We have investigated mechanisms involved in integrin-mediated signal transduction platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized adhesions fibroblasts, phosphorylated on normal Src-transformed fibroblasts. show that thrombin collagen causes an induction molecules isolated from activated display enhanced levels immune-complex assays....

Agonist-induced platelet activation causes fibrinogen binding to integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) and aggregation. This is associated with the phosphorylation of specific proteins on tyrosine residues. Since immobilized a solid matrix can bind GP IIb-IIIa without need for exogenous agonists, we examined whether adhesion induces phosphorylation. Platelets adhered in IIb-IIIa-dependent manner assumed spread morphology. change cell shape was multiple proteins, most...

Chicken integrin beta 1 cDNA and its site-directed mutants were cloned into a mammalian expression vector introduced mouse NIH 3T3 cells. Stable transfectants expressing the chicken subunit or identified by immunostaining with antibodies specific for subunit. The proteins expressed predominately in endoplasmic reticulum of to lesser degree plasma membrane. Immunoblots immunoprecipitations, using anti-chicken antibodies, revealed three different sizes (90, 95, 120 kD) 140-kD alpha...

Abstract FAK is a focal adhesion kinase that phosphorylated on tyrosine in activated platelets. Induction of phosphorylation requires both fibrinogen binding to integrin alpha IIb beta 3 and post-occupancy events during agonist-induced platelet aggregation or spreading matrix. To identify the signaling pathways necessary for FAK, we have examined conditions stimulate inhibit this platelets which were induced directly with an anti-beta Fab fragment (anti-LIBS6). Apyrase was added prevent...

α-Actinin is tyrosine-phosphorylated in activated human platelets (Izaguirre, G., Aguirre, L., Ji, P., Aneskievich, B., and Haimovich, B. (1999) J. Biol. Chem. 274, 37012–37020). Analysis of platelet RNA by reverse transcription-polymerase chain reaction revealed that α-actinin expressed identical to the cytoskeletal/non-muscle isoform. A construct this isoform containing a His6 tag at amino terminus was generated. Robust tyrosine phosphorylation recombinant protein detected cells treated...

Objectives: The intravenous administration of a bolus dose endotoxin to healthy human subjects triggers acute systemic inflammatory responses that include cytokine production and dynamic changes in gene expression peripheral blood leukocytes. This study sought determine the state clock leukocytes, leukocyte subpopulations, challenged with vivo at two circadian/diurnal phases clock. Design: Clinical laboratory investigation. Setting: University-based research clinical center. Subjects: Human...

Endotoxin is a potent inducer of systemic inflammatory responses in human and rodents. Here, we show that vivo endotoxin triggers rapid transient decline ATP concentration peripheral blood leukocytes murine liver, which associated with brief increase expression the autophagy indicator LC3-II. In both these tissues, reaches nadir, induced between 2 4 h post-endotoxin infusion, homeostasis restored within 12 h. Mouse liver SIRT1 AMP-activated protein kinase (AMPK) levels precipitously 10 min...

Two recent, independent, studies conducted novel metabolomics analyses relevant to human sepsis progression; one was a model of endotoxin (lipopolysaccharide (LPS)) challenge (experimental endotoxemia) and the other community acquired pneumonia outcome diagnostic study (CAPSOD). The purpose present assess concordance metabolic responses LPS community-acquired sepsis.We tested hypothesis that patterns response elicited by would agree with those in clinical sepsis. Alterations plasma...

Helicobacter pylori colonizes the human stomach, contributing to or causing several diseases. Translocation of CagA bacterial protein into gastric epithelial cells has been linked an increased risk peptic ulcer disease and carcinoma. Upon translocation, is tyrosine phosphorylated by Src family kinases (SFKs), which themselves become inactivated via a negative feedback loop. Here, we show that tyrosine-phosphorylated disrupts adhesion AGS extracellular matrix. Owing inactivation c-Src...

Vinculin is a conserved actin binding protein localized in focal adhesions and cell-cell junctions. Here, we report that vinculin tyrosine phosphorylated platelets spread on fibrinogen the phosphorylation Src kinases dependent. The of was reconstituted vanadate treated COS-7 cells coexpressing c-Src. sites were mapped to residues 100 1065. A phosphorylation-specific antibody directed against residue 1065 reacted with platelet but failed react from unstimulated lysates. Tyrosine located tail...

