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Results for “Extracellular Traps”

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PubMed1981

Localization of cell membrane components by surface diffusion into a "trap".

Randomly distributed cell membrane components may become localized toward a specific region of the surface as a results of cell-cell contact or the cell's exposure to extracellular ligands. The mechanism for this localization process is unknown. In the present study, we investigated the plausibility of a passive mechanisms, namely that a local region of the cell surface serves as a "trap" for diffusing membrane proteins. Based on a model of spherical cell with a single circular trap on the surface, we derived the equation describing the surface density distribution and the average lifetime of the trappable molecules in the trap-free region of the membrane. This surface-trap theory was then used to analyze our experimental finding on the rapid localization of muscle surface soybean agglutinin receptors induced by cell-cell contact in culture. THe result indicates that the rate of localization of these receptors toward the cell-cell contact site can be accounted for by assuming that the receptors possess a diffusion coefficient of about 2.5 X10(-9) cm2/s (range: 1.2-9.3X10(-9) cm2/s) before they are trapped at the contact site. Independent measurement of the rate of lateral diffusion of these receptors yielded a lateral diffusion coefficient of about 1.9 X 10(-9) cm2/s (range 1.2-2.7 X10(-9) cm2/s), a value within the range of that predicted by the rate of localization. We thus conclude that lateral diffusion of mobile membrane components toward a local trap is a plausible mechanism for their localization induced by local surface modulation.

Animals↗

PubMed1994

Copper resistance mechanisms in bacteria and fungi.

Copper is both an essential micronutrient and a toxic heavy metal for most living cells. The presence of high concentrations of cupric ions in the environment promotes the selection of microorganisms possessing genetic determinants for copper resistance. Several examples of chromosomal and plasmid copper-resistance systems in bacteria have been reported, and the mechanisms of resistance have started to be understood at the molecular level. Bacterial mechanisms of copper resistance are related to reduced copper transport, enhanced efflux of cupric ions, or copper complexation by cell components. Copper tolerance in fungi has also been ascribed to diverse mechanisms involving trapping of the metal by cell-wall components, altered uptake of copper, extracellular chelation or precipitation by secreted metabolites, and intracellular complexing by metallothioneins and phytochelatins; only the metallothionein chelation mechanism has been approached with molecular detail.

Bacteria↗

PubMed2005

Myocarditis and sacroiliitis: 2 previously unrecognized manifestations of tumor necrosis factor receptor associated periodic syndrome.

Tumor necrosis factor receptor associated periodic syndrome (TRAPS) is an autosomic-dominant periodic syndrome associated with mutations in the extracellular domain of the 55 kDa TNF receptor. Clinically, episodes of severe myalgia, arthralgia/arthritis, sterile peritonitis, scrotal inflammation, serositis, migratory rash, conjunctivitis, and recurrent fever are characteristic. We describe a 9-year-old African American boy with the P46L mutation of the TNF receptor who presented with 2 previously unrecognized manifestations: sacroiliitis and myocardiopathy, both showing a reversible course.

Arthritis↗

PubMed1987

A pharmacological approach to thromboxane receptor antagonism.

Thromboxane A2 (TxA2) appears to be an important mediator of ischemia and hypoxia. Despite its short half-life and the fact that it may not circulate in the blood until its values become quite high, TxA2 contributes to the pathogenesis of cardiopulmonary diseases (e.g., sudden death, myocardial ischemia, circulatory shock). It does so because it propagates its own formation by activating platelets and constricting blood vessels, thus activating more TxA2 and trapping it locally within an ischemic or hypoxic region. TxA2 concentrations in the extracellular fluid of lymph of ischemic regions may be much higher than that occurring in nonischemic, normally perfused regions. Specific and potent Tx receptor antagonists (TxRA) have recently become available for study. The TxRA are useful tools in the study of the pathophysiology of Tx-dependent disease processes and have been found to be effective in a variety of ischemic disorders including circulatory shock, myocardial ischemia, and sudden cardiopulmonary death. Moreover, inasmuch as early work indicates that these agents are both safe and effective in humans, Tx receptor antagonists may be employed as therapeutic agents in several cardiovascular disease states. Further investigation is necessary to clarify the role of TxRA as therapeutic agents.

