| 1. |
- Apitanyasai, Kantamas, et al.
(författare)
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Characterization of a hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus
- 2016
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Ingår i: Fish and Shellfish Immunology. - : Elsevier BV. - 1050-4648 .- 1095-9947. ; 58, s. 429-435
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Tidskriftsartikel (refereegranskat)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. |
- Benton, Jeanne, et al.
(författare)
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Cells from the Immune System Generate Adult-Born Neurons in Crayfish
- 2014
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Ingår i: Developmental Cell. - : Cell Press. - 1534-5807 .- 1878-1551. ; 30:3, s. 322-333
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Tidskriftsartikel (refereegranskat)abstract
- Neurogenesis is an ongoing process in the brains of adult decapod crustaceans. However, the first-generation precursors that produce adult-born neurons, which reside in a neurogenic niche, are not self-renewing in crayfish and must be replenished. The source of these neuronal precursors is unknown. Here, we report that adult-born neurons in crayfish can be derived from hemocytes. Following adoptive transfer of 5-ethynyl-2′-deoxyuridine (EdU)-labeled hemocytes, labeled cells populate the neurogenic niche containing the first-generation neuronal precursors. Seven weeks after adoptive transfer, EdU-labeled cells are located in brain clusters 9 and 10 (where adult-born neurons differentiate) and express appropriate neurotransmitters. Moreover, the number of cells composing the neurogenic niche in crayfish is tightly correlated with total hemocyte counts (THCs) and can be manipulated by raising or lowering THC. These studies identify hemocytes as a source of adult-born neurons in crayfish and demonstrate that the immune system is a key contributor to adult neurogenesis.
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| 3. |
- Hernandez-Perez, Ariadne, et al.
(författare)
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Environmental concentrations of sulfamethoxazole increase crayfish Pacifastacus leniusculus susceptibility to White Spot Syndrome Virus
- 2020
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Ingår i: Fish and Shellfish Immunology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 1050-4648 .- 1095-9947. ; 102, s. 177-184
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Tidskriftsartikel (refereegranskat)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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| 4. |
- Jearaphunt, Miti, et al.
(författare)
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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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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 10:4, s. e1004059-
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Tidskriftsartikel (refereegranskat)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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| 5. |
- Junkunlo, Kingkamon, et al.
(författare)
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PDGF/VEGF-related receptor affects transglutaminase activity to control cell migration during crustacean hematopoiesis
- 2017
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Ingår i: Stem Cells and Development. - : Mary Ann Liebert Inc. - 1547-3287 .- 1557-8534. ; 26:20, s. 1449-1459
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Junkunlo, Kingkamon, et al.
(författare)
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Reactive oxygen species affect transglutaminase activity and regulate hematopoiesis in a crustacean
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:34, s. 17593-17601
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Junkunlo, Kingkamon
(författare)
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Regulation of hematopoiesis in the freshwater crayfish, Pacifastacus leniusculus : role of transglutaminase
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 8. |
- Korkut, Gül Gizem, et al.
(författare)
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Expression of an active and inactive clip-domain Serine Protease Isolated from Signal Crayfish in an insect cell line
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In this study, we aimed to elucidate the cleavage specificity and endogenous substrate of a clip-domain serine protease from the crayfish Pacifastacus leniusculus (PlcSP). This serine protease can bind to white spot syndrome virus (WSSV) and is important for the entry of WSSV into the hematopoietic tissue (HPT). In order to understand its intrinsic role in the serine protease cascade, we wanted to use substrate phage display method to clarify cleavage specificity of PlcSP. Firstly, we aimed to make recombinant active and inactive forms of this PlcSP using human embryonic kidney cell line (HEK293) however PlcSP appeared to be toxic for mammalian cells and we were not able to express this crustacean protein. However, we were able to express these recombinantPlcSP both in their zymogenic and active forms in Sf9 insect cell line.
