Publications

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    ABSTRACT: In addition to its roles in hemostasis and wound repair, the blood clot plays an underappreciated role in innate immunity, where the established clot serves as a barrier to microbial penetration into the internal milieu and where the early clot entraps and immobilizes microbes that have entered wounds to the integuments. In this report we document the behavior of the pathogenic gram-negative bacterium Vibrio harveyi that has been entrapped in the fabric of the extracellular blood clot of one of its target organisms, the Pacific white shrimp, Litopenaeus vannamei. The freshly entrapped bacteria are held tightly by the clot, losing even Brownian motility, but by 1 h post-entrapment, a fraction of the bacteria have established small domains of fibrinolysis that enlarge progressively, enabling bacteria to escape from the clot's embrace. Escape is dependent on the actions of both serine- and metallo-proteases released from the bacterial cells.
    Biological Bulletin 04/2014; 226(2):102-10. · 1.23 Impact Factor
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    Peter B Armstrong
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    ABSTRACT: In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot) and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse) operate to capture lipopolysaccharide (LPS, endotoxin). The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL) test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.
    PLoS ONE 01/2013; 8(11):e80192. · 3.53 Impact Factor
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    ABSTRACT: Horseshoe crabs provide the important biomedical products, Limulus Amebocyte Lysate (LAL) and Tachypleus Amebocyte Lysate (TAL), which are produced by lysing the blood cells (amebocytes) using endotoxin-free distilled water. LAL/TAL is used to detect and quantify lipopolysaccharide (endotoxin), an important toxin of Gram-negative bacteria. Horseshoe crabs are the sole source for LAL/TAL and their fishery for the production of LAL/TAL is of concern to the sustained management of the species. This study investigated the mortality of the Malaysian horseshoe crab (Tachypleus gigas) related to blood extraction, with the aim of providing a basis for the sustainable management of the local fishery of this animal. We showed that the mortality rate of T. gigas due to the bleeding process is slightly higher than the values which has previously been reported for its conspecific, the American horseshoe crab (Limulus polyphemus). Overall mortalities of bled horseshoe crabs were 17.8% (male = 18.75%; female = 17.2%), while mortalities of unbled crabs were 3.5%. The quantification of the mortality due to the extent of bleeding commensurate with the commercial production of LAL/TAL provides a basis for the rational management of T. gigas in situations where harvest for LAL/TAL production is imposed on local populations of the animal.
    Marine behaviour and physiology 01/2011; 44(5):321-327.
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    Armstrong PB
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    ABSTRACT: Although proteases play essential roles in the lives of all organisms, they are also important agents of disease and pathogenesis in metazoans. Most notably, proteases are essential virulence factors for a broad array of prokaryote and eukaryote parasites. One strategy used by the immune system of metazoans to defend against parasitic attack is to neutralize the toxins and essential virulence factors that allow the parasite to gain entry to the host and to survive and proliferate in the internal environment of the metazoan host. The particular defense system of interest to the present review is the system of endogenous protease inhibitors that operate to inactivate the secreted proteases utilized by invading parasites during the infection cycle within the host. Protease inhibitors are of two broad classes, active-site inhibitors that bind to and inactivate the active sites of target proteases and the α2-macroglobulin class of inhibitors that operate as opsonins to bind and mark proteases in a manner that allows the subsequent endocytosis and intracellular proteolytic degradation of the α2-macroglobulin-protease complex. Members of the α2-macroglobulin class of inhibitors interact with target proteases by the novel process of enfolding the protease into a pocket within the interior of the α2-macroglobulin molecule, which is followed by binding of the complex to the α2-macroglobulin receptor at the surfaces of macrophages and other endocytotic cells and its endocytosis and degradation. In contrast to the active-site protease inhibitors, each of which is specialized to interact with a small subset of all endopeptidases, the α2-macroglobulin inhibitors are remarkably promiscuous, binding proteases of all enzymatic classes and origins. This characteristic allows α2-macroglobulin to play an important role in immune defense because this one protein is capable of binding and neutralizing the diverse array of proteases that function as virulence factors of the diverse array of parasites out there in the environment of metazoa.
