Association of annexin 2 with recycling endosomes requires either calcium- or cholesterol-stabilized membrane domains.

Annexin 2 is a Ca2+- and phospholipid-binding protein previously identified on endosomal membranes and the plasma membrane. Inferred from this location and its stimulatory effect on membrane transport annexin 2 has been proposed to play a role in the structural organization and dynamics of endosomal membranes. Validation of this view requires a detailed analysis of the distribution of annexin 2 over the endosomal compartment and a characterization of the parameters governing this distribution.

Transient tyrosine phosphorylation of human ryanodine receptor upon T cell stimulation.

The ryanodine receptor of Jurkat T lymphocytes was phosphorylated on tyrosine residues upon stimulation of the cells via the T cell receptor/CD3 complex. The tyrosine phosphorylation was transient, reaching a maximum at 2 min, and rapidly declined thereafter. In co-immunoprecipitates of the ryanodine receptor, the tyrosine kinases p56(lck) and p59(fyn) were detected.

Complex formation and submembranous localization of annexin 2 and S100A10 in live HepG2 cells.

The Ca(2+) and membrane binding protein annexin 2 can form a heterotetrameric complex with the S100A10 protein and this complex is thought to serve a bridging or scaffolding function in the membrane underlying cytoskeleton. To elucidate which of the subunits targets the complex to the subplasmalemmal region in live cells we employed YFP/CFP fusion proteins and live cell imaging in HepG2 cells. We show that monomeric annexin 2 is targeted to the plasma membrane whereas non-complexed S100A10 acquires a general cytosolic distribution.

Endothelial intracellular Ca2+ release following monocyte adhesion is required for the transendothelial migration of monocytes.

Although molecular changes accompanying leukocyte extravasation have been investigated intensively, the particular events following leukocyte adhesion and leading to the actual transendothelial migration process remain largely unknown. To characterize intraendothelial signals elicited by leukocyte adhesion and functionally required for their transmigration, we recorded endothelial free cytosolic intracellular Ca(2+)levels ([Ca(2+)]i) during the course of leukocyte adhesion. We show that monocyte and granulocyte adhesion induced Ca(2+)transients in either untreated or TNF-alpha-stimulated microvascular endothelial cells (HMEC-1).

BRCA1 and BRCA2 mutation status and cancer family history of Danish women affected with multifocal or bilateral breast cancer at a young age.

Introduction: A small fraction of breast cancer is the result of germline mutations in the BRCA1 and BRCA2 cancer susceptibility genes. Mutation carriers frequently have a positive family history of breast and ovarian cancer, are often diagnosed at a young age, and may have a higher incidence of double or multiple primary breast tumours than breast cancer patients in general.

Objectives: To estimate the prevalence and spectrum of BRCA1 and BRCA2 mutations in young Danish patients affected with bilateral or multifocal breast cancer and to determine the relationship of mutation status to family history of cancer.

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins.

Annexin II is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca(2+) in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes.

Intracellular Ca(2+) release mechanisms: multiple pathways having multiple functions within the same cell type?

The elevation of the cytosolic and nuclear Ca(2+) concentration is a fundamental signal transduction mechanism in almost all eukaryotic cells. Interestingly, three Ca(2+)-mobilising second messengers, D-myo-inositol 1,4,5-trisphosphate (InsP(3)), cyclic adenosine diphosphoribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP(+)) were identified in a phylogenetically wide range of different organisms. Moreover, in an as yet very limited number of cell types, sea urchin eggs, mouse pancreatic acinar cells, and human Jurkat T-lymphocytes, all three Ca(2+)-mobilising ligands have been shown to be involved in the generation of Ca(2+) signals.

Intact Ca(2+)-binding sites are required for targeting of annexin 1 to endosomal membranes in living HeLa cells.

