Interaction between platelet glycoprotein Ibalpha and filamin-1 is essential for glycoprotein Ib/IX receptor anchorage at high shear.

The interaction of the glycoprotein (GP) Ib-V-IX receptor complex with the membrane skeleton of platelets is dependent on a specific interaction between the cytoplasmic tail of GPIbalpha and filamin-1. This interaction has been proposed to regulate key aspects of platelet function, including the ligand binding of GPIb-V-IX and the ability of the cells to sustain adhesion to von Willebrand factor (vWf) under high shear. In this study we have examined sequences in the GPIbalpha intracellular domain necessary for interaction of the receptor with filamin-1. We have identified two adjacent sequences involving amino acids 557-568 and 569-579 of the GPIbalpha cytoplasmic domain that are critical for normal association between the receptor complex and filamin-1. Under flow conditions, Chinese hamster ovary (CHO) cells expressing these two mutant receptors exhibited an increase in translocation velocity that was associated with increased cell detachment from the vWf matrix at high shear. The shear-dependent acceleration in velocity of mutant Delta557-568 and Delta569-579 CHO cells was associated with a critical defect in receptor anchorage, evident from significant extraction of GPIb-IX from the CHO cell membrane at high shear. These studies define a critical role for amino acids within the 557-579 sequence of GPIbalpha for interaction with filamin-1.     

Identification and Characterization of Thioredoxin H-Type Gene Family in Triticum turgidum ssp. durum in Response to Natural and Environmental Factor-Induced Oxidative Stress

Plant Molecular Biology Reporter - Key message: Thioredoxin h-type isoforms are tissue-specific, differentially expressed in germinating seeds and under salinity stress and highly regulated by...     

Distinct glycoprotein Ib/V/IX and integrin alpha IIbbeta 3-dependent calcium signals cooperatively regulate platelet adhesion under flow.

We have investigated the calcium signaling relationship between the two major platelet adhesion receptors, glycoprotein Ib/V/IX (GPIb/V/IX) and integrin alpha(IIb)beta(3), involved in regulating platelet adhesion on von Willebrand factor (vWf) under flow. Our studies demonstrate that GPIb engagement of immobilized vWf elicits a transient calcium spike that may function to promote reversible arrest of translocating platelets. Subsequent integrin alpha(IIb)beta(3) engagement of vWf promotes sustained calcium oscillations that are essential for the maintenance of irreversible adhesion. GPIb-induced calcium spikes appear distinct from those initiated by integrin alpha(IIb)beta(3), in that the former are exclusively mediated through release of intracellular calcium stores via a signaling mechanism independent of PI 3-kinase. In contrast, integrin alpha(IIb)beta(3)-dependent calcium flux involves a PI 3-kinase-dependent signaling mechanism linked to intracellular calcium mobilization and subsequent transmembrane calcium influx. Studies employing the caged calcium chelator (o-nitrophenyl-EGTA) demonstrate that transient calcium spikes initiate a transient phase of platelet arrest that is converted to irreversible adhesion with the development of sustained oscillatory calcium flux. These studies demonstrate the existence of a dual step calcium signaling mechanism utilized by GPIb and integrin alpha(IIb)beta(3) that serves to regulate the dynamics of platelet adhesion under flow.     

Cytogenetic survey of 117 Tunisian patients with de novo myelodysplastic syndrome

Cytogenetic studies were performed on 117 Tunisian patients with de novo myelodysplastic syndromes (MDS). According to the French-American-British (FAB) criteria 40 patients presented with refractory anaemia (RA, 34%), eight with refractory anaemia with ringed sideroblasts (RARAS, 7%), 19 with refractory anaemia with excess of blasts (RAEB, 16%), 16 with refractory anaemia with excess of blasts in transformation (RAEB-t, 14%), 18 had chronic myelomonocytic leukaemia (CMML, 15%) and 16 unclassifiable MDS (14%). Seventy-five were men and forty-two were women. Five were children and 112 were adults with a median age of 58 years. Fifty-five per cent of the patients presented clonal chromosome abnormalities. Rates of abnormality varied from one FAB subtype to the other: 55% in RA, 75% in RARAS, 63% in RAEB, 75% in RAEB-t and 28% in CMML. The most frequent chromosome abnormalities were del(5q) (22 cases), monosomy 7 (12 cases), del(12p) (6 cases), and trisomy 8 (5 cases). Rare abnormalities were also found: ring of chromosome 12 and trisomy 15. Conventional cytogenetics remains the basic technique in identifying chromosomal abnormalities associated with MDS.     

Host adaptor proteins Gab1 and CrkII promote InlB-dependent entry of Listeria monocytogenes

The bacterial surface protein InlB mediates internalization of Listeria monocytogenes into mammalian cells through interaction with the host receptor tyrosine kinase, Met. InlB/Met interaction results in activation of the host phosphoinositide (PI) 3-kinase p85-p110, an event required for bacterial entry. p85-p110 activation coincides with tyrosine phosphorylation of the host adaptor Gab1, and formation of complexes between Gab1 and the p85 regulatory subunit of PI 3-kinase. When phosphorylated in response to agonists, Gab1 is known to recruit several Src-homology 2 (SH2) domain-containing proteins including p85, the tyrosine phosphatase Shp2 and the adaptor CrkII. Here, we demonstrate that Gab1.p85 and Gab1.CrkII complexes promote entry of Listeria. Overexpression of wild-type Gab1 stimulated entry, whereas Gab1 alleles unable to recruit all SH2 proteins known to bind wild-type Gab1 inhibited internalization. Further analysis with Gab1 alleles defective in binding individual effectors suggested that recruitment of p85 and CrkII are critical for entry. Consistent with this data, overexpression of wild-type CrkII stimulated bacterial uptake. Experiments with mutant CrkII alleles indicated that both the first and second SH3 domains of this adaptor participate in entry, with the second domain playing the most critical role. Taken together, these findings demonstrate novel roles for Gab1 and CrkII in Listeria internalization.     

