequential nOe connectivities (i, i + 1), indicative of a relative flexibility. The presence of HNi-HNi+1 cross-peaks and medium Halphai-HNi+1 connectivities, chemical shift deviations and temperature coefficient data provide, for the first time, experimental evidence that local folded structures around Gly residues occur in peptide segments of chorion proteins in solution. Simulated annealing calculations were used to examine the conformational space of the peptides and to probe the initial steps of amyloid fibril formation in the case of chorion proteins.     

Speciation pattern of Telestes souffia complex (Teleostei,Cyprinidae) in Europe using morphological and molecular markers*

Gilles, A., Costedoat, C., Barascud, B., Voisin, A., Banarescu, P., Bianco, P. G., Economidis, P. S., Mari?, D. & Chappaz, R. (2010). Speciation pattern of Telestes souffia complex (Teleostei, Cyprinidae) in Europe using morphological and molecular markers.—Zoologica Scripta, 39, 225–242. It is notorious that many species boundaries are erroneously defined. When molecular markers are used, misleading evidence can notably be due to the characteristics inherent to mitochondrial DNA and quantity of markers used but also because of a limited range distribution sampling. European cyprinids biodiversity inventory is still an ongoing task and surprising phylogeographic patterns and phylogenetic relationships are still recovered on account of methodological evolution. This is particularly obvious for the Telestes souffia complex. This species occurs in a fragmented range and species boundaries is greatly debated. In this study, we provide an updated delimitation of the different evolutionary entities constituting this T. souffia complex and propose a taxonomic revision. Morphological and molecular analyses were carried out on 520 specimens coming from 19 localities representing the complete geographical range of this species complex and six related sister species that could potentially interact with it (outgroup ‘sensu lato’). Phylogenetic reconstructions and multivariate analyses demonstrated that the T. souffia complex is constituted of at least three species (T. souffia, Telestes muticellus and Telestes montenigrinus). Data also suggested that T. souffia comprises three subspecies (T. s. souffia, T. s. agassii; T. s. rysela). We also confirm the splitting of the T. souffia sister species Telestes pleurobipunctatus into two distinct species. The Peloponnesian lineage will be referred as Telestes alfiensis and the continental lineage as T. pleurobipunctatus. Morphological and molecular markers displayed some degree of incongruence within T. souffia suggesting that introgressive hybridization has played a role in the evolution of the Telestes genus. However, discordance among data sets could also result from heterogeneous rate of morphological evolution. Finally, we demonstrated that T. muticellus was implicated in two categories of hybridization: an inter‐species hybridization (between T. muticellus and T. souffia) and an inter‐generic hybridization (between T. muticellus and Squalius lucumonis), a phenomenon rarely observed for a same species.     

gpDB: a database of GPCRs, G-proteins, effectors and their interactions

gpDB is a publicly accessible, relational database, containing information about G-proteins, G-protein coupled receptors (GPCRs) and effectors, as well as information concerning known interactions between these molecules. The sequences are classified according to a hierarchy of different classes, families and subfamilies based on literature search. The main innovation besides the classification of G-proteins, GPCRs and effectors is the relational model of the database, describing the known coupling specificity of GPCRs to their respective alpha subunits of G-proteins, and also the specific interaction between G-proteins and their effectors, a unique feature not available in any other database. AVAILABILITY: http://bioinformatics.biol.uoa.gr/gpDB CONTACT: shamodr@biol.uoa.gr SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.