| 1. |
- Apitanyasai, Kantamas, et al.
(author)
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Characterization of a hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus
- 2016
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In: Fish and Shellfish Immunology. - : Elsevier BV. - 1050-4648 .- 1095-9947. ; 58, s. 429-435
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Journal article (peer-reviewed)abstract
- Hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus has a distinct role from that of its homolog PmHHAP in the shrimp Penaeus monodon. Knockdown of PIHHAP in vitro using double-stranded RNA (dsRNA) had no effect on the cell morphology of hematopoietic tissue (HPT) cells. The total hemocyte number and caspase activity were unchanged after PIHHAP knockdown in vivo, in contrast to the results found in shrimp. Moreover, suppression of PIHHAP both in vitro and in vivo did not change the mRNA levels of some genes involved in hematopoiesis and hemocyte homeostasis. Interestingly, bacterial count and scanning electron microscope revealed that depletion of PIHHAP in intestine by RNAi resulted in higher number of bacteria in the crayfish intestine. Together, these results suggest that PIHHAP is not involved in hemocyte homeostasis in the crayfish P. leniusculus but appears to affect the bacterial number in the intestine through an unknown mechanism. Since PIHHAP has different functions from PmHHAP, we therefore named it HHAP-like protein.
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| 2. |
- Cerenius, Lage, 1956-, et al.
(author)
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High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans
- 2010
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In: Developmental and Comparative Immunology. - : Elsevier. - 0145-305X .- 1879-0089. ; 34:1, s. 69-75
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Journal article (peer-reviewed)abstract
- Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.
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| 3. |
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| 4. |
- Donpudsa, Suchao, et al.
(author)
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Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus
- 2010
-
In: Journal of Invertebrate Pathology. - : Elsevier BV. - 0022-2011 .- 1096-0805. ; 104:3, s. 234-238
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Journal article (peer-reviewed)abstract
- The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coil expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 mu M and 3.5-8 mu M, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.
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| 5. |
- Donpudsa, Suchao, et al.
(author)
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Proteinase inhibitory activities of two two-domain Kazal proteinase inhibitors from the freshwater crayfish Pacifastacus leniusculus and the importance of the P2 position in proteinase inhibitory activity
- 2010
-
In: Fish and Shellfish Immunology. - : Elsevier BV. - 1050-4648 .- 1095-9947. ; 29:5, s. 716-723
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Journal article (peer-reviewed)abstract
- Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors (KPIs), KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P. leniusculus, they are found specific to the hernocytes and contain an uncommon P-2 amino acid residue, Gly. To unveil their inhibitory activities, the two KPIs and their domains were over-expressed. By testing against subtilisin, trypsin, chymotrypsin and elastase, the KPI2 was found to inhibit strongly against subtilisin and weakly against trypsin, while the KPI8 was strongly active against only trypsin. With their P-1 Set and Lys residues, the KPI2_domain2 and KPI8_domain2 were responsible for strong inhibition against subtilisin and trypsin, respectively. Mutagenesis of KPI8_domain1 at P-2 amino acid residue from Gly to Pro, mimicking the P-2 residue of KPI8_domain2, rendered the KPI8_domain1 strongly active against trypsin, indicating the important role of P-2 residue in inhibitory activities of the Kazal-type serine proteinase inhibitors. Only the KPI2 was found to inhibit against the extracellular serine proteinases from the pathogenic oomycete of the freshwater crayfish, Aphanomyces astaci.
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| 6. |
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| 7. |
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| 8. |
- Ekblom, Charlotta, et al.
(author)
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Early Changes in Crayfish Hemocyte Proteins after Injection with a beta-1,3-glucan, Compared to Saline Injected and Naive Animals
- 2021
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In: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:12
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Journal article (peer-reviewed)abstract
- Early changes in hemocyte proteins in freshwater crayfish Pacifastacus leniusculus, in response to an injection with the fungal pattern recognition protein beta-1,3-glucan (laminarin) were investigated, as well as changes after saline (vehicle) injection and in naive animals. Injection of saline resulted in rapid recruitment of granular hemocytes from surrounding tissues, whereas laminarin injection on the other hand induced an initial dramatic drop of hemocytes. At six hours after injection, the hemocyte populations therefore were of different composition. The results show that mature granular hemocytes increase in number after saline injection as indicated by the high abundance of proteins present in granular cell vesicles, such as a vitelline membrane outer layer protein 1 homolog, mannose-binding lectin, masquerade, crustin 1 and serine protease homolog 1. After injection with the beta-1,3-glucan, only three proteins were enhanced in expression, in comparison with saline-injected animals and uninjected controls. All of them may be associated with immune responses, such as a new and previously undescribed Kazal proteinase inhibitor. One interesting observation was that the clotting protein was increased dramatically in most of the animals injected with laminarin. The number of significantly affected proteins was very few after a laminarin injection when compared to uninjected and saline-injected crayfish. This finding may demonstrate some problematic issues with gene and protein expression studies from other crustaceans receiving injections with pathogens or pattern recognition proteins. If no uninjected controls are included and no information about hemocyte count (total or differential) is given, expressions data for proteins or mRNAs are very difficult to properly interpret.
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| 9. |
- Guo, Enen, et al.
(author)
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A Pacifastacus leniusculus serine protease interacts with WSSV
- 2017
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In: Fish and Shellfish Immunology. - : Elsevier BV. - 1050-4648 .- 1095-9947. ; 68, s. 211-219
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Journal article (peer-reviewed)abstract
- Serine proteases are involved in many critical physiological processes including virus spread and replication. In the present study, we identified a new clip-domain serine protease (PIcSP) in the crayfish Pacifastacus leniusculus hemocytes, which can interact with the White Spot Syndrome Virus (WSSV) envelope protein VP28. It was characterized by a classic clip domain with six strictly conserved Cys residues, and contained the conserved His-Asp-Ser (H-D-S)motif in the catalytic domain. Furthermore, signal peptide prediction revealed that it has a 16-residue secretion signal peptide. Tissue distribution showed that it was mainly located in P. leniusculus hemocytes, and its expression was increased in hemocytes upon WSSV challenge. In vitro knock down of PIcSP decreased both the expression of VP28 and the WSSV copy number in hematopoietic stem (HPT) cells. Accordingly, these data suggest that the new serine protease may be of importance for WSSV infection into hematopoietic cells.
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| 10. |
- Hernandez-Perez, Ariadne, et al.