Annexin 1 is a Ca(2+)-regulated membrane binding protein and a major substrate of the epidermal growth factor receptor kinase. Because of its properties and intracellular distribution, the protein has been implicated in endocytic trafficking of the receptor, in particular in receptor sorting occurring in multivesicular endosomes. Up to now, however, the localization of annexin 1 to cellular membranes has been limited to subcellular fractionation and immunocytochemical analyses of fixed cells.

Identification of hydrophobic amino acid residues involved in the formation of S100P homodimers in vivo.

S100 proteins are small dimeric members of the EF-hand superfamily of Ca(2+) binding proteins thought to participate in mediating intracellular Ca(2+) signals by binding to and thereby regulating target proteins in a Ca(2+)-dependent manner. As dimer formation is crucial to S100 function, we applied a yeast two-hybrid approach in analyzing in vivo molecular aspects of S100 dimerization. We chose S100P, a member of the S100 family highly expressed in placenta, for detailed analysis and showed that S100P monomers strongly interact with one another but not with other S100 polypeptides, indicating that homodimer formation is obligatory for S100P.

A novel ligand of the formyl peptide receptor: annexin I regulates neutrophil extravasation by interacting with the FPR.

The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner.

Cyclic ADP-ribose.

The Ca2+-mobilizing natural compound cyclic ADP-ribose was discovered in sea urchin egg homogenates. Recently the involvement of cyclic ADP-ribose in Ca2+ signaling has been demonstrated in diverse biological systems spanning protozoa, plants, and cells from invertebrate, mammalian, and human sources. ADP-ribosyl cyclases synthesize cyclic ADP-ribose.

Plasma and urinary serotonin and 5-hydroxyindol-3-acetic acid in adults with migraine and tension-type headache.

Each headache can be a complex diagnostic, therapeutic, prognostic and social problem. The pain in the head can be connected with many organic and non-organic causes. In this work, the levels of plasma and urinary free 5-HT and 5-HIAA were investigated in eight migraine (aged 23-59 years) and ten tension-type headache suffers (aged 38-61 years).

Activation of the plasma kallikrein/kinin system on endothelial cell membranes.

For more than three decades, it has been known that the plasma kallikrein/kinin system becomes activated when exposed to artificial, negatively charged surfaces. The existence of an encompassing in vivo, negatively charged surface capable of activation of the plasma kallikrein/kinin system has, however, never been convincingly demonstrated. In this report, we describe current knowledge on how the proteins of the plasma kallikrein/kinin system assemble to become activated on cell membranes.

Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation.

GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man. All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins. The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated.

Activation of the plasma kallikrein/kinin system on endothelial cells.

For more than two decades, it has been known that activation of the plasma kallikrein/kinin system only occurs when it is exposed to artificial, negatively charged surfaces. The existence of physiological, negatively charged surfaces has, however, never been demonstrated in vivo. In this report, we describe current knowledge about how the proteins of the plasma kallikrein/kinin system interact with and become activated on cell membranes.

Interferon gamma regulates a unique set of proteins in fresh human bladder transitional cell carcinomas.

Poly(A) mRNA was isolated from human placental trophoblast cells stimulated with 100 U/mL of interleukin-2 and 5 microg/mL of phytohemagglutinin and reverse-transcribed. The cDNA coding for the mature interferon-gamma (IFN-gamma) protein was amplified using specific primers, cloned into the pGEX-4T2 vector, and expressed in Escherichia coli. Treatment of four fresh bladder transitional cell carcinoma (TCC) biopsies (TCCs 845-1, grade II, Ta; TCC 925-1, grade II, Ta; TCC 919-1, grade III, T1; TCC 950-1, grade III, T1) with the purified recombinant trophoblast IFN-gamma (50 U/mL, 20 h), followed by proteome analysis using two-dimensional gel electrophoresis, revealed several major proteins whose level of expression were affected by this cytokine.

Phospholipid vesicle binding and aggregation by four novel fish annexins are differently regulated by Ca2+.

