Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity and appear to act by coupling particular forms of FGF to appropriate FGF receptors, During neural development, one particular HS proteoglycan is able to rapidly switch its potentiating activity from FGF-2, as neural precursor cell proliferation occurs, to FGF-1, as neuronal differentiation occurs, Using various analytical techniques, including chemical and enzymatic cleavage, low pressure chromatography, and strong anion-exchange high performance Liquid chromatography, we have analyzed the different HSs expressed during these crucial developmental stages, There are distinct alterations in patterns of 6-O-sulfation, total chain length, and the number of sulfated domains of the HS from the more mature embryonic brain, These changes correlate with a switch in the ability of the HS to potentiate the actions of FGF-1 in triggering: cell differentiation It thus appears that each HS pool is designed to function in the modulation of an intricate interaction with a specific growth factor and its cognate receptor, and suggests tightly regulated expression of specific, bioactive disaccharide sequences, The data can be used to construct a simple model of controlled variations in HS chain structure which have functional consequences at a crucial stage of neuronal maturation

The association between blood glucose and oxidized lipoprotein(a) in healthy young women

<p>Abstract</p> <p>Background</p> <p>Oxidized lipoproteins play important roles in the atherosclerotic processes. Oxidized lipoprotein(a) (oxLp(a)) may be more potent in atherosclerotic pathophysiology than native Lp(a), a cardiovascular disease-relevant lipoprotein. Increased blood glucose concentrations can induce oxidative modification of lipoproteins. The aim of this study was to investigate the association between circulating oxLp(a) and cardiometabolic variables including blood glucose in healthy volunteers within the normal range of blood glucose.</p> <p>Methods</p> <p>Several cardiometabolic variables and serum oxLp(a) (using an ELISA system) were measured among 70 healthy females (mean age, 22 years).</p> <p>Results</p> <p>Lp(a) and glucose were significantly and positively correlated with oxLp(a) in simple correlation test. Furthermore, a multiple linear regression analysis showed oxLp(a) to have a weakly, but significantly positive and independent correlation with only blood glucose (<it>β </it>= 0.269, <it>P </it>< 0.05).</p> <p>Conclusions</p> <p>These results suggest that increased glucose may enhance the oxidization of Lp(a) even at normal glucose levels.</p

The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85

The Slp65 adaptor molecule is important for B-cell receptor signalling. Here, the interactomes of SLP65 in resting and activated B cells are resolved by mass spectrometry. A number of SLP65 interacting partners, including CIN85, continuously associate with SLP65 in a stimulation-independent manner. CIN85 recruits SLP65 to the membrane and it is required for SLP65 phosphorylation

Regulation of Cytokine Signaling by B Cell Antigen Receptor and Cd40-Controlled Expression of Heparan Sulfate Proteoglycans

Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell–derived factor (SDF)-1α, even though the affinities of HGF and SDF-1α for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation

REGULATION OF MEMBRANE BAND 3 TYR-PHOSPHORYLATION BY PROTEOLYSIS OF P72 AND POSSIBLE INVOLVEMENT IN SENESCENCE PROCESS

Erythrocyte senescence is characterized by exposure of cell surface epitopes on cell membrane proteins leading to immune mediated removal of red blood cells. One mechanism for antigen formation is tyrosine phosphorylation (Tyr-P) of the transmembrane protein band 3 by Syk kinase. Our aim was to test the hypothesis that proteolytic activation of Syk kinase by conversion from 72 kDa (p72Syk) to the 36 kDa (p36Syk) isoform enhances its phosphorylating activity independently of the association of Syk kinase with the cytoskeleton. Tyr-P assay was conducted using quantification of 32P uptake into the cytoplasmic domain of band 3 after addition of p72Syk or p36Syk. Effect of prephosphorylation of erythrocyte membrane band 3 protein by p36Syk on p72Syk-mediated phosphorylation and the effect of addition of a protease inhibitor (leupeptin) on p72Syk-mediated phosphorylation were studied by autoradiographic visualization of 32P uptake. Tyr-P by Syk isoforms of membrane skeletal and soluble fractions of band 3 was visualized by immunoblotting. It was found that p36Syk had a higher band 3 tyrosine phosphorylating activity compared with p72Syk. Pre-phosphorylation with p36Syk or p72Syk increased band 3 phosphorylating activity. Protease inhibition treatment reduced p72Syk but not p36Syk band 3 tyrosine phosphorylating activity significantly. Both soluble and membrane skeletal fractions of band 3 protein were equally tyrosine phosphorylated by each Syk isoform. In conclusion, we confirmed the hypothesis that proteolytic cleavage of p72Syk is an important regulatory step for band 3 Tyr-P and its independence of the association of band 3 with the cytoskeleton

HGF/SF and its receptor c-MET play a minor role in the dissemination of human B-lymphoma cells in SCID mice

