Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands

Data relating to the structural basis of ligand recognition by integrins are limited. Here we describe the physical requirements for high affinity binding of ligands to alpha v beta6. By combining a series of structural analyses with functional testing, we show that 20-mer peptide ligands, derived from high affinity ligands of alpha v beta6 (foot-and-mouth-disease virus, latency associated peptide), have a common structure comprising an Arg-Gly-Asp motif at the tip of a hairpin turn followed immediately by a C-terminal helix. This arrangement allows two conserved Leu/Ile residues at Asp(+1) and Asp(+4) to be presented on the outside face of the helix enabling a potential hydrophobic interaction with the alpha v beta6 integrin, in addition to the Arg-Gly-Asp interaction. The extent of the helix determines peptide affinity for alpha v beta6 and potency as an alpha v beta6 antagonist. A major role of this C-terminal helix is likely to be the correct positioning of the Asp(+1) and Asp(+4) residues. These data suggest an explanation for several biological functions of alpha v beta6 and provide a structural platform for design of alpha v beta6 antagonists.     

Regional localization of th human EGF-like growth factor CRIPTO gene (TDGF-1) to chromosome 3p21

The CRIPTO gene encodes a novel human growth factor structurally related to epidermal growth factor. We localized the CRIPTO gene to chromosome 3p21 by fluorescence in situ hybridization with a cosmid clone containing 40 kb of the CRIPTO genomic region (TDGF-1). To suppress hybridization to CRIPTO-related sequences, present in multiple copies in the human genome, hybridization was carried out in the presence of unlabeled CRIPTO cDNA in excess over the probe. Our finding confirms the provisional mapping of the CRIPTO gene to chromosome 3, and assigns it precisely to a chromosomal region involved in several rearrangements occurring in malignancy.CRIPTO-specific sequences are present in multiple copies in the human genome (Dono et al. 1991). Two genomic CRIPTO-encoding sequences, TDGF-1 and TDGF-3, have been isolated and characterized. TDGF-1 corresponds to the structural gene encoding the protein expressed in teratocarcinoma cells (Ciccodicola et al. 1989). TDGF-3, possibly a functional pseudogene, corresponds to a complete copy of the TDGF-1 mRNA that contains seven base changes representing both silent and replacement substitutions in the coding region (Dono et al. 1991). By somatic cell hybrid analysis TDGF-1 has been assigned to chromosome 3, and TDGF-3 to the Xq21–22 region (Dono et al. 1991).     

Nandrolone decanoate interferes with testosterone biosynthesis altering blood–testis barrier components

The aim of this study was to investigate whether nandrolone decanoate (ND) use affects testosterone production and testicular morphology in a model of trained and sedentary mice. A group of mice underwent endurance training while another set led a sedentary lifestyle and were freely mobile within cages. All experimental groups were treated with either ND or peanut oil at different doses for 6 weeks. Testosterone serum levels were measured via liquid chromatography–mass spectrometry. Western blot analysis and quantitative real‐time PCR were utilized to determine gene and protein expression levels of the primary enzymes implicated in testosterone biosynthesis and gene expression levels of the blood–testis barrier (BTB) components. Immunohistochemistry and immunofluorescence were conducted for testicular morphological evaluation. The study demonstrated that moderate to high doses of ND induced a diminished serum testosterone level and altered the expression level of the key steroidogenic enzymes involved in testosterone biosynthesis. At the morphological level, ND induced degradation of the BTB by targeting the tight junction protein‐1 (TJP1). ND stimulation deregulated metalloproteinase‐9, metalloproteinase‐2 (MMP‐2) and the tissue inhibitor of MMP‐2. Moreover, ND administration resulted in a mislocalization of mucin‐1. In conclusion, ND abuse induces a decline in testosterone production that is unable to regulate the internalization and redistribution of TJP1 and may induce the deregulation of other BTB constituents via the inhibition of MMP‐2. ND may well be considered as both a potential inducer of male infertility and a potential risk factor to a low endogenous bioavailable testosterone.     

Oxygen radical scavenging capacity of phenolic and non-phenolic compounds in red and white wines

The aim of the present study was the evaluation of the antioxidant content in phenolic and non-phenolic extracts of ten wine samples, trying to elucidate the potential role of unusual antioxidant compounds. Samples of wines processed from red and white grapes (Vitis vinifera L.), deprived of the volatile fraction at low temperature and buffered at physiological pH, were fractionated by C₁₈ into two fractions: FR1 and FR2. Non-phenolics, such as tartaric, malic, lactic, and succinic acids; glucose; fructose; and glycerin were mainly found in FR1, while polyphenols were present exclusively in FR2. Peroxyl radical quenching was assayed by the ORAC method, while superoxide and hydroxyl radical scavenging activity were assayed by electron paramagnetic resonance. In the ORAC and superoxide assays, most of the activity was found in FR2, while in hydroxyl radical assay, the activity was found in FR1. Model solutions were used to attribute a role to the single compounds in the evaluation of wine’s ROS scavenging capacity: the ORAC and superoxide anion scavenging effects were mainly attributed to the polyphenols, averaging 94.8%, with some contribution from glycerin, particularly in white wines. Unexpectedly, the main chemical responsible for hydroxyl radical scavenging activity was glycerin (56.1%), with the polyphenols scavenging at 18.1%.     

Linear array of conserved sequence motifs to discriminate protein subfamilies: study on pyridine nucleotide-disulfide reductases

Background  

The pyridine nucleotide disulfide reductase (PNDR) is a large and heterogeneous protein family divided into two classes (I and II), which reflect the divergent evolution of its characteristic disulfide redox active site. However, not all the PNDR members fit into these categories and this suggests the need of further studies to achieve a more comprehensive classification of this complex family.     

Fluoro-edenite fibres induce lung cell apoptosis: an in vivo study

We previously showed that apoptosis in the lungs of sheep exposed to fluoro-edenite fibres is induced via the receptor pathway. The present study was performed to gain further insights into the mechanisms of activation of programmed cell death induced by the fibres. Fluoro-edenite fibres are similar in size and morphology to some amphibolic asbestos fibres. They have been found in benmoreitic lavas, in the local stone quarry, in building materials and in road paving at Biancavilla, a town in eastern Sicily (Italy), where epidemiological surveys revealed a cluster of mortality from pleural mesothelioma. Inhalation of asbestos fibres can cause chronic inflammation and carcinogenesis. Since fluoro-edenite has been shown to activate the apoptotic process, we set out to characterise the expression of apoptosis-regulating proteins in fluoro-edenite-exposed lung disease and sought to determine if apoptosis results from fluoro-edenite exposure. Lung tissue from apparently healthy sheep habitually grazing near Biancavilla was processed for immunohistochemical localisation of bcl-2 and bax. Results showed epithelial and interstitial bax overexpression, especially in cells directly in contact with the fibres, and negative bcl-2 immunoexpression. TUNEL-positive cells were detected in alveoli and connective tissue. The integrity of alveolar epithelium and alveolar apoptosis are critical determinants in the pathways that initiate fibrogenesis in the lung and fibroblastic foci are usually found close to abnormal or denuded alveolar epithelium. Our results are consistent with the hypothesis that apoptosis is an important mechanism for removing cells with irreparable fluoro-edenite-induced genetic changes that predispose them to a neoplastic evolution.