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Anchalee Tassanakajon - One of the best experts on this subject based on the ideXlab platform.
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Alpha-2-macroglobulin is a modulator of prophenoloxidase System in pacific white shrimp Litopenaeus vannamai
Fish & shellfish immunology, 2016Co-Authors: Sirikwan Ponprateep, Anchalee Tassanakajon, Tipachai Vatanavicharn, Vichien RimphanitchayakitAbstract:The shrimp multifunctional protein alpha-2-macroglobulin (A2M) is abundantly expressed in plasma, highly up-regulated upon microbial infection and involved in several immune pathways such as blood Clotting System, phagocytosis and melanization. Herein, the function of LvA2M from Litopenaeus vannamei on the prophenoloxidase (proPO) System is reported. The recombinant (r)LvA2M produced strongly and specifically inhibited trypsin and the PO activity in shrimp plasma in a dose-dependent manner. Silencing of LvA2M led to an increase in the PO activity in shrimp plasma although the expression of proPO-associated genes, proPO-activating enzyme (PPAE) and prophenoloxidase (proPO) but not the proPO-activating factor (PPAF) was down-regulated. In Vibrio parahaemolyticus AHPND-infected shrimp, the LvA2M activity was suppressed in an early phase of infection while the PO activity was increased. Thus, the proPO-activating System was regulated by the LvA2M.
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shrimp mirnas regulate innate immune response against white spot syndrome virus infection
Developmental and Comparative Immunology, 2016Co-Authors: Napol Kaewkascholkul, Kulwadee Somboonviwat, Shuichi Asakawa, Ikuo Hirono, Anchalee Tassanakajon, Kunlaya SomboonwiwatAbstract:MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5'UTR, ORF, and 3'UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood Clotting System, apoptosis-related proteins, proteins in prophenoloxidase System, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3'UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response.
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identification of immune related genes in hemocytes of black tiger shrimp penaeus monodon
Marine Biotechnology, 2002Co-Authors: Premruethai Supungul, Ikuo Hirono, Takashi Aoki, Sirawut Klinbunga, Sarawut Jitrapakdee, Rath Pichyangkura, Anchalee TassanakajonAbstract:An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 x 10(5). Of these, 615 clones having inserts larger than 500 bp were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the Clotting System and the prophenoloxidase-activating System, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.
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identification of immune related genes in hemocytes of black tiger shrimp penaeus monodon
Marine Biotechnology, 2002Co-Authors: Premruethai Supungul, Ikuo Hirono, Takashi Aoki, Sirawut Klinbunga, Sarawut Jitrapakdee, Rath Pichyangkura, Anchalee TassanakajonAbstract:An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 × 105. Of these, 615 clones having inserts larger than 500 by were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the Clotting System and the prophenoloxidase-activating System, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.
Peter B Armstrong - One of the best experts on this subject based on the ideXlab platform.
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blood collection from the american horseshoe crab limulus polyphemus
Journal of Visualized Experiments, 2008Co-Authors: Peter B Armstrong, Mara ConradAbstract: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.
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response of the blood Clotting System of the american horseshoe crab limulus polyphemus to a novel form of lipopolysaccharide from a green alga
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2006Co-Authors: Mara L Conrad, R L Pardy, Norman R Wainwright, Alice Child, Peter B ArmstrongAbstract: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.
Ikuo Hirono - One of the best experts on this subject based on the ideXlab platform.
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shrimp mirnas regulate innate immune response against white spot syndrome virus infection
Developmental and Comparative Immunology, 2016Co-Authors: Napol Kaewkascholkul, Kulwadee Somboonviwat, Shuichi Asakawa, Ikuo Hirono, Anchalee Tassanakajon, Kunlaya SomboonwiwatAbstract:MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5'UTR, ORF, and 3'UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood Clotting System, apoptosis-related proteins, proteins in prophenoloxidase System, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3'UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response.
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increased bacterial load in shrimp hemolymph in the absence of prophenoloxidase
FEBS Journal, 2009Co-Authors: Fernand F Fagutao, Takashi Koyama, Akihiro Kaizu, Tatsuo Saitotaki, Hidehiro Kondo, Takashi Aoki, Ikuo HironoAbstract:Invertebrates rely on their innate immune responses to protect themselves from pathogens, one of which is melanization of bacteria mediated by the activation of phenoloxidase (PO). Furthermore, invertebrate hemolymph, even that of healthy individuals, has been shown to contain bacterial species. The mechanisms that prevent these bacteria from proliferating and becoming deleterious to the host are, however, poorly understood. Here, we show that knocking down the activity of the inactive precursor of PO [prophenoloxidase (proPO)] by RNA interference resulted in a significant increase in the bacterial load of kuruma shrimp, Marsupenaeus japonicus, even in the absence of a bacterial or viral challenge. Silencing of proPO also led to a sharp increase in shrimp mortality. In addition, the hemolymph of proPO-depleted shrimp had significantly lower hemocyte counts and PO activity than control samples. Microarray analysis after proPO silencing also showed a decrease in the expression of a few antimicrobial peptides, but no effect on the expression of the genes involved in the Clotting System. Treatment with antibiotics prior to and after proPO dsRNA injection, to counteract the loss of proPO, resulted in a significant increase in shrimp survival. Our results therefore show that the absence of proPO renders the shrimp incapable of controlling bacteria present in the hemolymph, and that proPO is therefore essential for its survival.
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identification of immune related genes in hemocytes of black tiger shrimp penaeus monodon
Marine Biotechnology, 2002Co-Authors: Premruethai Supungul, Ikuo Hirono, Takashi Aoki, Sirawut Klinbunga, Sarawut Jitrapakdee, Rath Pichyangkura, Anchalee TassanakajonAbstract:An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 x 10(5). Of these, 615 clones having inserts larger than 500 bp were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the Clotting System and the prophenoloxidase-activating System, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.