Animals↗

PubMed1990

Extracellular ATP-induced production of hydrogen peroxide in porcine thyroid cells.

Hydrogen peroxide (H2O2) is an essential substrate for the peroxidase reaction in thyroid hormone biosynthesis. We demonstrated the production of H2O2 from porcine thyroid cells stimulated with extracellular ATP, using a scopoletin-horseradish peroxidase (HRP) system. Incubation of isolated cells for 1 day in the presence of 10% (v/v) newborn calf serum was necessary for the detection of induction by ATP of H2O2 production. The rate of H2O2 production induced by the addition of ATP increased in a dose-dependent manner, and the concentration of ATP required for half-maximum stimulation was about 10 mumol/l. ADP and GTP were also effective, but only at higher concentrations than ATP. In the absence of extracellular Ca2+, the production rate was very low. Production of H2O2 from thyroid cells was also measured by a method which discriminated between H2O2 and superoxide anion (O2-); in this, diacetyldeuteroheme-substituted HRP was employed as the trapping agent for both O2 metabolites. The thyroid cells produced H2O2, but not O2-, when the cells were stimulated by extracellular ATP.

Adenosine Triphosphate↗

PubMed1995

A method to trap IgG antibodies within functional membrane vesicles.

Monoclonal antibodies have been used extensively to study the roles of specific extracellular proteins in cellular activation. Due to the large size of these molecules, their use for the study of receptor-mediated activation of intracellular processes has been limited. This report describes a method to introduce whole IgG antibody molecules into large functional membrane vesicles (ghosts) derived from rat basophilic leukemia (RBL) cells. Furthermore, an IgG1 mouse monoclonal antibody (JRK) directed against the cytoplasmic portion of the beta subunit of the high affinity receptor for IgE (Fc epsilon RI) can partially inhibit Fc epsilon RI-mediated PI hydrolysis through modest effects on both basal and receptor-mediated activation of phospholipase C.

Animals↗

PubMed2006

Gene trap disruption of the mouse heparan sulfate 6-O-endosulfatase gene, Sulf2.

Heparan sulfate (HS) chains are found in the extracellular matrix, covalently linked to core proteins collectively termed heparan sulfate proteoglycans (HSPGs). A wealth of data has demonstrated roles for HSPGs in the regulation of many cell surface signaling pathways that are crucial during development. Variations in the sulfation pattern along the HS chains influence their ability to interact with molecules such as growth factors, chemokines, morphogens, and adhesion molecules. Sulf1 and Sulf2 are members of a class of recently identified genes that encode heparan sulfate 6-O-endosulfatases (Sulf genes). The removal of 6-O-sulfate from HS via SULF activity influences the function of many factors, including Wnt, fibroblast growth factor, hepatocyte growth factor, heparin-binding epidermal growth factor, and bone morphogenetic protein. Given their possible developmental roles, we have examined Sulf gene expression during mouse embryogenesis. The two Sulf genes are expressed in a broad range of tissues throughout development with largely nonoverlapping expression patterns. Sulf2 transcripts are expressed in the lung, heart, placenta, and ribs. We generated a mouse line possessing a gene trap disruption of the Sulf2 gene. Mice homozygous for the Sulf2 gene trap allele are viable and fertile and have no major developmental defects on several genetic backgrounds. However, we observed strain-specific, nonpenetrant defects affecting viability, lung development, and growth in Sulf2 homozygous animals. These data suggest that Sulf2 may have roles in several tissues but that there is compensation by and/or redundancy with Sulf1.

Animals↗

PubMed2002

A novel mutation in the third extracellular domain of the tumor necrosis factor receptor 1 in a Finnish family with autosomal-dominant recurrent fever.