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| 9. |
- Korkut, Gül Gizem, 1986-
(författare)
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Interaction between crayfish and some microorganisms; Effect of temperature
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Innate immunity, which constitutes the first line of defense in vertebrates, is the only immune system that invertebrates rely on to protect themselves from pathogens. The invertebrate immune system is composed of cellular and humoral components. Cellular immunity is phagocytosis, opsonization and encapsulation. The humoral part is mainly composed of the events taking place upon secretion of granules and the enzymes within that lead to the lysis of the pathogen by antimicrobial peptides (AMPs) and the melanization cascade. The Prophenoloxidase (proPO) activating system is an important pathway that is stored in the granules of semigranular and granular hemocytes (blood cells). These cells will degranulate and release the proPO system when activated upon pathogen recognition. This cascade results in the melanization reaction and to trap and eliminate pathogens. White spot syndrome virus (WSSV) is a deadly pathogen mainly targeting crustaceans and causing huge economic losses since its first emergence in 1992 in Taiwan. It is known that WSSV disables the immune system of the host by interfering with the proPO cascade. Temperature is a restricting factor for the WSSV infections however it is not known if its affects are on host immunity or on the virus itself.With the aim of elucidating WSSV infection, we studied the virus entry mechanisms. By crosslinking WSSV with the hemocytes we showed that a new clip-domain serine protease (PlcSP) plays an important role during the WSSV infection in crayfish by means of interacting with WSSV envelope protein VP28. Moreover, we have shown that the viral entry is inhibited at cold temperatures due to temperature’s inhibitory effect on PlcSP expression. We also showed that by slowing down of the host’s metabolism hence proliferation in host tissue either by low temperature or cell cycle inhibitors, we could inhibit WSSV replication once it has entered the host cell. We tested if the temperature effects host or pathogen, or both, we investigated the mortalities, phagocytosis, bacterial clearance, total hemocyte counts, degranulation and melanization rate of crayfish under a cold and warm temperature by using two strains of gram-negative bacteria and LPS. It is apparent that the cellular immunity is more effective at low temperature while the humoral immunity can become overactivated and toxic for the host at higher temperature. Furthermore, we aimed to study the cleavage specificity for PlcSP since it is predicted to be secreted from hemocytes and takes part in the serine protease cascade during melanization reaction.
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| 10. |
- Korkut, Gül Gizem, et al.
(författare)
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The effect of temperature on bacteria-host interactions in the freshwater crayfish, Pacifastacus leniusculus
- 2018
-
Ingår i: Journal of Invertebrate Pathology. - : Elsevier BV. - 0022-2011 .- 1096-0805. ; 157, s. 67-73
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Tidskriftsartikel (refereegranskat)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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| 11. |
- Korkut, Gül Gizem, et al.
(författare)
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The effect of temperature on White Spot Disease progression in a crustacean, Pacifastacus leniusculus
- 2018
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 89, s. 7-13
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Tidskriftsartikel (refereegranskat)abstract
- The effects of temperature on the progression of White Spot Disease (WSD) have been studied in the freshwater crayfish Pacifastacus leniusculus. In this study, we aimed to understand the reason for previously observed low mortalities with white spot syndrome virus (WSSV) infected crayfish at low temperatures. The susceptibility of freshwater crayfish to WSSV was studied at different temperatures. The mortality rate at 6°C was zero, meanwhile the animals kept at 22°C developed WSD symptoms and died in a few days after WSSV injections, however upon transfer of animals from 6°C to 22°C the mortality reached 100% indicating that the virus is not cleared. Moreover, the VP28 expression at 6°C was significantly lower compared to animals kept at 22°C. We injected animals with demecolcine, an inhibitor that arrests the cell cycle in metaphase, and observed a delayed mortality. Furthermore, the VP28 expression was found to be lower in these animals receiving both injections with WSSV and demecolcine since cell proliferation was inhibited by demecolcine. We quantified WSSV copy numbers and found that virus entry was blocked at 6°C, but not in demecolcine treatments. We supported this result by quantifying the expression of a clip domain serine protease (PlcSP) which plays an important role for WSSV binding, and we found that the PlcSP expression was inhibited at 6°C. Therefore, our hypothesis is that the WSSV needs proliferating cells to replicate, and an optimum temperature to enter the host hematopoietic stem cells successfully.
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| 12. |
- Korkut, Gül Gizem, et al.
(författare)
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The effect of temperature on Pacifastacus leniusculus immunity
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Tidskriftsartikel (refereegranskat)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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| 13. |
- Noonin, Chadanat, et al.