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identification of immune related genes in hemocytes of black tiger shrimp penaeus monodon
Marine Biotechnology, 2002Co-Authors: Premruethai Supungul, Ikuo Hirono, Takashi Aoki, Sirawut Klinbunga, Sarawut Jitrapakdee, Rath Pichyangkura, Anchalee TassanakajonAbstract:An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 × 105. Of these, 615 clones having inserts larger than 500 by were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the Clotting System and the prophenoloxidase-activating System, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.
Harold F Dvorak - One of the best experts on this subject based on the ideXlab platform.
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Delayed-type hypersensitivity skin reactions in congenital afibrinogenemia lack fibrin deposition and induration
2015Co-Authors: Robert B Colvin, Michael W Mosesson, Harold F DvorakAbstract:delayed-type hypersensitivity skin reactions and is the usual measure of their intensity. The precise basis of induration has not been established, although activation ofthe Clotting System with consequent fibrin deposition has been clearly implicated. In this study, two subjects with congenital afibrinogenemia, a genetic defect in fibrinogen synthesis, were skin tested with standard microbial antigens: streptokinase-streptodornase, mo-nilia, mumps, and tuberculin purified protein derivative. One positive delayed reaction from each subject was biopsied at 40-48 h and compared with 23 biopsies of similar skin tests in normal volunteers. The eight skin tests in the afibrinogenic subjects lacked induration, although the erythema was similar in size (10-34 mm in diameter), intensity, and time-course to those in normals. Biopsies from the two strongest reactions from the afibrinogenemic subjects showed a typical perivascular mononuclear infiltrate. No more than traces of fibrin/fibrinogen were detected by im-munofluorescence, in striking contrast to the abundant fibrin/fibrinogen deposition in 23 positive, indurated reactions in normal subjects. These findings indicate that fibrinogen itself is essential for the development of induration in delayed-type skin reactions in man. As judged by 1-,um sections and fluorescence, this is probably a result of the formation of an extravascular fibrin gel
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vascular hyperpermeability angiogenesis and stroma generation
Cold Spring Harbor Perspectives in Medicine, 2012Co-Authors: Janice A Nagy, Ann M Dvorak, Harold F DvorakAbstract:It has been known for more than half a century that the tumor microvasculature is hyperpermeable to plasma proteins. However, the identity of the leaky vessels and the consequences of vascular hyperpermeability have received little attention. This article places tumor vascular hyperpermeability in a broader context, relating it to (1) the low-level “basal” permeability of the normal vasculature; (2) the “acute,” short-term hyperpermeability induced by vascular permeability factor/vascular endothelial growth factor (VPF/VEGF-A) and other vascular permeabilizing agents; and (3) the “chronic” hyperpermeability associated with longer-term exposure to agents such as VPF/VEGF-A that accompanies many types of pathological angiogenesis. Leakage of plasma protein-rich fluids is important because it activates the Clotting System, depositing an extravascular fibrin gel provisional matrix that serves as the first step in stroma generation.
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vascular permeability factor vascular endothelial growth factor and the significance of microvascular hyperpermeability in angiogenesis
Current Topics in Microbiology and Immunology, 1999Co-Authors: Harold F Dvorak, Janice A Nagy, Ann M Dvorak, Lawrence F. Brown, Dian FengAbstract:Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) was originally discovered in the late 1970s because of its capacity to increase the permeability of microvessels to plasma and plasma proteins (Dvorak et al. 1979a,b). Using plastic-embedded, light microscopic section and, subsequent, immunohistochemistry, we noted that transplantable tumors growing in guinea pigs and rodents exhibit substantial deposits of fibrin in their stroma. Fibrin results from the Clotting of fibrinogen, a 340kDa plasma protein which, under normal circumstances, is retained almost quantitatively within the blood vasculature. For fibrin to be deposited outside of blood vessels in tumor stroma, it was necessary that two requirements be met; namely, (1) that microvessels be abnormally hyperpermeable to permit the escape of fibrinogen and other plasma proteins necessary for blood Clotting and (2) that there be a mechanism in place for activating the Clotting System. In fact, both requirements were found to be met in tumors. The microvessels supplying tumors were hyperpermeable to fibrinogen and other plasma proteins, and both tumor cells and host stromal cells were capable of initiating extravascular coagulation via the tissue-factor pathway. Encouraged by these findings, we initiated a search for a tumor product that could account for tumor-vessels hyperpermeability. A potent vascular permeabilizing protein was soon found in serum-free tumor culture supernatants (Dvorak et al. 1979a,b) and was subsequently purified to homogeneity and given the name vascular permeability factor (VPF) (Senger et al. 1983, 1986, 1987, 1990).
Premruethai Supungul - One of the best experts on this subject based on the ideXlab platform.
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identification of immune related genes in hemocytes of black tiger shrimp penaeus monodon
Marine Biotechnology, 2002Co-Authors: Premruethai Supungul, Ikuo Hirono, Takashi Aoki, Sirawut Klinbunga, Sarawut Jitrapakdee, Rath Pichyangkura, Anchalee TassanakajonAbstract:An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 x 10(5). Of these, 615 clones having inserts larger than 500 bp were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the Clotting System and the prophenoloxidase-activating System, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.
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identification of immune related genes in hemocytes of black tiger shrimp penaeus monodon
Marine Biotechnology, 2002Co-Authors: Premruethai Supungul, Ikuo Hirono, Takashi Aoki, Sirawut Klinbunga, Sarawut Jitrapakdee, Rath Pichyangkura, Anchalee TassanakajonAbstract:An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 × 105. Of these, 615 clones having inserts larger than 500 by were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the Clotting System and the prophenoloxidase-activating System, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.