OBJECTIVE: To investigate the presence of TRAPS (tumor necrosis factor receptor-associated periodic syndrome), which is a recently defined, dominantly inherited autoinflammatory syndrome caused by mutations in the tumor necrosis factor receptor superfamily 1A gene (TNFRSF1A, CD120a), in a Finnish family with recurrent fever. METHODS: The TNFRSF1A gene was sequenced in both affected and unaffected family members. Flow cytometry and enzyme-linked immunosorbent assay analyses were used to assess membrane expression and serum levels of the TNFRSF1A protein, respectively. RESULTS: A missense mutation in exon 4, located in the third extracellular domain of TNFRSF1A and resulting in an amino acid substitution (F112I) close to a conserved cysteine, was found in all 4 affected family members and in 1 asymptomatic individual. The mutation was clearly associated with low levels of soluble TNFRSF1A as well as with the clinical symptoms of recurrent fever and abdominal pain. Impaired shedding of TNFRSF1A after phorbol myristate acetate stimulation was detected in blood granulocytes and monocytes from the 3 adult family members with the mutation, but in the child bearing the mutation and showing clinical symptoms of recent onset, the shedding defect was less marked. CONCLUSION: TRAPS should be suspected in any patient who presents with a history of intermittent fever accompanied by unexplained abdominal pain, arthritis, or skin rash, particularly in the presence of a family history of such symptoms. Screening for low serum levels of soluble TNFRSF1A identifies individuals who are likely to have TNFRSF1A mutations.

Adult↗

PubMed2000

Contributions of the extracellular and cytoplasmic domains of platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) in regulating cell-cell localization.

PECAM-1/CD31, a vascular cell adhesion/signaling molecule that has been implicated in a number of vascular functions (including angiogenesis and the transmigration of leukocytes through endothelium) is highly enriched at the cell-cell borders of adjacent endothelial cells. To identify the mechanisms responsible for this localization, a series of PECAM-1 mutants and chimeric PECAM-1 molecules were transfected into non-PECAM-expressing cells and the ability of the constructs to move to cell-cell borders of adjacent cells was determined using immunohistochemistry and confocal microscopy. Although neither the extracellular domain, by itself, nor the cytoplasmic domain, by itself, was sufficient to direct cell-cell localization, the combination of the extracellular and transmembrane domains with a small group of highly charged amino acids in a membrane proximal region of the cytoplasmic domain was sufficient to direct efficient localization of the molecule to cell-cell borders. Importantly, only constructs that supported PECAM-1 mediated adhesion localized to cell-cell borders. Our data are consistent with a 'diffusion trapping' model in which movement of PECAM-1 in the cell membrane occurs relatively freely until the 'stablized' extracellular domain of the molecule encounters its ligand on an adjacent cell. When this occurs, the complex is 'captured' at the cell-cell interface leading to localization at cell-cell borders.

Cell Membrane↗

PubMed2006

The layered fold of the TSR domain of P. falciparum TRAP contains a heparin binding site.

Thrombospondin-related anonymous protein, TRAP, has a critical role in the hepatocyte invasion step of Plasmodium sporozoites, the transmissible form of the parasite causing malaria. The extracellular domains of this sporozoite surface protein interact with hepatocyte surface receptors whereas its intracellular domain acts as a link to the sporozoite actomyosin motor system. Liver heparan sulfate proteoglycans have been identified as potential ligands for TRAP. Proteoglycan binding has been associated with the A- and TSR domains of TRAP. We present the solution NMR structure of the TSR domain of TRAP and a chemical shift mapping study of its heparin binding epitope. The domain has an elongated structure stabilized by an array of tryptophan and arginine residues as well as disulfide bonds. The fold is very similar to those of thrombospondin type-1 (TSP-1) and F-spondin TSRs. The heparin binding site of TRAP-TSR is located in the N-terminal half of the structure, the layered side chains forming an integral part of the site. The smallest heparin fragment capable of binding to TRAP-TSR is a tetrasaccharide.

Animals↗

PubMed1998

Voltage sensor-trapping: enhanced activation of sodium channels by beta-scorpion toxin bound to the S3-S4 loop in domain II.