    Invertebrate Survival Journal. 01/2010;
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    ABSTRACT: The serum-amyloid-P-component-like pentraxin from Limulus polyphemus, a recently discovered pentraxin species and important effector protein of the hemolymph immune system, displays two distinct doubly stacked cyclic molecular aggregations, heptameric and octameric. The refined three-dimensional structures determined by X-ray crystallography, both based on the same cDNA sequence, show that each aggregate is constructed from a similar dimer of protomers, which is repeated to make up the ring structure. The native octameric form has been refined at a resolution of 3 A, the native heptameric form at 2.3 A, and the phosphoethanolamine (PE)-bound octameric form at 2.7 A. The existence of the hitherto undescribed heptameric form was confirmed by single-particle analysis using cryo-electron microscopy. In the native structures, the calcium-binding site is similar to that in human pentraxins, with two calcium ions bound in each subunit. Upon binding PE, however, each subunit binds a third calcium ion, with all three calcium ions contributing to the binding and orientation of the bound phosphate group within the ligand-binding pocket. While the phosphate is well-defined in the electron density, the ethanolamine group is poorly defined, suggesting structural and binding variabilities of this group. Although sequence homology with human serum amyloid P component is relatively low, structural homology is high, with very similar overall folds and a common affinity for PE. This is due, in part, to a "topological" equivalence of side-chain position. Identical side chains that are important in both function and fold, from different regions of the sequence in human and Limulus structures, occupy similar space within the overall subunit fold. Sequence and structure alignment, based on the refined three-dimensional structures presented here and the known horseshoe crab pentraxin sequences, suggest that adaptation and refinement of C-reactive-protein-mediated immune responses in these ancient creatures lacking antibody-based immunity are based on adaptation by gene duplication.
    Journal of Molecular Biology 04/2009; 386(5):1240-54. · 3.91 Impact Factor
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    ABSTRACT: C-reactive protein (CRP) from the American horseshoe crab, Limulus polyphemus, exhibits complex membrane activities. Here, we describe the behavior of protein and lipid as CRP interacts with model liposomes and bacterial membranes. Limulus C-reactive protein (L-CRP) forms extended fibrilar structures that encapsulate liposomes in the presence of Ca(2+). We have observed structures consistent in size and shape with these fibers bound to the surface of Gram-negative bacteria. The membranes of Limulus CRP-treated bacteria exhibit significantly different mechano-elastic properties than those of untreated bacteria. In vitro, bilayer lipids undergo a rigidification and reorganization of small domains. We suggest that these interactions reflect the protein's role as a primary defense molecule, functioning in the entrapment and killing of potential pathogens.
    FEBS letters 03/2009; 583(6):1001-5. · 3.54 Impact Factor
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    ABSTRACT: The pentraxins are a family of highly conserved plasma proteins of metazoans known to function in immune defence. The canonical members, C-reactive protein and serum amyloid P component, have been identified in arthropods and humans. Mammalian pentraxins are known to bind lipid bilayers, and a pentraxin representative from the American horseshoe crab, Limulus polyphemus, binds and permeabilizes mammalian erythrocytes. Both activities are Ca(2+)-dependent. Utilizing model liposomes and planar lipid bilayers, in the present study we have investigated the membrane-active properties of the three pentraxin representatives from Limulus and show that all of the Limulus pentraxins permeabilize lipid bilayers. Mechanistically, Limulus C-reactive protein forms transmembrane pores in asymmetric planar lipid bilayers that mimic the outer membrane of Gram-negative bacteria and exhibits a Ca(2+)-independent form of membrane binding that may be sufficient for pore formation.
    Biochemical Journal 08/2008; 413(2):305-13. · 4.65 Impact Factor
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    John M. Harrington, Matthias Leippe, Peter B. Armstrong
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    ABSTRACT: The cuticle of the American horseshoe crab, Limulus polyphemus, is largely free of the macroscopic epibionts that rapidly colonize most solid surfaces exposed to sea water. Here, we describe The cuticle of the American horseshoe crab, Limulus polyphemus, is largely free of the macroscopic epibionts that rapidly colonize most solid surfaces exposed to sea water. Here, we describe a viscous surface secretion that coats the carapace of the horseshoe crab. We report methods for stimulating production of a viscous surface secretion that coats the carapace of the horseshoe crab. We report methods for stimulating production of the substance, identify hemolytic and liposome-permeabilizing activities and provide a partial biochemical characterization. the substance, identify hemolytic and liposome-permeabilizing activities and provide a partial biochemical characterization. We propose that this secretion functions as an anti-fouling agent protecting the chitinized epithelium of the cuticle from We propose that this secretion functions as an anti-fouling agent protecting the chitinized epithelium of the cuticle from colonization by deleterious epibionts. colonization by deleterious epibionts.