(author)
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Environmental concentrations of sulfamethoxazole increase crayfish Pacifastacus leniusculus susceptibility to White Spot Syndrome Virus
- 2020
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In: Fish and Shellfish Immunology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 1050-4648 .- 1095-9947. ; 102, s. 177-184
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Journal article (peer-reviewed)abstract
- Antibiotics used for humans and livestock are emerging as pollutants in aquatic environments. However, little is known about their effect on aquatic organisms, especially in crustaceans. In the present study, the freshwater crayfish Pacifastacus leniusculus was exposed during 21 days to environmental concentrations of sulfamethoxazole (SMX) (100 ng/L and 1 mu g/L). Subsequently, the crayfish susceptibility to infection was evaluated by using White Spot Syndrome Virus (WSSV) challenge, a well-known crustacean pathogen. The median survival time of the infected crayfish exposed to 100 ng/L SMX was one day, whereas the control and the group exposed to 1 mu g/L SMX survived for two and three days, respectively. In order to elucidate the effect of SMX upon the crayfish immune response, new sets of crayfish were exposed to the same SMX treatments to evaluate mRNA levels of immune-related genes which are expressed and present in hemocytes and intestine, and to perform total and differential hemocyte counts. These results show a significant down-regulation of the antimicrobial peptide (AMP) Crustin 3 in hemocytes from the 100 ng/L SMX group, as well as a significant up-regulation of the AMP Crustin 1 in intestines from the 1 mu g/L SMX group. Semigranular and total hemocyte cell number were observed to be significantly lower after exposure to 100 ng/L SMX in comparison with the control group. The present study demonstrates that environmentally relevant SMX concentrations in the water at 100 ng/L led to an increased WSSV susceptibility, that may have been caused by a reduction of circulating hemocytes. Nevertheless, SMX concentrations of 1 mu g/L could marginally and for a few days have an immunostimulatory effect.
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| 11. |
- Hernandez-Perez, Ariadne, et al.
(author)
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Gut microbiome alterations in the crustacean Pacifastacus leniusculus exposed to environmental concentrations of antibiotics and effects on susceptibility to bacteria challenges*
- 2022
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In: Developmental and Comparative Immunology. - : Elsevier. - 0145-305X .- 1879-0089. ; 126
-
Journal article (peer-reviewed)abstract
- Gut-associated microbiota in crustaceans are recognized as a key element for maintaining homeostasis and health in the animal. Since the richness of these microbial communities is strongly influenced by the local environment, especially in aquatic organisms, it is important to address to what extent environmental variations can affect these communities. In the present study, we used high-throughput 16S rRNA sequencing technology to study the composition of gut-associated microbiota of the crayfish Pacifastacus leniusculus after exposure to environmentally-relevant concentrations of an antibiotic, namely sulfamethoxazole. Also, we examined if alterations of microbiota caused by environmentally-relevant concentrations of this antibiotic affected the host susceptibility to bacterial diseases, including Vibrio species. As a result, we found high individual variability of bacterial abundance and composition in the intestinal microbiome of crayfish, in both antibiotic-exposed and antibiotic-free crayfish. However, an increase of chitinolytic bacteria including Vibrio spp. was detected in some animals exposed to the antibiotic. Moreover, when crayfish susceptibility to bacterial infections was tested, the antibiotic-exposed crayfish survived longer than the control crayfish group. This study represents the first approach for investigating the interplay between crayfish and intestinal bacteria during antibiotic-pollution scenarios. Results herein should be considered by scientists before planning experiments under laboratory conditions, especially to study environmental effects on aquatic animals' intestinal health and immune status.
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| 12. |
- Hernández-Pérez, Ariadne, et al.
(author)
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Vibrio areninigrae as a pathogenic bacterium in a crustacean
- 2021
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In: Journal of Invertebrate Pathology. - : Elsevier. - 0022-2011 .- 1096-0805. ; 178
-
Journal article (peer-reviewed)abstract
- The occurrence of infectious diseases poses a significant threat to the aquaculture industry worldwide. Therefore, characterization of potentially harmful pathogens is one of the most important strategies to control disease outbreaks. In the present study, we investigated for the first time the pathogenicity of two Vibrio species, Vibrio metschnikovii, a foodborne pathogen that causes fatalities in humans, and Vibrio areninigrae, a bacteria isolated from black sand in Korea, using a crustacean model, the signal crayfish Pacifastacus leniusculus. Mortality challenges indicated that injection of V. metschnikovii (108 CFU/crayfish) has a mortality percentage of 22% in crayfish. In contrast, injection of P. leniusculus with 108 or 107 CFU of V. areninigrae resulted in 100% mortality within one and two days post-injection, respectively. V. areninigrae was successfully re-isolated from hepatopancreas of infected crayfish and caused 100% mortality when reinjected into new healthy crayfish. As a consequence of this infection, histopathological analysis revealed nodule formation in crayfish hepatopancreas, heart, and gills, as well as sloughed cells inside hepatopancreatic tubules and atrophy. Moreover, extracellular crude products (ECP’s) were obtained from V. areninigrae in order to investigate putative virulence factors. In vivo challenges with ECP’s caused >90% mortalities within the first 24 h. In vitro challenges with ECP’s of hemocytes induced cytotoxicity of hemocytes within the first hour of exposure. These findings represent the first report that V. areninigrae is a highly pathogenic bacterium that can cause disease in crustaceans. On the contrary, V. metschnikovii could not represent a threat for freshwater crayfish.
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| 13. |
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| 14. |
- Jearaphunt, Miti, et al.
(author)
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Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity
- 2014
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In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 10:4, s. e1004059-
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Journal article (peer-reviewed)abstract
- Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.
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| 15. |
- Jiravanichpaisal, Pikul, et al.
(author)
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Characterization of white spot syndrome virus replication in in vitro-cultured haematopoietic stem cells of freshwater crayfish, Pacifastacus leniusculus
- 2006
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In: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 87:Pt 4, s. 847-854
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Journal article (peer-reviewed)abstract
- Replication of White spot syndrome virus (WSSV) was investigated in haematopoietic cells (hpt cells) derived from haematopoietic tissue (hpt) of freshwater crayfish, Pacifastacus leniusculus. Temperature and type of inoculum for virus replication were studied. The cell culture remained viable at a wide range of temperatures ranging from 4 to 25 degrees C. WSSV replicated in cells, as evidenced by in situ hybridization, RT-PCR and by the presence of virions visualized with an electron microscope. Moreover, the results showed that the infectivity of WSSV to hpt cells is dependent on temperature and a supplemented growth factor (cytokine) astakine. WSSV replicated more rapidly at higher temperatures than at lower temperatures. No virus replication was observed at 4 degrees C. Detectable WSSV-infected cells were present as early as 36 In post-inoculation, demonstrated by in situ hybridization or RT-PCR of VP28 expression at 25 degrees C. Hot cells can survive a few weeks at 25 or 16 degrees C and longer than several months at 4 degrees C.
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| 16. |
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| 17. |
- Jiravanichpaisal, Pikul, et al.
(author)
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Expression of immune-related genes in larval stages of the giant tiger shrimp, Penaeus monodon
- 2007
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In: Fish and Shellfish Immunology. - : Elsevier BV. - 1050-4648 .- 1095-9947. ; 23:4, s. 815-824
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Journal article (peer-reviewed)abstract
- Shrimp undergo several morphologically different stages during development and therefore the expression of some immune-related genes such as prophenoloxidase (proPO), peroxinectin (Prx), crustin (Crus), penaeidin (Pen), transglutaminase (TGase), haemocyanin (Hc) and astakine (Ak) were determined during larval development of the shrimp (Penaeus monodon), i.e. nauplius 4 (N4), protozoea 1 and 3 (Z1 and 3), mysis 3 (My 3), post-larvae 3 (PL3) and also in haemocytes of juveniles. Semi-quantitative RT-PCR analysis showed that all transcripts were already present in the early larval stage of N4 but at different levels. The transcript of proPO was found to be extremely low or even absent at N4, whereas Prx, Crus, Pen, TGase, Hc and Ak were significantly expressed at all larval stages. Up to now expression of proPO and Prx has only been reported from haemocytes in crustaceans and in this study Prx also appeared to be expressed in stages which appear to lack haemocytes. Thus, this may suggest that Prx is expressed in other cells than haemocytes. It is well known among invertebrates that the proPO system plays a crucial role as an immune effector molecule against microbes. However, in this study, the transcript of proPO was low during the larval stages and hardly present at all at N4. This might indicate that the development of immune-competent haemocytes during the larval stages is not completed and as a consequence they are likely to be more susceptible to infectious diseases during these stages.