Polypeptide neurotoxins alter ion channel gating by binding to extracellular receptor sites, even though the voltage sensors are in their S4 transmembrane segments. By analysis of sodium channel chimeras, a beta-scorpion toxin is shown here to negatively shift voltage dependence of activation and enhance closed state inactivation by binding to a receptor site that requires glycine 845 (Gly-845) in the S3-S4 loop at the extracellular end of the S4 segment in domain II of the alpha subunit. Toxin action requires prior depolarization to drive the S4 voltage sensors outward, but these effects are lost in the mutant G845N. The results reveal a voltage sensor-trapping model of toxin action in which the IIS4 voltage sensor is trapped in its outward, activated position by toxin binding.

Amino Acid Sequence↗

PubMed1994

Relaxant and contractile responses of porcine pulmonary arteries to a thrombin receptor activating peptide (TRAP).

Recent studies on cloning of the thrombin receptor, which belongs to the family of G-protein-coupled receptors, suggest that thrombin cleaves a peptide from the extracellular N-terminus. A synthetic peptide of 14 amino acids corresponding to the sequence of the newly generated N-terminus was found to possess thrombin-like activity in several cells endowed with thrombin receptors. The relaxant and contractile effects of this thrombin receptor activating peptide (TRAP, Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe) were investigated in porcine pulmonary arteries and compared with the action of thrombin. In PGF2 alpha-precontracted vessels with intact endothelium, TRAP (0.3-10 mumol/l) caused reversible transient and concentration-dependent relaxation which was absent after mechanical removal of the endothelium. Preincubation of the vessels with NG-nitro-L-arginine (200 mumol/l) markedly reduced the relaxation. The TRAP-induced relaxation was associated with an increase in cGMP in the arteries. In comparison to thrombin, TRAP (EC50: 0.8 mumol/l) was less potent by more than three orders of magnitude. In endothelium-denuded pulmonary arteries TRAP (1-20 mumol/l) caused a concentration-dependent contraction which was reversible after washout. The TRAP-induced contractile response was preceded by an increase in generation of inositol 1,4,5-triphosphate (IP3); the peak of IP3 accumulation was reached after 30 s. Compared with the contractile effect of thrombin, that of TRAP was weaker by three of magnitude. The vascular effect of TRAP was not inhibited by the thrombin inhibitors hirudin or heparin while the protein kinase C inhibitor staurosporine (0.1 mumol/l) preferentially inhibited the tonic phase of contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMed1996

Rapid dispersal of clustered postsynaptic nuclei following dissociation of skeletal muscle fibers.

The vertebrate neuromuscular junction is a highly specialized structure containing many unique proteins and an underlying cluster of nuclei. Part of this specialization results from the expression of the genes for these proteins in nuclei clustered in the postsynaptic region. Contractile activity, as well as molecules located in the synaptic extracellular matrix (ECM), have been implicated in the induction of gene expression in these clustered nuclei. The present experiments were aimed at examining whether the presence of the synaptic ECM and presynaptic cells play a role in maintaining the clustering of the nuclei. We describe the normal distribution of nuclei clustered in the synaptic region of intact adult frog, Rana pipiens, skeletal muscle fibers and show that innervation is not required to maintain the nuclear clusters. Even after long-term (4 week) denervation, the clusters remain unchanged. Dissociation of the muscle fibers with proteases that remove ECM, Schwann cells and other satellite cells from the synaptic sites is followed by a rapid (within approximately 1.5 h) and almost complete dispersal of the clustered nuclei. Attempts to recluster the postsynaptic nuclei by the application of ECM components to muscle fibers in vitro were not successful. We propose that a factor or factors, localized in the synaptic ECM as a result of synapse formation and acting via the transmembrane or cytoplasmic domains of their respective receptors, induces the formation of a specialized cytoskeleton in the postsynaptic region that is capable of pulling in or 'trapping' nuclei. The removal of these factors from the ECM by proteases brings about the disorganization of the cytoskeleton and the freeing of the 'trapped' nuclei.

Animals↗

PubMed1998

The tubular vacuolation process in amphibian skeletal muscle.