The MET protooncogene, c-MET, encodes a cell surface tyrosine kinase receptor. The ligand for c-MET is hepatocyte growth factor (HGF), also known as scatter factor (SF), which is known to affect proliferation and motility of primarily epithelial cells. Recently, HGF/SF was also shown to affect haemopoiesis. Studies with epithelial and transfected NIH3T3 cells indicated that the HGF/SF–c-MET interaction promotes invasion in vitro and in vivo. We previously demonstrated that HGF/SF induces adhesion of c-MET-positive B-lymphoma cells to extracellular matrix molecules, and promoted migration and invasion in in vitro assays. Here, the effect of HGF/SF on tumorigenicity of c-MET-positive and c-MET-negative human B-lymphoma cell lines was studied in C.B-17 scid/scid (severe combined immune deficient) mice. Intravenously (i.v.) injected c-MET-positive (BJAB) as well as c-MET-negative (Daudi and Ramos cells) B-lymphoma cells formed tumours in SCID mice. The B-lymphoma cells invaded different organs, such as liver, kidney, lymph nodes, lung, gonads and the central nervous system. We assessed the effect of human HGF/SF on the dissemination of the B-lymphoma cells and found that administration of 5 μg HGF/SF to mice, injected (i.v.) with c-MET-positive lymphoma cells, significantly (P = 0.018) increased the number of metastases in lung, liver and lymph nodes. In addition, HGF/SF did not significantly influence dissemination of c-MET-negative lymphoma cells (P = 0.350 with Daudi cells and P = 0.353 with Ramos cells). Thus the effect of administration of HGF/SF on invasion of lymphoma cells is not an indirect one, e.g. via an effect on endothelial cells. Finally, we investigated the effect of HGF/SF on dissemination of c-MET-transduced Ramos cells. In response to HGF/SF, c-MET-transduced Ramos cells showed an increased migration through Matrigel in Boyden chambers compared to wild-type and control-transduced Ramos cells. The dissemination pattern of c-MET-transduced cells did not differ from control cells in in vivo experiments using SCID mice. Also no effect of HGF/SF administration could be documented, in contrast to the in vitro experiments. From our experiments can be concluded that the HGF/SF–c-MET interaction only plays a minor role in the dissemination of human B-lymphoma cells. © 1999 Cancer Research Campaig

GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation

The Listeria monocytogenes protein InlB promotes intracellular invasion by activating the receptor tyrosine kinase Met. Earlier studies have indicated that the LRR fragment of InlB is sufficient for Met activation, but we show that this is not the case unless the LRR fragment is artificially dimerized through a disulphide bond. In contrast, activation of Met proceeds through monomers of intact InlB and, at physiologically relevant concentrations, requires coordinated action in cis of both InlB N-terminal LRR region and C-terminal GW domains. The GW domains are shown to be crucial for potentiating Met activation and inducing intracellular invasion, with these effects depending on association between GW domains and glycosaminoglycans. Glycosaminoglycans do not alter the monomeric state of InlB, and are likely to enhance Met activation through a receptor-mediated mode, as opposed to the ligand-mediated mode observed for the LRR fragment. Surprisingly, we find that gC1q-R, a host protein implicated in InlB-mediated invasion, specifically antagonizes rather than enhances InlB signalling, and that interaction between InlB and gC1q-R is unnecessary for bacterial invasion. Lastly, we demonstrate that HGF, the endogenous ligand of Met, substitutes for InlB in promoting intracellular invasion, suggesting that no special properties are required of InlB in invasion besides its hormone-like mimicry of HGF

Purification and characterization of a protein-tyrosine kinase from lymphocytes

The major NaCl-stimulated protein-tyrosine kinase activity has been isolated from a soluble extract of bovine thymus based on its ability to phosphorylate the tyrosine-containing peptide angiotensin I. The enzyme was purified by sequential column chromatography on DEAE-cellulose, heparin-agarose, casein-agarose, butyl-agarose, and Sephadex G-75. The purified enzyme (p40) is a monomer of 40,000 Daltons. The p40 kinase contains an ATP-binding site as shown by its specific labeling with photoaffinity analogs of ATP. Furthermore, the kinase catalyzes an intramolecular autophosphorylation reaction that leads to the modification of tyrosine residues. Polyclonal antisera raised against p40 cross-react with a 72 kDa protein-tyrosine kinase (p72) from spleen and thymus. Like p40 the 72 kDa protein is phosphorylated on tyrosine in vitro and phosphopeptide maps indicate that the proteins are also structurally related. The following observations suggest a precursor-product relationship between p72 and p40. The recovery of p40 from spleen homogenates is reduced, while the recovery of p72, is enhanced if high concentrations of leupeptin or soybean trypsin inhibitor are added to the homogenization buffer. The generation of p40 in spleen homogenates occurs with a concomitant increase in protein-tyrosine kinase activity. Activated catalytic fragments of 38-43 kDa can be generated by the treatment of partially purified p72 with exogenously added proteases. Western blotting studies reveal the highest levels of p72 are in spleen, thymus and lung, tissues that also have high protein-tyrosine kinase activity and generate high levels of p40 after homogenization. Consistent with this, we have observed high levels of p72 in certain B and T cell-derived cell lines. The presence of immunoreactive p72 in NIH3T3 cells suggests that the kinase may be expressed in cells other than those of hematopoietic origin. p40 is of higher specific activity and has been more stringently characterized than its partially purified precursor (p72), however, preliminary experiments reveal many similarities between the two enzymes. For example, both kinases strongly prefer Mn\sp {+2} over Mg\sp {+2} in in vitro autophosphorylation reactions, show an enhanced ability to phosphorylate angiotensin I in the presence of high concentrations of NaCl and exhibit the same preference for substrates, with the cytoplasmic domain of erythrocyte band 3 serving as the best substrate for both kinases