    Marine Biology 02/2008; 153(6):1165-1171. · 2.39 Impact Factor
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    Peter Armstrong, Mara Conrad
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    ABSTRACT: The horseshoe crab has the best-characterized immune system of any long-lived invertebrate. The study of immunity in horseshoe crabs has been facilitated by the ease in collecting large volumes of blood and from the simplicity of the blood. Horseshoe crabs show only a single cell type in the general circulation, the granular amebocyte. The plasma has the salt content of sea water and only three abundant proteins, hemocyanin, the respiratory protein, the C-reactive proteins, which function in the cytolytic destruction of foreign cells, including bacterial cells, and alpha2-macroglobulin, which inhibits the proteases of invading pathogens. Blood is collected by direct cardiac puncture under conditions that minimize contamination by lipopolysaccharide (a.k.a., endotoxin, LPS), a product of the Gram-negative bacteria. A large animal can yield 200 - 400 mL of blood. For the study of the plasma, blood cells are immediately removed from the plasma by centrifugation and the plasma can then be fractionated into its constituent proteins. The blood cells are conveniently studied microscopically by collecting small volumes of blood into LPS-free isotonic saline (0.5 M NaCl) under conditions that permit direct microscopic examination by placing one of more LPS-free coverglasses on the culture dish surface, then mounting those coverglasses in simple observation chambers following cell attachment. A second preparation for direct observation is to collect 3 - 5 mL of blood in a LPS-free embryo dish and then explanting fragments of aggregated amebocytes to a chamber that sandwiches the tissue between a slide and a coverglass. In this preparation, the motile amebocytes migrate onto the coverglass surface, where they can readily be observed. The blood clotting system involves aggregation of amebocytes and the formation of an extracellular clot of a protein, coagulin, which is released from the secretory granules of the blood cells. Biochemical analysis of washed blood cells requires that aggregation and degranulation does not occur, which can be accomplished by collecting blood into 0.1 volumes of 2% Tween-20, 0.5 M LPS-free NaCl, followed by centrifugation of the cells and washing with 0.5 M NaCl.
    Journal of Visualized Experiments 02/2008;
  • JAMES P. QUIGLEY, PETER B. ARMSTRONG
    Annals of the New York Academy of Sciences 12/2006; 712(1):131 - 145. · 4.38 Impact Factor
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    ABSTRACT: Lipopolysaccharide (LPS) (a.k.a., endotoxin) is an essential component of the outer leaflet of the outer membrane of gram-negative bacteria and is a potent activator of the innate immune system of animals. Lipid A, the glycolipid core of LPS, is the agent responsible for disease and death from gram-negative sepsis, an important cause of human mortality and morbidity. Although it is generally accepted that lipid A is restricted to the prokaryotes, recent efforts to purify molecules from green algae with structural features unique to lipid A have met with success. Furthermore, the vascular plant Arabidopsis thaliana has been found to contain genes that encode all of the enzymes of the biosynthetic pathway for lipid A. It is not known whether vascular plants synthesize lipid A or where lipid A might be located in the tissues. For the present study, we used affinity reagents for lipid A to probe green alga and tissues of the garden pea for a light microscopic localization of lipid A in these eukaryote cells. We find staining for lipid A in free-living and endosymbiotic green algae and in the chloroplasts of vascular plants, indicating that this molecule is not restricted to prokaryotes, but is found also in select eukaryotes.