The exposure of amphibian muscle to osmotic shock through the introduction and subsequent withdrawal of extracellular glycerol causes 'vacuolation' in the transverse tubules. Such manoeuvres can also electrically isolate the transverse tubules from the surface ('detubulation'), particular if followed by exposures to high extracellular [Ca2+] and/or gradual cooling. This study explored factors influencing vacuolation in Rana temporaria sartorius muscle. Vacuole formation was detected using phase contrast microscopy and through the trapping or otherwise of lissamine rhodamine dye fluorescence within such vacuoles. The preparations were also examined using electron microscopy, for penetration into the transverse tubules and tubular vacuoles of extracellular horseradish peroxidase introduced following the osmotic procedures. These comparisons distinguished for he first time two types of vacuole, 'open' and 'closed', whose lumina were respectively continuous with or detached from the remaining extracellular space. The vacuoles formed closed to and between the Z-lines, but subsequently elongated along the longitudinal axis of the muscle fibres. This suggested an involvement of tubular membrane material; the latter appeared particularly concentrated around such Z-lines in the electron-micrograph stereopairs of thick longitudinal sections. 'Open' vacuoles formed following osmotic shock produced by extracellular glycerol withdrawal from a glycerol-loaded fibre at a stage when one would expect a net water entry to the intracellular space. This suggests that vacuole formation requires active fluid transport into the tubular lumina in response to fibre swelling. 'Closed' vacuoles only formed when the muscle was subsequently exposed to high extracellular [Ca/+] and/or gradual cooling following the initial osmotic shock. Their densities were similar to those shown by 'open' vacuoles in preparations not so treated, suggesting that both vacuole types resulted from a single process initiated by glycerol withdrawal. However, vacuole 'closure' took place well after formation of 'open' vacuoles, over 25 min after glycerol withdrawal. Its time course closely paralleled the development of detubulation reported recently. It was irreversible, in contrast to the reversibility of 'open' vacuole formation. These findings identify electrophysiological 'detubulation' of striated muscle with 'closure' of initially 'open' vacuoles. The reversible formation of open vacuoles is compatible with some normal membrane responses to some physiological stresses such as fatigue, whereas irreversible formation of closed vacuoles might only be expected in pathological situations as in dystrophic muscle.

Animals↗

PubMed2003

Current transients associated with BK channels in human glioma cells.

We have previously demonstrated the expression of BK channels in human glioma cells. There was a curious feature to the whole-cell currents of glioma cells seen during whole-cell patch-clamp: large, outward current transients accompanied repolarization of the cell membrane following an activating voltage step. This transient current, Itransient, activated and inactivated rapidly (approximately 1 ms). The I-V relationship of Itransient had features that were inconsistent with simple ionic current through open ion channels: (i) Itransient amplitude peaked with a -80 mV voltage change and was invariant over a 200 mV range, and (ii) Itransient remained large and outward at -140 mV. We provide evidence for a direct relationship of Itransient to glioma BK currents. They had an identical time course of activation, identical pharmacology, identical voltage-dependence, and small, random variations in the amplitude of the steady-state BK current and Itransient seen over time were often perfectly in phase. Substituting intracellular K+ with Cs+, Li+, or Na+ ions reversibly reduced Itransient and BK currents. Itransient was not observed in recordings of other BK currents (hbr5 expressed in HEK cells and BK currents in rat neurons), suggesting Itransient is unique to BK currents in human glioma cells. We conclude that Itransient is generated by a mechanism related to the deactivation, and level of prior activation, of glioma BK channels. To account for these findings we propose that K+ ions are "trapped" within glioma BK channels during deactivation and are forced to exit to the extracellular side in a manner independent of membrane potential.

Adaptation, Physiological↗

PubMed1989

Macromolecular transport in rabbit blastocysts: evidence for a specific uteroglobin transport system.