Five monoclonal antibodies specific for the 260 kDa subunit of rat skeletal muscle sodium channel were used to probe distribution this in adult muscle. All reacted with surface membrane fast- and slow-twitch fibers anterior tibial soleus muscles. Immunoreactivity was also present endplate region; significantly more intense than that surrounding extrajunctional membrane. At electron microscopic level, junctional immunoreactivity could be traced uniformly throughout secondary folds...

Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer adhesion by a beta(1)-integrin-dependent mechanism requires an intact cytoskeleton activation focal kinase (FAK) Src. alpha-Actinin facilitates formation physically links integrin-associated complexes with the cytoskeleton. We therefore hypothesized alpha-actinin may necessary for...

Platelets express a single low affinity receptor for immunoglobulin, FcγRII, that triggers multiple cellular responses upon interaction with multivalent immune complexes. In this study we show immobilized IgG is also potent stimulant of platelet activation triggering adhesion, aggregation, massive dense granule secretion, and thromboxane production. Platelet adhesion to was blocked by the FcγRII receptor-specific monoclonal antibody, IV.3. Pretreatment platelets cytochalasin D inhibit actin...

Antibodies were raised in rabbits against the purified voltage-dependent sodium channel from rat skeletal muscle sarcolemma. The resultant antiserum reacted with a solid-phase radioimmunoassay and precipitated crude mixture of solubilized membrane proteins. Crude proteins separated according to size under nondenaturing conditions by chromatography on Sepharose CL-6B contained single peak immunoreactivity that coincided native channel. On immunoblots sarcolemmal proteins, predominantly...

Leukocyte signaling in patients with systemic insulin resistance is largely unexplored. We recently discovered the presence of multiple Toll-like receptor 4 (TLR4) intermediates leukocytes from type 2 diabetes or acute associated cardiopulmonary bypass surgery. extend this work to show that addition matrix metalloproteinase 9, hypoxia-inducible factor 1α, and cleaved AMPKα, patient also express IRS-1 phosphorylated on Ser(312), Akt Thr(308), elevated TLR4 expression. Similar were detected...

Abstract LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPKα HIF-1α expression in leukocytes. In human leukocytes, more specifically neutrophils, yields 55- 35-kDa protein fragments. this study, we address the mechanism by which is cleaved its relevance to health. Our data indicate that linked MMP9 both are required for mammalian target rapamycin complex-1 S6K1 LPS-stimulated mice Three key observations support conclusion. First, no changes...

Shigella flexneri is a gram-negative bacterium that causes bacillary dysentery in humans characterized by an acute inflammatory response of the colon. The fate phagocytes are infected vitro with virulent has been subject some investigation and debate. In this study we found caused rapid increase cell membrane permeability human monocyte-derived macrophages (HMDM) but not monocytes, as demonstrated uptake fluorescent vital dyes. Within 2 h infection, 59% +/- 6% HMDM </=4% monocytes were...

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates binding site for Src family kinases. phosphorylates the cytoskeletal protein alpha-actinin Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) an phosphatase. PTP 1B-dependent dephosphorylation was seen in COS-7 cells 1B-null fibroblasts reconstituted with 1B. Furthermore, show coexpression wild-type causes FAK. No observed coexpressing phosphorylation...

A sequential extraction procedure of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS) buffer followed by RIPA or Laemmli sample was developed to define two distinct subpopulations beta-1 integrins in primary chicken embryo fibroblasts. Extraction cells culture revealed an association adhesion plaque-localized integrin with the CHAPS-insoluble fraction. Phosphorylated were found both fractions, but specific phosphorylation 12-fold higher CHAPS insoluble The evenly...

Purpose Ischemia/reperfusion (I/R) during partial nephrectomy (PN) contributes to acute kidney injury (AKI), which is inaccurately assessed using existent clinical markers of renal function. We evaluated I/R-related changes in expression hypoxia inducible factor 1α (HIF-1α) and toll-like receptor 4 (TLR4), within tissue peripheral blood leukocytes (PBL) a porcine model PN. Materials Methods Three adult pigs each underwent unilateral hilar cross clamping for 180 min followed by 15...

An endotoxin challenge, sepsis, and injury/trauma, trigger significant changes in human peripheral blood leukocytes (PBL) gene expression. In this study, we have sought to test the hypothesis that Toll-like receptor 4 (TLR4) induced transcription patterns elicited humans exposed vivo would parallel expression observed trauma patients with initial non-infectious injury. addition, identify functional modules are commonly affected by these two insults of differing magnitude duration. PBL were...