Focal adhesion kinase regulation by oxidative stress in different cell types

Focal adhesion kinase (FAK) is a tyrosine kinase ubiquitously expressed in cells. It was initially shown to be the initiator of focal adhesion formation in adherent cells, after its binding to integrins which induce its autophosphorylation. However, it can be also activated by a great variety of other stimuli able to act on different intracellular signaling. Reactive oxygen species (ROS), which have been shown to act as external or internal cell stimuli, induce tyrosine phosphorylation of FAK. Its autophosphorylation is followed by a submembranous localization which is crucial for many of the biological roles of FAK, including cell spreading, cell migration, cell proliferation, and prevention of apoptosis. It plays an important role in development of tumor cells, its regulation could be thus a way of impairing cell proliferation in cancer. We describe in this review the structure, activity, and functions of FAK in different cells and how ROS are able, like other stimuli, to induce its phosphorylation and modification of cell morphology and structure. The link between ROS and FAK activation could explain the role of ROS in mediating cell proliferation, cell migration, or apoptosis.     

Holoprosencephaly

Cardiac tumours are benign or malignant neoplasms arising primarily in the inner lining, muscle layer, or the surrounding pericardium of the heart. They can be primary or metastatic. Primary cardiac tumours are rare in paediatric practice with a prevalence of 0.0017 to 0.28 in autopsy series. In contrast, the incidence of cardiac tumours during foetal life has been reported to be approximately 0.14%. The vast majority of primary cardiac tumours in children are benign, whilst approximately 10% are malignant. Secondary malignant tumours are 10–20 times more prevalent than primary malignant tumours. Rhabdomyoma is the most common cardiac tumour during foetal life and childhood. It accounts for more than 60% of all primary cardiac tumours. The frequency and type of cardiac tumours in adults differ from those in children with 75% being benign and 25% being malignant. Myxomas are the most common primary tumours in adults constituting 40% of benign tumours. Sarcomas make up 75% of malignant cardiac masses. Echocardiography, Computing Tomography (CT) and Magnetic Resonance Imaging (MRI) of the heart are the main non-invasive diagnostic tools. Cardiac catheterisation is seldom necessary. Tumour biopsy with histological assessment remains the gold standard for confirmation of the diagnosis. Surgical resection of primary cardiac tumours should be considered to relieve symptoms and mechanical obstruction to blood flow. The outcome of surgical resection in symptomatic, non-myxomatous benign cardiac tumours is favourable. Patients with primary cardiac malignancies may benefit from palliative surgery but this approach should not be recommended for patients with metastatic cardiac tumours. Surgery, chemotherapy and radiotherapy may prolong survival. The prognosis for malignant primary cardiac tumours is generally extremely poor.     

Duplicated Leptin Receptors in Two Species of Eel Bring New Insights into the Evolution of the Leptin System in Vertebrates

Since its discovery in mammals as a key-hormone in reproduction and metabolism, leptin has been identified in an increasing number of tetrapods and teleosts. Tetrapods possess only one leptin gene, while most teleosts possess two leptin genes, as a result of the teleost third whole genome duplication event (3R). Leptin acts through a specific receptor (LEPR). In the European and Japanese eels, we identified two leptin genes, and for the first time in vertebrates, two LEPR genes. Synteny analyses indicated that eel LEPRa and LEPRb result from teleost 3R. LEPRb seems to have been lost in the teleost lineage shortly after the elopomorph divergence. Quantitative PCRs revealed a wide distribution of leptins and LEPRs in the European eel, including tissues involved in metabolism and reproduction. Noticeably, leptin1 was expressed in fat tissue, while leptin2 in the liver, reflecting subfunctionalization. Four-month fasting had no impact on the expression of leptins and LEPRs in control European eels. This might be related to the remarkable adaptation of silver eel metabolism to long-term fasting throughout the reproductive oceanic migration. In contrast, sexual maturation induced differential increases in the expression of leptins and LEPRs in the BPG-liver axis. Leptin2 was strikingly upregulated in the liver, the central organ of the reproductive metabolic challenge in teleosts. LEPRs were differentially regulated during sexual maturation, which may have contributed to the conservation of the duplicated LEPRs in this species. This suggests an ancient and positive role of the leptin system in the vertebrate reproductive function. This study brings new insights on the evolutionary history of the leptin system in vertebrates. Among extant vertebrates, the eel represents a unique case of duplicated leptins and leptin receptors as a result of 3R.     

Expression, linkage, and polymorphism of MHC-related genes in rainbow trout, Oncorhynchus mykiss.