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| 18. |
- Jiravanichpaisal, Pikul, et al.
(author)
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Inflammation in Arthropods
- 2010
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In: Current pharmaceutical design. - 1381-6128 .- 1873-4286. ; 16:38, s. 4166-4174
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Research review (peer-reviewed)abstract
- The inflammatory process in arthropods includes primarily the recruitment of circulating hemocytes to wounds or sites of microbial infections. Melanization, capsule formation and clotting reactions will finally result in the sealing of wounds. In this review we will focus on recent research about hemolymph clotting and melanization reactions, and the recruitment of hemocytes to wounds and infections. We further describe in more detail new knowledge about crustacean hematopoiesis that is crucial for hemocyte recruitment to the site of an infection and there develop an inflammatory response Moreover, we pay special attention to the gut as an important route of infection in arthropods. Since the gastrointestinal tract provides a first line of defense and regulation of the indigenous bacteria and the intestine often harbors loads of potential pathogenic microorganisms. Therefore the integrity of intestinal epithelium and to maintain the correct flora is crucial to animal health.
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| 19. |
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| 20. |
- Jiravanichpaisal, Pikul, 1962-
(author)
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White Spot Syndrome Virus Interaction with a Freshwater Crayfish
- 2005
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Doctoral thesis (other academic/artistic)abstract
- Viruses are very abundant in water and hence diseases caused by viruses are common in marine organisms. These diseases create great problems for the commercial farming of crustaceans and mussels. One of the most common and most disastrous diseases for shrimp is caused by the white spot syndrome virus (WSSV), which is spread all around the world and also is infecting many different species of crustaceans including freshwater crayfish. Although during recent years knowledge has been gathered on the ways in which invertebrates defend themselves against bacteria and fungi virtually nothing is known about the defence processes elicited by virus. The aim of this work was to develop a model to use for studies of virus-host interactions in vivo and in vitro. Temperature was found to be important for the virus infectivity and at lower temperature the virus apparently did not replicate, but if animals kept at low temperature for more than 40 days were transferred to higher temperatures they died quickly due to an increased virus replication. In crayfish infected with the virus it was found that hemocytes did not degranulate and the melanization reaction was also inhibited in the hemocyes. Thus it is apparent that this virus interacts with the immune system and hemocytes in particular and to be able to study this in some greater detail it was necessary to develop a cell culture to study virus-host interactions at the molecular level. Hence, we have developed a stem cell culture from the hematopoietic tissue (hpt) that will differentiate and mature into hemocytes and which can be used to replicate the WSSV in the presence of an endogenous cytokine, astakine. Astakine is the first cytokine like-factor described which is directly involved in hematopoiesis in an invertebrate.
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| 21. |
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| 22. |
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| 23. |
- Ju, Jin Sung, et al.
(author)
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A novel 40-kDa protein containing six repeats of an epidermal growth factor-like domain functions as a pattern recognition protein for lipopolysaccharide
- 2006
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In: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 177:3, s. 1838-1845
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Journal article (peer-reviewed)abstract
- Determination of structures and functions of pattern recognition proteins are important for understanding pathogen recognition mechanisms in host defense and for elucidating the activation mechanism of innate immune reactions. In this study, a novel 40-kDa protein, named LPS recognition protein (LRP), was purified to homogeneity from the cell-free plasma of larvae of the large beetle, Holotrichia diomphalia. LRP exhibited agglutinating activities on Escherichia coli, but not on Staphylococcus aureus and Candida albicans. This E. coli-agglutinating activity was preferentially inhibited by the rough-type LPS with a complete core oligosaccharide. LRP consists of 317 aa residues and six repeats of an epidermal growth factor-like domain-Recombinant LRP expressed in a baculovirus system also showed E. coli agglutination activity in vitro and was able to neutralize LPS by inhibition of LPS-induced IL-6 production in mouse bone marrow mast cells. Furthermore, E. coli coated with the purified LRP were more rapidly cleared in the Holotrichia larvae than only E. coli, indicating that this protein participates in the clearance of E. coli in vivo. The three amino-terminal epidermal growth factor-like domains of LRP, but not the three carboxyl epidermal growth factor-like domains, are involved in the LPS-binding activity. Taken together, this LRP functions as a pattern recognition protein for LPS and plays a role as an innate immune protein.
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| 24. |
- Junkunlo, Kingkamon, et al.
(author)
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A transcription factor glial cell missing (Gcm) in the freshwater crayfish Pacifastacus leniusculus
- 2020
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In: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 113
-
Journal article (peer-reviewed)abstract
- The transcription factor glial cell missing, Gcm, is known to be an important protein in the determination of glial cell fate as well as embryonic plasmatocyte differentiation in Drosophila melanogaster. So far, no function for Gcm in crustaceans has been reported. In this study, we show the cDNA sequence of a Gcm homologue in the freshwater crayfish Pacifastacus leniusculus. The P. leniusculus Gcm transcript is expressed exclusively in brain and nervous tissue, and by in situ hybridization we show that the expression is restricted to a small number of large cells with morphology similar to neurosecretory cells. Furthermore, we show that the expression of Gcm coincides with the expression of a Repo homologue, that is induced in expression by Gcm in Drosophila. Moreover, the Gcm transcript is increased shortly and transiently after injection of cystamine, a substance that inhibits transglutaminase and also strongly affects the movement behavior of crayfish. This finding of Gcm transcripts in a subpopulation of brain cells in very low numbers may enable more detailed studies about Gcm in adult crustaceans.
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| 25. |
- Junkunlo, Kingkamon, et al.
(author)
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Clotting protein : An extracellular matrix (ECM) protein involved in crustacean hematopoiesis
- 2018
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In: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 78, s. 132-140
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Journal article (peer-reviewed)abstract
- Hematopoietic progenitor cells in crustaceans are organized in lobule-like structures surrounded by different types of cells and extracellular matrix (ECM) proteins in a Hematopoietic tissue (HPT). Here we show that the clotting protein (CP) is part of the ECM in HPT and is secreted during HPT cell culture. The formation of a filamentous network of CP was observed in HPT cell culture. A high amount of CP protein was detected at the surfaces of undifferentiated cells (round-shaped) compared with migrating cells (spindle shaped). Co-localization of the CP protein and TGase activity was observed on the cell surface and filamentous network between cells. A role for CP together with collagen was revealed in a 3D culture in which a collagen-I matrix was immobilized with CP or supplemented with CP. The results showed possible functions of CP, collagen, TGase and the cytokine Ast1 in the regulation of HPT progenitor cell behavior. This is the first study to provide insight into the role of CP, which probably not only participates in clot formation but also functions as an ECM component protein controlling hematopoietic stem cell behavior.