    The FASEB Journal 11/2006; 20(12):2145-6. · 5.70 Impact Factor
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    ABSTRACT: The embryonic epidermis of amniotes is a two-cell layer sheet with a periderm positioned superficial to the basal cell layer which, itself, attaches apically to the basal surface of the periderm and basally to the basal lamina. The presence of the periderm is essential to maintain the basal layer as a two-dimensional monolayer. Wounds to the epidermis that remove selectively just the periderm are healed by a stacking of the basal layer cells that constitute the wound bed. Basal cell stacking involves the desertion of the basal lamina by many of the cells so as to increase their contact area with other basal layer cells. This suggests that a preferential adhesion to the planar basal lamina is not important for the monolayered organization of the basal layer but, instead, association with inner surface of the planar periderm is the principal process that maintains the basal layer as a monolayer. The conversion of the basal layer from monolayer to multilayer during wound healing diminishes its planar area, resulting in movement of the wound borders toward the center of the wound. This is a novel scenario for wound healing.
    Experimental Cell Research 09/2006; 312(13):2415-23. · 3.56 Impact Factor
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    ABSTRACT: Lipopolysaccharide (LPS, endotoxin) is a component of Gram-negative bacteria and is the principal indicator to the innate immune systems of higher animals of a Gram-negative bacterial invasion. LPS activates the blood clotting system of the American horseshoe crab, Limulus polyphemus. By stimulating blood cell degranulation, LPS triggers the release of the proteins of the clotting system from the cells, and by activating a protease cascade that converts coagulogen, a soluble zymogen, to coagulin, the structural protein of the clot, LPS triggers the production of the fibrillar coagulin blood clot. Although originally thought to be restricted to the Gram-negative bacteria and the cyanobacteria, LPS, or a very similar molecule, has recently been described from a eukaryotic green alga, Chlorella. Here we show that, like LPS from Gram-negative bacteria, the algal molecule stimulates exocytosis of the Limulus blood cell and the clotting of coagulin. The coagulin clot efficiently entraps the cells of Chlorella in a network of fibrils. Invasion and erosion of the carapace by green algae is an important cause of mortality of Limulus, and it is suggested that the cellular response to aLPS may contribute to defense against this pathogen.
    Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 09/2006; 144(4):423-8. · 2.17 Impact Factor
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    Peter B Armstrong
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    ABSTRACT: The immune system is the collection of effector molecules and cells of the host that act against invading parasites and their products. Secreted proteases serve important roles in parasitic metabolism and virulence and the several families of protein protease inhibitors of the plasma and blood cells play an important role in immunity by inactivating and clearing the protease virulence factors of parasites. The protease inhibitors are of two classes, the active-site inhibitors and the alpha2-macroglobulins. Inhibitors for the first class bind and inactivate the active site of the target protease. Proteins of the second class bind proteases by a unique molecular trap mechanism and deliver the bound protease to a receptor-mediated endocytic system for degradation in secondary lysosomes. Proteins of the alpha2-macroglobulin family are present in a variety of animal phyla, including the nematodes, arthropods, mollusks, echinoderms, urochordates, and vertebrates. A shared suite of unique functional characteristics have been documented for the alpha2-macroglobulins of vertebrates, arthropods, and mollusks. The alpha2-macroglobulins of nematodes, arthropods, mollusks, and vertebrates show significant sequence identity in key functional domains. Thus, the alpha2-macroglobulins comprise an evolutionarily conserved arm of the innate immune system with similar structure and function in animal phyla separated by 0.6 billion years of evolution.
    Immunobiology 02/2006; 211(4):263-81. · 2.81 Impact Factor
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    Peter B. Armstrong
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    ABSTRACT: The active motility of the cells of the yolk sac of the living Fundulus embryo was studied by time-lapse cinemicrography with phase contrast optics. In the teleost, the yolk sac lies peripheral to the body of the embryo proper and consists of a fluid-filled space bounded above by a superficial epithelium, the enveloping layer (EVL), and below by the yolk syncytial layer (YSL). The cell types treated in the present study are the enveloping layer epithelial cell, the stellate cell which lies in a layer flattened on the inner surface of the EVL, the epithelioid deep cell, the yolk sac amoebocyte, the yolk sac endothelial cell and the yolk sac melanoblast. The most actively motile cells examined in the present study are the yolk sac amebocyte and the melanoblast, which emigrates from the embryo proper at stages 19–21. The amoebocytes are compact rounded cells that move very rapidly by the extension of lamellipods with scalloped margins. The amoebocytes wander over the yolk sac in an apparently undirected fashion and invade the embryo proper when they happen to encounter it, moving between cells of the lateral mesoderm. The melanoblasts migrate by the gradual extension of elongated branching processes. Cells are sometimes monopodial, with movement being parallel to the long axis of the cell. Alternatively, movement may be perpendicular to the predominant long axis, with processes being extended alternatively from opposite ends of the cell obliquely forward, so the path described is a zig-zag to either side of the overall direction of movement. Although the melanoblasts show irregularity in their movement, the predominant direction of initial movement is away from the embryo proper. The major yolk sac blood vessels form in situ by the collective activities of presumptive endothelial cells that enclose volumes of the yolk sac space with sheet-like processes from the cell body and from the extensions that connect cells into networks.