Though uterine proteins are found within the blastocoel of the rabbit blastocyst, the mechanisms involved in protein entry into the blastocoel have not been studied. To investigate potential avenues of protein entry into the blastocoel, we have monitored uptake of a fluorescent, fluid-phase marker (lucifer yellow) into the rabbit blastocyst trophectodermal cell. In addition, we have measured the transtrophectodermal permeabilities of several uterine proteins (uteroglobin (UTG), rabbit serum albumin, rabbit IgG), and radiolabeled fluid-phase markers (sucrose, polyethylene glycol, dextran) in the 6- and 7-day post-coitus rabbit blastocyst. Extrablastocoelic lucifer yellow (LY) was rapidly endocytosed by the trophectodermal cell and was subsequently trapped in a perinuclear compartment for at least 30 min after removal of the extracellular dye. The slow rate of LY turnover within the endocytic compartments implies that the rate of fluid-phase transcytosis was negligible. Permeability coefficients of the fluid-phase markers and proteins, with the exception of UTG, decreased with increasing molecular weight of each compound tested. These data are consistent with the conclusion that these compounds traverse the trophectoderm via a 'leak' pathway, as opposed to a transcytotic pathway. In contrast, permeability of 125I-UTG when compared to the other compounds, was 10-fold greater than would be predicted based on its molecular weight. Furthermore, the flux of radioiodinated UTG displayed saturation kinetics with a Km of 19.5 micrograms/ml and a Vmax of 132 ng.cm-2.h-1. Sodium dodecylsulfate-polyacrylamide gel electrophoresis demonstrated that the radioiodinated protein recovered from the blastocoel was of a lower molecular weight than native UTG and was not immunoreactive with goat anti-UTG antibody. Our data are consistent with the idea that UTG is transported across the trophectoderm by a receptor-mediated system, and UTG is modified intracellularly during transport to a protein of a lower molecular weight.

Animals↗

PubMed1994

Intermediates in synaptic vesicle recycling revealed by optical imaging of Drosophila neuromuscular junctions.

We show that uptake and release of the styryl dye FM1-43 may be used to monitor synaptic vesicle exocytosis and recycling at Drosophila larval neuromuscular junctions. At Drosophila nerve terminals, FM1-43 specifically labels subsynaptic domains enriched in synaptotagmin, in a manner that requires Ca2+, membrane depolarization, and shibire (shi) function. Endocytosis rates, very low in unstimulated synapses, are induced severalfold by the exocytosis of synaptic vesicles. Using shi(ts)1 mutant synapses to separate synaptic vesicle fusion and recycling temporally, we show that recycling events subsequent to the shi block do not require extracellular Ca2+. We suggest that two distinct intermediate stages in vesicle recycling may be trapped and analyzed at Drosophila neuromuscular junctions.

Animals↗

PubMed2003

Mechanical forces impeding exocytotic surfactant release revealed by optical tweezers.

The release of surfactant from alveolar type II cells is essential to lower the surface tension in the lung and to facilitate inspiration. However, the factors controlling dispersal and diffusion of this hydrophobic material are still poorly understood. Here we report that release of surfactant from the fused vesicle, termed lamellar body (LB), resisted mechanical forces applied by optical tweezers: At constant trapping force, the probability to expand LB contents, i.e., to "pull" surfactant into the extracellular fluid, increased with time after LB fusion with the plasma membrane, consistent with slow fusion pore expansion in these cells. Elevations of the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) had a similar effect. Inasmuch as surfactant did not disintegrate in the extracellular space, this method permitted for the first time the determination of elastic and recoil properties of the macromolecular complex, yielding a spring constant of approximately 12.5 pN/ micro m. This is the first functional evidence that release of hydrophobic material is mechanically impeded and occurs in an "all-or-none" fashion. This mode of release is most probably the result of cohesive forces of surfactant, combined with adhesive forces and/or retaining forces exerted by a constrictive fusion pore acting as a regulated mechanical barrier, withstanding forces up to 160 pN. In independent experiments equiaxial strain was exerted on cells without optical tweezers. Strain facilitated surfactant release from preexisting fused vesicles, consistent with the view of mechanical impediments during the release process, which can be overcome by cell strain.

Animals↗