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| 26. |
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| 27. |
- Junkunlo, Kingkamon, et al.
(author)
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PDGF/VEGF-related receptor affects transglutaminase activity to control cell migration during crustacean hematopoiesis
- 2017
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In: Stem Cells and Development. - : Mary Ann Liebert Inc. - 1547-3287 .- 1557-8534. ; 26:20, s. 1449-1459
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Journal article (peer-reviewed)abstract
- The platelet-derived growth factor (PDGF) receptor, a tyrosine kinase (TK) receptor whose ligand is PDGF, is crucial in the transduction of extracellular signals into cells and mediates numerous processes, such as cell proliferation, differentiation, survival, and migration. We demonstrate the important roles of a receptor TK related to the PDGF/VEGF family protein (PVR) in controlling hematopoietic progenitor cell migration by affecting extracellular transglutaminase (TGase) activity. Pl_PVR1, GenBank accession No. KY444650, is highly expressed in hemocytes and the hematopoietic tissue (HPT). Sunitinib malate was used to block the PVF/PVR downstream pathway in HPT cell culture. The addition of Sunitinib also caused the HPT cells to increase in size and begin spreading. An increase in extracellular TGase activity on the HPT cell membrane was observed in a dose-dependent manner after treatment with Sunitinib malate. The presence of crude Ast1 provided a combinatorial beneficial effect that enhanced the number of spreading cells after inhibition of the Pl_PVR downstream signaling cascade. In addition, an increased immunoreactivity for beta-tubulin and elongation of beta-tubulin filaments were found in Pl_PVR signaling-inhibited cells. The potential roles of PVF/PVR signaling in controlling progenitor cell activity during hematopoiesis in crayfish were investigated and discussed.
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| 28. |
- Junkunlo, Kingkamon, et al.
(author)
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Reactive oxygen species affect transglutaminase activity and regulate hematopoiesis in a crustacean
- 2016
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:34, s. 17593-17601
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Journal article (peer-reviewed)abstract
- Reactive oxygen species (ROS) serve as a prime signal in the commitment to hematopoiesis in both mammals and Drosophila. In this study, the potential function of ROS during hematopoiesis in the crayfish Pacifastacus leniusculus was examined. The antioxidant N-acetylcysteine (NAC) was used to decrease ROS in both in vivo and in vitro experiments. An increase in ROS was observed in the anterior proliferation center (APC) after LPS injection. In the absence of NAC, the LPS-induced increase in ROS levels resulted in the rapid restoration of the circulating hemocyte number. In the presence of NAC, a delay in the recovery rate of the hemocyte number was observed. NAC treatment also blocked the spread of APC and other hematopoietic tissue (HPT) cells, maintaining these cells at an undifferentiated stage. Extracellular transglutaminase (TGase) has been shown previously to play a role in maintaining HPT cells in an undifferentiated form. In this study, we show that extracellular TGase activity increased when the ROS level in HPT or APC cells was reduced after NAC treatment. In addition, collagen, a major component of the extracellular matrix and a TGase substrate were co-localized on the HPT cell surface. Taken together, the results of this study show that ROS are involved in crayfish hematopoiesis, in which a low ROS level is required to maintain hematopoietic progenitor cells in the tissue and to reduce hemocyte release. The potential roles of TGase in this process are investigated and discussed.
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| 29. |
- Junkunlo, Kingkamon
(author)
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Regulation of hematopoiesis in the freshwater crayfish, Pacifastacus leniusculus : role of transglutaminase
- 2017
-
Doctoral thesis (other academic/artistic)abstract
- The freshwater crayfish, Pacifastacus leniusculus, has been used as a model for studying hematopoiesis or blood cell production or hematopoiesis and immunity. The work of this thesis aims to investigate the impact of factors such as ROS signaling, Ast1, and the PVF/PVR signaling pathway in controlling stem cell behavior during hematopoiesis and specifically the role of the crosslinking enzyme transglutaminase (TGase) in regulation of hematopoiesis.The role of ROS in crayfish hematopoiesis was characterized by using the antioxidant named NAC to inhibit ROS production. Low ROS level resulted in a prolonged decrease in hemocyte numbers and a combined injection of LPS and NAC caused a slower rate of new hemocyte production. A low ROS level in cell cultures supplemented with crude Ast1 was found to inhibit cell spreading and a high extracellular TGase activity was detected on the surfaces of APC and HPT cells. We suggest that ROS serves as a prime signal to control proliferation and differentiation of progenitor cells by affecting extracellular TGase activity. We reported an inhibitory effect of Ast1 on TGase enzyme activity and on its crosslinking activity and consequently Ast1 affects the clot formation and thus coagulation by inhibiting the crosslinking activity of the TGase enzyme. Secretion of the clot protein (CP) and the production of CP filament network between spreading cells were observed in HPT cell cultures in vitro. In the presence of CP together with Ast1 in 3D-collagen-I cultures, HPT cells were found to be more elongated and they formed chains of cells throughout the surrounding matrix. In the HPT tissue, CP was located around the HPT cells or around the lobules of HPT, and thus, CP was demonstrated to be a part of ECM and to possibly function together with collagen in generating a suitable environment for HPT progenitor cells. The inhibition of PVF/PVR downstream signaling pathway by Sunitinib malate resulted in a dramatic change of cell morphology and induction of an increase cell surface area during cell culture. The addition of crude Ast1 into the cell cultures in vitro enhanced this effect. Consequently, cell migration was stimulated and a high extracellular TGase activity on HPT cell surface was found after this inhibition. In conclusion, the work in this thesis provides new insight in understanding the role of the extracellular matrix (ECM) and extracellular TGase activity in controlling stem cell activity.
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| 30. |
- Junkunlo, Kingkamon, et al.
(author)
-
Transglutaminase 1 and 2 are localized in different blood cells in the freshwater crayfish Pacifastacus leniusculus
- 2020
-
In: Fish and Shellfish Immunology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 1050-4648 .- 1095-9947. ; 104, s. 83-91
-
Journal article (peer-reviewed)abstract
- In the present study we show that hemocytes in the freshwater crayfish Pacifastacus leniusculus express two different transglutaminases. We describe the sequence of a previously unknown TGase (Pl_TGase1) and named this as Pl_TGase2 and compared this sequence with similar sequences from other crustaceans. The catalytic core domain is similar to the previously described TGase in P. leniusculus, but Pl_TGase2 has significant differences in the N-terminal and C-terminal domains. Further, we show conclusive evidences that these different transglutaminases are specific for different hemocyte types so that Pl_TGase1 is expressed in the hematopoietic tissue and in the cytoplasm of semigranular hemocytes, while Pl_TGase2 is expressed in vesicles in the granular hemocytes. By in situ hybridization we show that both Pl_TGase1 and Pl_TGase2 mRNA are present only in a subset of the respective hemocyte population. This observation indicates that there may be different subtypes of semigranular as well as granular hemocytes which may have different specific functions.