    Journal of Morphology 02/2005; 165(1):13 - 29. · 1.60 Impact Factor
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    Margaret T. Armstrong, Peter B. Armstrong
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    ABSTRACT: The effect of specific components of the extracellular matrix on the motility of tissue cells was studied using organ-cultured aggregates of embryonic fibroblasts. Spherical aggregates of chick embryo heart and skin fibroblasts were fused with [3H]-thymidine-labeled aggregates of the identical cell type. The movement of labeled cells into the unlabeled partner aggregate served as an estimate of cell motility in the cultured tissue-like aggregates. Collagenase treatment decreased the collagen content of heart fibroblast aggregates and increased cell motility; ascorbic acid treatment increased the collagen content of skin fibroblast aggregates and decreased cell motility. Reduction of the glycosaminoglycan content with testicular hyaluronidase had no measurable effect on cell motility in heart fibroblast aggregates.
    Cell Motility and the Cytoskeleton 02/2005; 1(1):99 - 112. · 4.19 Impact Factor
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    Acta Crystallographica Section A - ACTA CRYSTALLOGR A. 01/2005; 61.
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    ABSTRACT: The American horseshoe crab Limulus polyphemus contains α2-macroglobulin (α2M) in the hemolymph plasma and hemocytes. α2M from Limnulus shows many of the typical characteristics of mammalian α2M, including the presence of an internal thiol-ester, reactivity with a diversity of endopeptidases, a unique proteinase-trapping mechanism, and reactivity with the mammalian α2M receptor. Additionally, Limulusα2M has the unique property that it regulates the limulin-based hemolytic system of the plasma. A cDNA encoding Limulusα2M has been obtained from a hemocyte cDNA library. The open reading frame encodes an N-terminal signal sequence of 25 amino acid residues and a mature protein of 1482 residues. The entire amino acid sequence is similar to those of the mammalian α2Ms (28–29% identity) and contains common features found in mammalian α2Ms, a bait region, an internal thiol-ester site, and a receptor-binding domain. However, the N-terminal portion (positions 24–105) has no sequence similarity with those of mammalian α2Ms, and it is structurally related to that of the human complement factor C8γ chain, consistent with a role for Limulus α2M in host defense. The component sugar analysis of Limulus α2M showed the existence of a complex type of oligosaccharide chain similar to those of mammalian α2M. However, unlike mammalian α2M, no sialic acid was detected in Limulusα2M and it contained approximately 3 mol/mol N-acetylgalactosamine, suggesting the presence of O-linked sugar chains, which have not been found in mammalian α2M.Expression of α2M was detected in hemocyies, but not in hepatopancreas, heart, stomach, intestine, coxal gland, brain and skeletal muscle. Furthermore, immunoblotting of large and small granules of the hemocytes with antiserum against α2M indicated the presence of the α2M in large granules. Trypsin-treated Limulusα2M, but not the native α2M, displaced methylamine-treated human 125I-α2M from the human α2M receptor with a Kd of 30 nM, suggesting conservation of the proteinase-clearance mechanisms between mammalian and arthropod evolutionary lineages.
    European Journal of Biochemistry. 07/2004; 242(3):822 - 831.
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    Peter B Armstrong, Margaret T Armstrong
    Biological Bulletin 11/2003; 205(2):201-3. · 1.23 Impact Factor

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