Mutational analysis of the mitochondrial 12S rRNA and tRNASer(UCN) genes in Tunisian patients with nonsyndromic hearing loss

We explored the mitochondrial 12S rRNA and the tRNASer(UCN) genes in 100 Tunisian families affected with NSHL and in 100 control individuals. We identified the mitochondrial A1555G mutation in one out of these 100 families and not in the 100 control individuals. Members of this family harbouring the A1555G mutation showed phenotypic heterogeneity which could be explained by an eventual nuclear-mitochondrial interaction. So, we have screened three nuclear genes: GJB2, GJB3, and GJB6 but we have not found correlation between the phenotypic heterogeneity and variants detected in these genes. We explored also the entire mitochondrial 12S rRNA and the tRNASer(UCN) genes. We detected five novel polymorphisms: T742C, T794A, A813G, C868T, and C954T, and 12 known polymorphisms in the mitochondrial 12S rRNA gene. None of the 100 families or the 100 controls were found to carry mutations in the tRNASer(UCN) gene. We report here the first mutational screening of the mitochondrial 12S rRNA and the tRNASer(UCN) genes in the Tunisian population which describes the second family harbouring the A1555G mutation in Africa and reveals novel polymorphisms in the mitochondrial 12S rRNA gene.     

Contribution of PTPN22, CD28, CTLA-4 and ZAP-70 variants to the risk of type 1 diabetes in Tunisians

Type 1 diabetes (T1D) is caused by an immune-mediated destruction of the insulin-producing β-cells. Several studies support the involvement of T cell activation molecules. In order to underline the role of the genes involved in this pathway, we investigated, using the Sequenom MassARRAY platform, polymorphisms of sixteen single-nucleotide polymorphisms (SNPs) belonging to PTPN22, CD28, CTLA-4, and ZAP-70 genes in 76 T1D patients and 162 unrelated healthy controls from Southern Tunisia.     

Effect of IL-6 trans-signaling on the pro-remodeling phenotype of airway smooth muscle

Increased levels of IL-6 are documented in asthma, but its contribution to the pathology is unknown. Asthma is characterized by airway wall thickening due to increased extracellular matrix deposition, inflammation, angiogenesis, and airway smooth muscle (ASM) mass. IL-6 binds to a specific membrane-bound receptor, IL-6 receptor-alpha (mIL-6Ralpha), and subsequently to the signaling protein gp130. Alternatively, IL-6 can bind to soluble IL-6 recpetor-alpha (sIL-6Ralpha) to stimulate membrane receptor-deficient cells, a process called trans-signaling. We discovered that primary human ASM cells do not express mIL-6Ralpha and, therefore, investigated the effect of IL-6 trans-signaling on the pro-remodeling phenotype of ASM. ASM required sIL-6Ralpha to activate signal transducer and activator 3, with no differences observed between cells from asthmatic subjects compared with controls. Further analysis revealed that IL-6 alone or with sIL-6Ralpha did not induce release of matrix-stimulating factors (including connective tissue growth factor, fibronectin, or integrins) and had no effect on mast cell adhesion to ASM or ASM proliferation. However, in the presence of sIL-6Ralpha, IL-6 increased eotaxin and VEGF release and may thereby contribute to local inflammation and vessel expansion in airway walls of asthmatic subjects. As levels of sIL-6Ralpha are increased in asthma, this demonstration of IL-6 trans-signaling in ASM has relevance to the development of airway remodeling.     

MAGUKs: beyond ionic channel anchoring

A family of anchoring proteins named MAGUK (for membrane associated guanylate kinase) has emerged as a key element in the organization of protein complexes in specialized membrane regions. These proteins are characterized by the presence of multipe protein-protein interaction domains including PDZ and SH3 domains. The MAGUK family comprises the post-synaptic density 95 (PSD-95) protein and closely related molecules such as chapsyn-110, synapse-associated protein 102 (SAP-102), and SAP-97. These are located either on the pre- and/or post-synaptic sides of synapses or at cell-cell adhesion sites of epithelial cells. MAGUK proteins interact with glutamate receptors and various ionic channels. For instance, an interaction has been reported between the first two PDZ domains of MAGUK proteins and several channels via a consensus sequence Thr/Ser-X-Val/Leu usually located at their carboxy terminus. The role of these anchoring proteins in channel function is not fully understood. MAGUK proteins enhance the current density by increasing the number of functional channels to the sarcolemma. They can also facilitate signaling between channels and several enzymes or G protein-dependent signaling pathways. In the heart also, MAGUK proteins are abundantly expressed and they interact with various channels including Shaker Kv1.5 and connexins.