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| 31. |
- Junkunlo, Kingkamon, et al.
(author)
-
Transglutaminase inhibition stimulates hematopoiesis and reduces aggressive behavior of crayfish, Pacifastacus leniusculus
- 2019
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; :2, s. 708-715
-
Journal article (peer-reviewed)abstract
- Transglutaminase (TGase) is a Ca2+-dependent cross-linking enzyme, which has both enzymatic and nonenzymatic properties. TGase is involved in several cellular activities, including adhesion, migration, survival, apoptosis, and extracellular matrix (ECM) organization. In this study, we focused on the role of the TGase enzyme in controlling hematopoiesis in the crayfish, Pacifastacus leniusculus. We hypothesized that a high TGase activity could mediate an interaction of progenitor cells with the ECM to maintain cells in an undifferentiated stage in the hematopoietic tissue (HPT). We found here that the reversible inhibitor cystamine decreases the enzymatic activity of TGase from crayfish HPT, as well as from guinea pig, in a concentration-dependent manner. Cystamine injection decreased TGase activity in HPT without affecting production of reactive oxygen species. Moreover, the decrease in TGase activity in the HPT increased the number of circulating hemocytes. Interestingly the cystamine-mediated TGase inhibition reduced aggressive behavior and movement in crayfish. In conclusion, we show that cystamine-mediated TGase inhibition directly releases HPT progenitor cells from the HPT into the peripheral circulation in the hemolymph and strongly reduces aggressive behavior in crayfish.
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| 32. |
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| 33. |
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| 34. |
- Korkut, Gül Gizem, et al.
(author)
-
The effect of temperature on bacteria-host interactions in the freshwater crayfish, Pacifastacus leniusculus
- 2018
-
In: Journal of Invertebrate Pathology. - : Elsevier BV. - 0022-2011 .- 1096-0805. ; 157, s. 67-73
-
Journal article (peer-reviewed)abstract
- Water temperature is known to affect many aspects of aquatic life including immune responses and susceptibility to diseases. In this context, we studied the effect of temperature on the defense system of the freshwater crayfish Pacifastacus leniusculus. Animals were challenged with two pathogenic Gram-negative bacteria, Aeromonas hydrophila and Pseudomonas gessardii, as well as the bacterial cell wall component lipopolysaccharide (LPS) at two different temperatures, cold (6 °C) and room temperature (22 °C). The immune responses were compared by means of differences in mortality, phagocytosis, bacterial clearance, and the melanization reaction of the hemolymph at these two temperatures. We observed that crayfish survival was higher at cold temperature. The mortality rate was zero at 6 °C following A. hydrophila or LPS injections. Furthermore, the bacteria were completely cleared from crayfish after they had been held at 6 °C for more than 9 days. We also observed a strong melanization reaction of hemolymph at 22 °C when stimulated with LPS, as well as with bacteria. Taken together, our results suggest that the cellular immunity is more effective at low temperature in this cold-adapted animal and pathogens are efficiently removed from the body by mean of phagocytosis.
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| 35. |
- Korkut, Gül Gizem, et al.
(author)
-
The effect of temperature on Pacifastacus leniusculus immunity
-
Journal article (peer-reviewed)abstract
- Global climate change is an upcoming threat to marine as well as freshwater invertebrates andthe temperature around the globe is expected to rise with up to 4°C in the next decade. Thewater temperature is known to affect many aspects of aquatic life including immuneresponses and susceptibility to diseases. In this context, we studied the effect of temperatureon the defense system of the freshwater crayfish Pacifastacus leniusculus. Animals werechallenged with two pathogenic Gram-negative bacteria Aeromonas hydrophila andPseudomonas gessardii, as well as the bacterial cell wall component lipopolysaccharide(LPS) at two different temperatures, one cold and one room temperature. The immuneresponses were compared by means of differences in mortality, phagocytosis, bacterialclearance, and the melanization reaction of the hemolymph at these two temperatures. Weobserved that crayfish survived infections better at cold temperatures. The mortality rate waszero at 6°C following A. hydrophila or LPS injections. Furthermore, the bacteria werecompletely cleared from crayfish after they had been kept at this low temperature for morethan 9 days. We also observed a strong melanization reaction of hemolymph at 22°C whenstimulated with LPS, as well as with bacteria. Taken together, our results suggest that thecellular immunity is more effective at low temperature in this cold-adapted animal andpathogens are efficiently removed from the body by means of phagocytosis.
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| 36. |
- Lin, Xionghui, 1976-, et al.
(author)
-
Ancient Cytokines, the Role of Astakines as Hematopoietic Growth Factors
- 2010
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:37, s. 28577-28586
-
Journal article (peer-reviewed)abstract
- Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins (PKs) are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus, astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1, we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named as astakine 2. Both crustacean astakines lack the N-terminal AVIT motif, which is present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. We have found astakine-like sequences in 19 different invertebrate species, and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis, and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation. Astakine 1 can stimulate proliferation of hematopoietic tissue (Hpt) cells (precursor of hemocytes) as well as specifically induce differentiation of Hpt cells along the semigranular cell lineage, whereas astakine 2 plays a role in granular cell differentiation. Moreover, we discuss the impact of the putative structures of different astakines in comparison with the vertebrate prokineticins.
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| 37. |
- Lin, Xionghui, 1976-, et al.
(author)
-
EVOLUTION OF HEMATOPOIESIS: AN ASTAKINE INDUCED NOVEL HEMATOPOIETIC FACTOR
-
Other publication (other academic/artistic)abstract
- We report here the cloning and initial characterization of a novel invertebrate hematopoietic factor. This factor was identified from the SSH library with the aim to find the downstream genes of an invertebrate cytokine, astakine 1 in the freshwater crayfish Pacifastacus leniusculus. This Pacifastacus Hematopoietic Factor (PHF) was found to be induced in the primary cell culture of crayfish Hpt cells (precursor of crayfish blood cells) by treatment with astakine 1. Silencing PHF did not affect the renewal of Hpt cells in vitro, but induced the apoptosis rate of Hpt cells. PHF is dominantly present in the blood lineage of crayfish (Hpt cells and blood cells), and in vivo RNAi experiment shows that knockdown of this gene results in severe loss of blood cells in the animal. Our data suggest that crayfish PHF is critical for the survival of not only hemocytes but also the Hpt cells by preventing their apoptosis, thus it plays an important role in the hematopoiesis in crayfish. PHF is a small cysteine rich protein (ca. 9 kDa) with high similarity with the N-terminal region of vertebrate CRIM1 and both of them contains an IGFBP variant motif with unknown function. Our study of PHF may also shed light on the function of this untypical IGFBP motif located in the N-terminal of vertebrate CRIM1.