The architecture of the MHC in teleost fish, which display a lack of linkage between class I and II genes, differs from all other vertebrates. Because rainbow trout have been examined for a variety of immunologically relevant genes, they present a good teleost model for examining both the expression and organization of MHC-related genes. Full-length cDNA and partial gDNA clones for proteasome delta, low molecular mass polypeptide (LMP) 2, TAP1, TAP2A, TAP2B, class Ia, and class IIB were isolated for this study. Aside from the expected polymorphisms associated with class I genes, LMP2 and TAP2 are polygenic. More specifically, we found a unique lineage of LMP2 (LMP2/delta) that shares identity to both LMP2 and delta but is expressed like the standard LMP2. Additionally, two very different TAP2 loci were found, one of which encodes polymorphic alleles. In general, the class I pathway genes are expressed in most tissues, with highest levels in lymphoid tissue. We then analyzed the basic genomic organization of the trout MHC in an isogenic backcross. The main class Ia region does not cosegregate with the class IIB locus, but LMP2, LMP2/delta, TAP1A, and TAP2B are linked to the class Ia locus. Interestingly, TAP2A (second TAP2 locus) is a unique lineage in sequence composition that appears not to be linked to this cluster or to class IIB. These results support and extend the recent findings of nonlinkage between class I and II in a different teleost order (cyprinids), suggesting that this unique arrangement is common to all teleosts.     

Association between coding variability in the LRP gene and the risk of late-onset Alzheimer’s disease

We have sequenced the entire (89 exons) open reading frame of the LRP gene in 12 cases of Alzheimer’s disease (AD) from Northern France. We have found no novel changes but confirm the occurrence of a polymorphism in exon 6 of the gene (A216V). This polymorphism is rare (2.8% of controls) and is in linkage equilibrium with previously reported polymorphisms. The V216 allele is negatively associated with the disease in a large case-controlled series. These data suggest that the LRP receptor may be involved in the pathobiology of AD, but the association that we report here cannot explain the previously reported genetic data implicating the LRP gene in AD. If the LRP gene is a major site of genetic variability leading to AD, there must be other biologically relevant variability in promoter or other regulatory elements of this large gene. Received: 28 December 1998 / Accepted: 1 March 1999     

Compartmentalization of GABA Synthesis by GAD67 Differs between Pancreatic Beta Cells and Neurons

The inhibitory neurotransmitter GABA is synthesized by the enzyme glutamic acid decarboxylase (GAD) in neurons and in pancreatic β-cells in islets of Langerhans where it functions as a paracrine and autocrine signaling molecule regulating the function of islet endocrine cells. The localization of the two non-allelic isoforms GAD65 and GAD67 to vesicular membranes is important for rapid delivery and accumulation of GABA for regulated secretion. While the membrane anchoring and trafficking of GAD65 are mediated by intrinsic hydrophobic modifications, GAD67 remains hydrophilic, and yet is targeted to vesicular membrane pathways and synaptic clusters in neurons by both a GAD65-dependent and a distinct GAD65-independent mechanism. Herein we have investigated the membrane association and targeting of GAD67 and GAD65 in monolayer cultures of primary rat, human, and mouse islets and in insulinoma cells. GAD65 is primarily detected in Golgi membranes and in peripheral vesicles distinct from insulin vesicles in β-cells. In the absence of GAD65, GAD67 is in contrast primarily cytosolic in β-cells; its co-expression with GAD65 is necessary for targeting to Golgi membranes and vesicular compartments. Thus, the GAD65-independent mechanism for targeting of GAD67 to synaptic vesicles in neurons is not functional in islet β-cells. Therefore, only GAD65:GAD65 homodimers and GAD67:GAD65 heterodimers, but not the GAD67:GAD67 homodimer gain access to vesicular compartments in β-cells to facilitate rapid accumulation of newly synthesized GABA for regulated secretion and fine tuning of GABA-signaling in islets of Langerhans.     

A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm.

We present a novel method that predicts transmembrane domains in proteins using solely information contained in the sequence itself. The PRED-TMR algorithm described, refines a standard hydrophobicity analysis with a detection of potential termini ('edges', starts and ends) of transmembrane regions. This allows one both to discard highly hydrophobic regions not delimited by clear start and end configurations and to confirm putative transmembrane segments not distinguishable by their hydrophobic composition. The accuracy obtained on a test set of 101 non-homologous transmembrane proteins with reliable topologies compares well with that of other popular existing methods. Only a slight decrease in prediction accuracy was observed when the algorithm was applied to all transmembrane proteins of the SwissProt database (release 35). A WWW server running the PRED-TMR algorithm is available at http://o2.db.uoa. gr/PRED-TMR/     

Les centres d’investigation clinique–innovation technologique : des instruments pour les technologies pour la santé

This paper describes the clinical investigation centres devoted to technological innovation. Their objectives and the topic areas are detailed and also the main actors involved in the process of socio-economic development (research and industrial grants, certification organisms, clinical evaluation, etc.). An overall tour of the present instruments at the disposal of academic laboratories and companies is therefore made. National and international contextual elements of health technologies are provided as well.     

Reactive oxygen species activate focal adhesion kinase, paxillin and P130CAS tyrosine phosphorylation in endothelial cells

Reactive oxygen species (ROS), particularly hydroxyl radical (HO), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 μM) and herbimycin A (0.9 μM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120–130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO. Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125^FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 μM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO, which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.