Four members of the annexin family, herein referred to as max (for medaka annexin) 1-4, have recently been identified through hybridization cloning in the killifish Oryzias latipes (D. Osterloh, J. Wittbrodt and V.

Characterization and developmentally regulated expression of four annexins in the killifish medaka.

Annexins are Ca2+-regulated membrane binding proteins implicated in a wide range of membrane-related and signal transduction events, including the endocytosis of membrane receptors and Ca2+-regulated as well as constitutive secretion. To date, 10 unique members of this multigene family have been identified in a variety of cell types and tissues of higher vertebrates, with different members showing distinct tissue distributions in the adult organisms. To establish whether annexins also function in embryonic development, we analyzed the expression pattern during vertebrate morphogenesis using the medaka fish Oryzias latipes as a model system.

Hydrophobic residues in the C-terminal region of S100A1 are essential for target protein binding but not for dimerization.

S100 proteins are a family of small dimeric proteins characterized by two EF hand type Ca2+ binding motifs which are flanked by unique N- and C-terminal regions. Although shown unequivocally in only a few cases S100 proteins are thought to function by binding to, and thereby regulating, cellular target proteins in a Ca2+ dependent manner. To describe for one member of the family, S100A1, structural requirements underlying target protein binding, we generated specifically mutated S100A1 derivatives and characterized their interaction with the alpha subunit of the actin capping protein CapZ shown here to represent a direct binding partner for S100A1.

Paraoxonase PON1 polymorphism leu-Met54 is associated with carotid atherosclerosis: results of the Austrian Stroke Prevention Study.

Background And Purpose: Genetic polymorphism at the paraoxonase locus is associated with serum concentration and activity of paraoxonase and with increased risk for coronary heart disease. Two frequent polymorphisms present at the paraoxonase gene are the methionine (M allele) leucine (L allele) interchange at position 54 and the arginine (B allele) glutamine (A allele) interchange at position 191. This is the first study to determine the effect of these polymorphisms on carotid atherosclerosis.

Disruption of endothelial microfilaments selectively reduces the transendothelial migration of monocytes.

The transendothelial migration of leukocytes (diapedesis) is a central event in inflammatory and immunological processes. Although leukocyte-endothelium interactions occurring during diapedesis have been investigated intensively, little is known about the actual transmigration and the molecular mechanisms involved. Toward this end we analyzed whether the endothelial cytoskeleton plays a direct role during the transendothelial migration of monocytes.

Retinol-induced secretion of human retinol-binding protein in yeast.

Retinol-binding protein (RBP) functions as a transporter for retinol (vitamin A) in plasma in higher eukaryotes. We have successfully expressed human RBP in Saccharomyces cerevisiae, and its secretion was found to be induced by retinol also in this lower eukaryote. Reduced induction of secretion by retinol in a temperature-sensitive sec18-1 mutant that is blocked in secretion at the restricted temperature suggests that as in mammalian cells, RBP can be released from the endoplasmic reticulum upon addition of retinol.

Beta-fibrinogen gene polymorphism (C148-->T) is associated with carotid atherosclerosis: results of the Austrian Stroke Prevention Study.

Polymorphisms at the beta-fibrinogen locus have been shown to be associated with plasma concentration of fibrinogen and coronary heart disease. The effect of the genetic heterogeneity of fibrinogen on carotid atherosclerosis has not been determined so far. We examined the influence of the C148 --> T polymorphism on carotid disease in a large cohort of middle-aged to elderly subjects without evidence of neuropsychiatric disease.

In vitro interactions of oxidatively modified LDL with type I, II, III, IV, and V collagen, laminin, fibronectin, and poly-D-lysine.

The accumulation of LDL in the arterial intima is considered a key event in atherogenesis. We investigated the binding of oxidized LDL (ox-LDL) to microtiter plates coated with type I or II collagen, laminin, fibronectin, or poly-D-lysine. Oxidation of LDL, 125I-LDL, or Eu(3+)-LDL was performed with CuCl2, varying the time of oxidation.