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| 38. |
- Lin, Xionghui, 1976-
(author)
-
Hematopoiesis in a Crustacean
- 2010
-
Doctoral thesis (other academic/artistic)abstract
- Hemocytes (blood cells) play an important role in the immune response in invertebrates, and thus the regulation of hemocyte homeostasis (hematopoiesis) is essential for the host survival against pathogens. Astakine 1, a homologue to vertebrate prokineticins, was first identified in the freshwater crayfish Pacifastacus leniusculus as a cytokine, and was found to be necessary for new hemocyte synthesis and release in vivo, and also to induce spreading and proliferation of Hematopoietic tissue cells (Hpt cells, precursor of hemocytes) in vitro. The work of this thesis is aimed to further our understanding of the molecular mechanisms involved in astakine 1 induced hematopoiesis.Crayfish transglutaminase (Tgase) has been identified in the hemocytes, and is essential for the coagulation reaction. Interestingly this enzyme is exceedingly abundant in the Hpt cells, and the spreading of Hpt cells induced by astakine 1 was accompanied by sequential loss of TGase activity from the surface of these cells. This loss of TGase activity may be an important effect of astakine 1, resulting in recruiting new hemocytes into the circulatory system. Although astakine 1 contain a prokineticin domain, it lacks the conserved N-terminal AVIT motif present in its vertebrate homologues. This motif is important for vertebrate prokineticins to interact with their receptors, indicating a different receptor interaction for crayfish astakine 1. Astakine 1 was indeed found to interact with a completely different receptor, the β-subunit of ATP synthase, on a portion of Hpt cells, and subsequently block its extracellular ATP formation. Surface ATP synthase has been reported on numerous mammalian cells, but now for the first time in an invertebrate. The activity of ATP synthase on the Hpt cells may be important for the survival and proliferation of Hpt cells, but the underlying mechanisms remain further study. With the finding of a second type of astakine in crayfish, invertebrate astakines can be divided into two groups: astakine 1 and astakine 2. The properties of astakine 2 are different from those of astakine 1 both in structure and function. In primary cell culture of Hpt cells, only astakine 1 can promote proliferation as well as differentiation into semigranular cells, whereas astakine 2 may play a potential role in the maturation of granular cells. Moreover, a novel cysteine rich protein, Pacifastacus hematopoiesis factor (PHF), was found to be one target gene of astakine 1 in Hpt cells. Down regulation of PHF results in increased apoptosis in Hpt cells in vitro, and in vivo silencing PHF leads to a severe loss of hemocytes in the animal. Therefore astakine 1 acquires the anti-apoptosis ability by inducing its downstream gene PHF in the Hpt cells. With its ability to promote the survival, proliferation and differentiation of Hpt cells, astakine 1 is proven to be an important hematopoietic growth factor.
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| 39. |
- Lin, Xionghui, et al.
(author)
-
Identification and properties of a receptor for the invertebrate cytokine astakine, involved in hematopoiesis
- 2009
-
In: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 315:7, s. 1171-1180
-
Journal article (peer-reviewed)abstract
- We have recently isolated an invertebrate cytokine from a freshwater crayfish, which we named astakine 1. Interestingly this protein is expressed exclusively in hemocytes and hematopoietic tissue and is essential for the release of new hemocytes into the open circulatory system of these animals. This astakine has a prokineticin (PK) domain but lacks the N-terminal AVIT amino acids and hence receptor binding may differ from vertebrate PKs. Accordingly, here we report that a receptor for astakine 1 on hematopoietic tissue (Hpt) cells is identical to the beta-subunit of F1ATP synthase. In this study we have used several different methods to clearly demonstrate that ATP-synthase is located on the plasma membrane of a subpopulation of Hpt cells and there may function as a receptor for astakine, whereas mature blood cells (hemocytes) do not have any ATP-synthase on the outside of their plasma membranes. Our results clearly show that ATP synthase beta subunits are present on the cell surface of Hpt cells and highlight the need for more detailed studies on intracellular traffic connections between mitochondria and other membrane compartments.
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| 40. |
- Lin, Xionghui, 1976-, et al.
(author)
-
Invertebrate astakines - regulators of differentiation in hematopoietic tissues
-
Other publication (other academic/artistic)abstract
- Hematopoiesis is the process by which hemocytes mature and subsequently enter the circulation. Vertebrate prokineticins are known to take part in this process, as are the invertebrate prokineticin domain proteins, astakines. In Pacifastacus leniusculus astakine 1 is essential for the release of new hemocytes into the open circulatory system of these animals. In addition to astakine 1 we have now cloned a homologue of astakine 1 with an insert of 13 amino acids, named astakine 2. Common to both crustacean astakines is the lack of the N-terminal AVIT amino acids present in vertebrate PKs, and hence receptor binding differs from that of vertebrate PKs. Now we have found astakine-like sequences in 19 different invertebrate species and the sequences show that some motifs are conserved among invertebrate groups. Previously we showed that astakine 1 is directly involved in hematopoiesis and now we show that astakine 1 and astakine 2 have different roles in hemocyte lineage differentiation and that astakine 1 specifically induce differentiation along the semigranular cell lineage. Further we discuss the impact of the putative structure of different astakines in comparison with the vertebrate prokineticins.
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| 41. |
- Lin, Xionghui, et al.
(author)
-
Invertebrate Hematopoiesis : An Astakine-Dependent Novel Hematopoietic Factor
- 2011
-
In: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 186:4, s. 2073-2079
-
Journal article (peer-reviewed)abstract
- A novel factor, named crustacean hematopoietic factor (CHF), was identified from a library of suppression subtractive hybridization with the aim to find downstream genes of an invertebrate cytokine, astakine 1, in the freshwater crayfish Pacifastacus leniusculus. CHF is a small cysteine-rich protein (∼9 kDa) with high similarity to the N-terminal region of vertebrate CRIM1 in containing an insulin growth factor binding protein variant motif with unknown function. CHF was found to be induced in primary cell cultures of crayfish hematopoietic tissue (Hpt) cells (precursors of crayfish blood cells) after treatment with astakine 1. Silencing of CHF did not affect the renewal of Hpt cells in vitro, but induced apoptosis of Hpt cells. CHF is exclusively expressed in the blood cell lineage of crayfish (Hpt cells and blood cells), and in vivo RNA interference experiments show that knockdown of this gene results in severe loss of blood cells and a higher apoptotic rate in Hpt. Our data further suggest that crayfish CHF is critical for the survival of hemocytes and Hpt cells by preventing their apoptosis, thus it plays an important role in hemocyte homeostasis in crayfish. Our study of CHF may also shed light on the function of this untypical insulin growth factor binding protein motif located in the N-terminal of vertebrate CRIM1.
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| 42. |
- Lin, Xionghui, et al.
(author)
-
Transglutaminase activity in the hematopoietic tissue of a crustacean, Pacifastacus leniusculus, importance in hemocyte homeostasis
- 2008
-
In: BMC Immunology. - : Springer Science and Business Media LLC. - 1471-2172. ; 9:58, s. 1-11
-
Journal article (peer-reviewed)abstract
- BACKGROUND: Transglutaminases (TGases) form a group of enzymes that have many different substrates and among the most well known are fibrin for Factor XIIIa and the clotting protein in crustaceans. We also found that TGase is an abundant protein in the hematopoietic tissue (Hpt) cells of crayfish and hence we have studied the possible function of this enzyme in hematopoiesis. RESULTS: TGase is one of the most abundant proteins in the Hpt and its mRNA expression as well as enzyme activity is very high in the Hpt cells, lesser in the semi-granular hemocytes and very low in the granular cells. In cultured hematopoietic tissues, high activity was present in cells in the centre of the tissue, whereas cells migrating out of the tissue had very low TGase activity. RNAi experiments using dsRNA for TGase completely knocked down the transcript and as a result the cell morphology was changed and the cells started to spread intensely. If astakine, a cytokine directly involved in hematopoiesis, was added the cells started to spread and adopt a morphology similar to that observed after RNAi of TGase. Astakine had no effect on TGase expression, but after a prolonged incubation for one week with this invertebrate cytokine, TGase activity inside and outside the cells was completely lost. Thus it seems as if astakine addition to the Hpt cells and RNAi of TGase in the cell culture will lead to the same results, i.e. loss of TGase activity in the cells and they start to differentiate and spread. CONCLUSION: The results of this study suggest that TGase is important for keeping the Hpt cells in an undifferentiated stage inside the hematopoietic tissue and if expression of TGase mRNA is blocked the cells start to differentiate and spread. This shows a new function for transglutaminase in preventing hematopoietic stem cells from starting to differentiate and migrate into the hemolymph, whereas their proliferation is unaffected. Astakine is also important for the hematopoiesis, since it induces hemocyte synthesis in the Hpt but now we also show that it in some unknown way participates in the differentiation of the Hpt cells.
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| 43. |
- Liu, Haipeng, et al.
(author)
-
Antilipopolysaccharide factor interferes with white spot syndrome virus replication in vitro and in vivo in the crayfish Pacifastacus leniusculus
- 2006
-
In: Journal of Virology. - 0022-538X .- 1098-5514. ; 80:21, s. 10365-10371
-
Journal article (peer-reviewed)abstract
- In a study of genes expressed differentially in the freshwater crayfish Pacifastacus leniusculus infected experimentally with the white spot syndrome virus (WSSV), one protein, known as antilipopolysaccharide factor (ALF), was chosen, among those whose transcript levels increased upon viral infection, for further studies. ALF RNA interference (RNAi) experiments in whole animals and in cell cultures indicated that ALF can protect against WSSV infection, since knockdown of AILF by RNAi specifically resulted in higher rates of viral propagation. In a cell culture of hematopoietic tissue (Hpt) from P. leniusculus, quantitative PCR showed that knockdown of ALF by RNAi resulted into WSSV levels that were about 10-fold higher than those treated with control double-stranded RNA (dsRNA). In addition, RNAi experiments with other crayfish genes that had been found to be up-regulated by a WSSV infection did not result in any changes of viral loads. Thus, the cell culture does not respond to dsRNA in a similar manner, as shown earlier for dsRNA injected into shrimp, which gave a higher degree of resistance to WSSV infection. If ALF transcription in whole animals was stimulated by the administration of LTV-treated WSSV, a partial protection against a subsequent challenge with the active virus was conferred to the host. This is the first crustacean gene product identified with the capacity to interfere with replication of this important pathogen.
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| 44. |
- Liu, Haipeng, 1976-
(author)
-
Functional Studies of Some Immune Relevant Genes in a Crustacean
- 2008
-
Doctoral thesis (other academic/artistic)abstract
- The freshwater crayfish, Pacifastacus leniusculus, mounts a strong innate immune response against microbes such as viruses and bacteria. In this thesis, a novel RNA interference (RNAi) method mediated with histone H2A was developed and applied in crayfish hematopoietic tissue cell cultures for gene functional studies. Further, the interactions between host (crayfish) and pathogens (white spot syndrome virus and Aeromonas hydrophila, respectively) were studied using RNAi technology in live animals. An antilipopolysaccharide factor isolated from viral challenged crayfish by suppression subtractive hybridization was shown to interfere with the propagation of white spot syndrome virus both in vivo and in vitro in crayfish, suggesting an important role of this factor in antiviral defense. Besides, RNAi of phenoloxidase, a critical immune effector involved in melanization, revealed that phenoloxidase activity is necessary for crayfish immune defense against a highly pathogenic bacterial infection in crayfish. In addition, RNAi was also employed to study a marker protein gene involved in hemocyte maturation in crayfish. Taken together, these studies may provide more insights into the immune responses against pathogen invasion as well as hemocyte ontogenesis in crustaceans.
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| 45. |
- Liu, Haipeng, et al.
(author)
-
Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?
- 2011
-
In: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 35:1, s. 51-61
-
Journal article (peer-reviewed)abstract
- Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30 kDa protein from hemocytes of the freshwater crayfish, Pacifastocus leniusculus, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type pepticloglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the Pl-SPH1 and lipopolysaccharide- and beta-1,3-glucan-bincling protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two Pl-SPlis are involved in this activation possibly by forming a complex with LGBP and without a PGRP.
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| 46. |
- Liu, Haipeng, et al.
(author)
-
Phenoloxidase is an important component of the defense against Aeromonas hydrophila infection in a crustacean, Pacifastacus leniusculus
- 2007
-
In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:46, s. 33593-33598
-
Journal article (peer-reviewed)abstract
- The melanization cascade, in which phenoloxidase is the terminal enzyme, appears to play a key role in recognition of and defense against microbial infections in invertebrates. Here, we show that phenoloxidase activity and melanization are important for the immune defense toward a highly pathogenic bacterium, Aeromonas hydrophila, in the freshwater crayfish, Pacifastacus leniusculus. RNA interference-mediated depletion of crayfish prophenoloxidase leads to increased bacterial growth, lower phagocytosis, lower phenoloxidase activity, lower nodule formation, and higher mortality when infected with this bacterium. In contrast, if RNA interference of pacifastin, an inhibitor of the crayfish prophenoloxidase activation cascade, is performed, it results in lower bacterial growth, increased phagocytosis, increased nodule formation, higher phenoloxidase activity, and delayed mortality. Our data therefore suggest that phenoloxidase is required in crayfish defense against an infection by A. hydrophila, a highly virulent and pathogenic bacterium to crayfish.
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| 47. |
- Noonin, Chadanat, et al.
(author)
-
Crayfish hematopoietic tissue as a model for stem cell development in arthropods
- 2012
-
Conference paper (peer-reviewed)abstract
- Arthropods, are suitable model animals to study the regulation of blood cell synthesis and differentiation of the innate immune system, since they lack the lymphocytes, and oxygen-carrying erythrocytes. In contrast to most insects, many crustaceans have a long life span and need to continuously synthesize blood cells. Crayfish hematopoiesis takes place in the hematopoietic tissue (HPT). The HPT of Pacifastacus leniusculus provides a simple model to study hematopoiesis because the tissue can be isolated and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. This tissue was earlier shown to be localized at the dorsal part of stomach. Here, we show that the HPT extends towards the anterior part the animal and link to the brain. Staining of HPT sections revealed that the most anterior of the tissue close to the brain contains higher percentage of cells with loose chromatin, whereas most of the cells in the posterior part have dark nuclear staining with condense chormatin. BrdU incorporation and immunostaining for phospho-histone H3 indicates that the actively proliferating cells occupy the anterior part of the tissue especially in the area close to the brain, proposed stem cell center (SCC). In contrast the more differentiated cells reside in the posterior part. Injection of LPS, which induced blood loss mimicking a bacterial infection, stimulated HPT cell proliferation especially in the anterior part of the tissue. High ROS level was found close to proliferating SCC and the brain, and laminarin-induced hemocyte loss caused induction of ROS level in SCC. This indicates the involvement of ROS in crayfish hematopoiesis. Isolated cells from SCC actively divide and form cell clusters whereas the cells from the remaining HPT from monolayer in in vitro culture. Collagen-I-matrix gel provided an appropriate environment for HPT cell culture and exhibited a suitable system to study HPT cell proliferation and differentiation indicating by induction of hemocyte marker transcripts. Being easily isolated and studied both in vitro and in vivo on stem cell proliferation as well as differentiation into mature hemocytes suggests that crayfish HPT provides an alternative simple model system to study hematopoiesis in arthropods. Moreover, the discovery of the astakine cytokines and antiapoptotic factor CHF offers an opportunity to explore the regulation of invertebrate hematopoiesis and its connection to the central nervous system as well as give new information on the evolution of different blood cell lineages.
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| 48. |
- Noonin, Chadanat, et al.
(author)
-
Invertebrate hematopoiesis : an anterior proliferation centre as a link between the hematopoietic tissue and the brain
- 2012
-
In: Stem Cells and Development. - : Mary Ann Liebert Inc. - 1547-3287 .- 1557-8534. ; 21:17, s. 3173-3186
-
Journal article (peer-reviewed)abstract
- During evolution, the innate and adaptive immune systems developed to protect organisms from nonself substances. The innate immune system is phylogenetically more ancient and is present in most multicellular organisms, whereas adaptive responses are restricted to vertebrates. Arthropods, lack the blood cells of the lymphoid lineage, and oxygen-carrying erythrocytes, making them suitable model animals to study the regulation of the blood cells of the innate immune system. Many crustaceans have a long life span and need to continuously synthesize blood cells, in contrast to many insects. The hematopoietic tissue (HPT) of Pacifastacus leniusculus provides a simple model to study hematopoiesis because the tissue can be isolated and the proliferation of stem cells and their differentiation can be studied both in vivo and in vitro. Here we demonstrate new findings of a physical link between the HPT and the brain. Actively proliferating cells were localized to an anterior proliferation centre (APC) in the anterior part of the tissue near the area linking the HPT to the brain, whereas more differentiated cells were detected in the posterior part. The central areas of HPT expand in response to lipopolysaccharide-induced blood loss. Cells isolated from the APC divide rapidly and form cell clusters in vitro; conversely, the cells from the remaining HPT form monolayers, and they can be induced to differentiate in vitro. Our findings offer an opportunity to learn more about invertebrate hematopoiesis and its connection to the central nervous system and thereby to obtain new information about the evolution of different blood and nerve cell lineages.
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| 49. |
- Noonin, Chadanat
(author)
-
Melanization and Hemocyte Homeostasis in the Freshwater Crayfish, Pacifastacus leniusculus
- 2013
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Doctoral thesis (other academic/artistic)abstract
- Blood cells or hemocytes play important roles in immunity. They are a major source of many immune-related molecules such as antibodies in adaptive immunity of vertebrates and prophenoloxidase (proPO) in invertebrates. In the crayfish Pacifastacus leniusculus, the proPO-system has been reported to be an important component of immune responses against microorganisms. In this study, several mutant strains of Aeromonas hydrophila were used to reveal that LPS (lipopolysaccharide) is an important factor for the pathogenicity of A. hydrophila, strongly inducing the proPO system and melanization. This proPO activating system is a multistep process, which has to be tightly controlled to avoid the harmful side effects of toxic intermediates. Many regulating factors have been reported to fine-tune the proPO-system. In this study, the cleavage of caspase-1-like activity was shown to be a novel negative regulator of PO activity in crayfish. Moreover, the fragments obtained by cleavage of proPO by the proPO-activating enzyme and caspase-1-like protein increased bacterial clearance. Thus, the peptides generated also have important biological functions.In addition to being a source of immune proteins, hemocytes also participate in phagocytosis, encapsulation, and nodulation. An infection normally causes a reduction of hemocyte numbers. Consequently, hemocyte homeostasis is important for maintaining appropriate hemocyte numbers in the circulation of the animal. This study shows that the reactive oxygen species level in the anterior proliferation center of crayfish hematopoietic tissue (HPT), together with cell proliferation, was increased during infection. Pl-β-thymosins were proposed to be involved in hemocyte homeostasis by increasing stem cell migration and thus increasing the circulating hemocyte number. Crayfish hemocyte numbers, as well astakine (Ast1 and Ast2) expression in hemocytes and HPT, were previously shown to be under circadian regulation. Here, we show that Ast1, Ast2, and proPO exhibit rhythmic expression in the crayfish brain similarly to their orthologs, prokineticin 1, prokineticin 2 and tyrosinase, respectively, in the zebrafish brain. Tyrosinase expression was detected in zebrafish brain cells while PO-positive cells were identified as hemocytes that had infiltrated into the crayfish brain. Therefore, this information suggests a close relationship between crayfish hemocytes and the crayfish brain as well as vertebrate neurons.
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- Noonin, Chadanat, et al.
(author)
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Melanization and Pathogenicity in the Insect, Tenebrio molitor, and the Crustacean, Pacifastacus leniusculus, by Aeromonas hydrophila AH-3
- 2010
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:12, s. e15728-
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Journal article (peer-reviewed)abstract
- Aeromonas hydrophila is the most common Aeromonas species causing infections in human and other animals such as amphibians, reptiles, fish and crustaceans. Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Several mutant strains of A. hydrophila AH-3 were initially used to study their virulence in two animal species, Pacifastacus leniusculus (crayfish) and Tenebrio molitor larvae (mealworm). The AH-3 strains used in this study have mutations in genes involving the synthesis of flagella, LPS structures, secretion systems, and some other factors, which have been reported to be involved in A. hydrophila pathogenicity. Our study shows that the LPS (O-antigen and external core) is the most determinant A. hydrophila AH-3 virulence factor in both animals. Furthermore, we studied the immune responses of these hosts to infection of virulent or non-virulent strains of A. hydrophila AH-3. The AH-3 wild type (WT) containing the complete LPS core is highly virulent and this bacterium strongly stimulated the prophenoloxidase activating system resulting in melanization in both crayfish and mealworm. In contrast, the ΔwaaE mutant which has LPS without O-antigen and external core was non-virulent and lost ability to stimulate this system and melanization in these two animals. The high phenoloxidase activity found in WT infected crayfish appears to result from a low expression of pacifastin, a prophenoloxidase activating enzyme inhibitor, and this gene expression was not changed in the ΔwaaE mutant infected animal and consequently phenoloxidase activity was not altered as compared to non-infected animals. Therefore we show that the virulence factors of A. hydrophila are the same regardless whether an insect or a crustacean is infected and the O-antigen and external core is essential for activation of the proPO system and as virulence factors for this bacterium.
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