(författare)
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Circadian regulation of melanization and prokineticin homologues is conserved in the brain of freshwater crayfish and zebrafish
- 2013
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 40:2, s. 218-226
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Tidskriftsartikel (refereegranskat)abstract
- Circadian clock is important to living organisms to adjust to the external environment. This clock has been extensively studied in mammals, and prokineticin 2 (Prok2) acts as one of the messenger between the central nervous system and peripheral tissues. In this study, expression profiles of Prok1 and Prok2 were investigated in a non-mammalian vertebrate brain, zebrafish, and the expression was compared to the Prok homologues, astakines (Ast1 and Ast2) in crayfish. These transcripts exhibited circadian oscillation in the brain, and Ast1 had similar pattern to Prok2. In addition, the expression of tyrosinase, an enzyme which expression is regulated by E-box elements like in Prok2, was also examined in zebrafish brain and was compared with the expression of prophenoloxidase (proPO), the melanization enzyme, in crayfish brain. Interestingly, the expressions of both Tyr and proPO displayed circadian rhythm in a similar pattern to Prok2 and Ast1, respectively. Therefore, this study shows that circadian oscillation of prokineticin homologues and enzymes involved in melanization are conserved.
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| 14. |
- Noonin, Chadanat, et al.
(författare)
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Crayfish hematopoietic tissue as a model for stem cell development in arthropods
- 2012
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Konferensbidrag (refereegranskat)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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| 15. |
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| 16. |
- Noonin, Chadanat, et al.
(författare)
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Invertebrate hematopoiesis : an anterior proliferation centre as a link between the hematopoietic tissue and the brain
- 2012
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Ingår i: Stem Cells and Development. - : Mary Ann Liebert Inc. - 1547-3287 .- 1557-8534. ; 21:17, s. 3173-3186
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Tidskriftsartikel (refereegranskat)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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| 17. |
- Noonin, Chadanat
(författare)
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Involvement of Serotonin in crayfish hematopoiesis
- 2018
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 86, s. 189-195
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Tidskriftsartikel (refereegranskat)abstract
- Serotonin (5-HT) is a conserved monoamine neurotransmitter that has several physiological functions both in vertebrates and invertebrates. In addition to its well-known function in the central nervous system, 5-HT also participates in peripheral system. However, in crustaceans, the knowledge about peripheral functions of 5-HT is limited. In this study, a role of 5-HT in hematopoiesis in crayfish, Pacifastacus leniusculus, was investigated. The presence of 5-HT in crayfish plasma and the effect of 5-HT injection on hemocyte number were examined. The effects of 5-HT on hematopoietic tissue (HPT) cell proliferation and secretion of the hematopoietic cytokine, astakine 1 (Ast 1) were determined in vitro. The results from this study suggest that 5-HT has no direct effect on HPT cell proliferation, but it participates in crayfish hematopoiesis through stimulating Ast 1 cytokine release from crayfish hemocytes, and thereby affects release of new hemocytes into the circulation.
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| 18. |
- Noonin, Chadanat
(författare)
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Melanization and Hemocyte Homeostasis in the Freshwater Crayfish, Pacifastacus leniusculus
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 19. |
- Noonin, Chadanat, et al.
(författare)
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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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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 5:12, s. e15728-
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Tidskriftsartikel (refereegranskat)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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| 20. |
- Saelee, Netnapa, et al.
(författare)
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beta-Thymosins and Hemocyte Homeostasis in a Crustacean
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:4, s. e60974-
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Tidskriftsartikel (refereegranskat)abstract
- Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (T beta 4) has been associated with biological activities in tissue repair and cell migration via interaction with ATP-synthase in vertebrates, while the information of similar thymosin functions in invertebrates is limited. We have shown previously that ATP-synthase is present on the surface of crayfish hematopoietic tissue (HPT) cells, and that astakine 1 (Ast1, an invertebrate cytokine) was found to interact with this beta-subunit of ATP synthase. Here, we identified five different beta-thymosins from Pacifastacus leniusculus, designated Pl-beta-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-beta-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the b-subunit of ATP-synthase, and Pl-beta-thymosin1, but not Pl-beta-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-beta-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-beta-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-beta-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-beta-thymosin2 had a similar but transient effect. Both Pl-beta-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-beta-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-beta-thymosin1 and Pl-beta-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.
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| 21. |
- Sirikharin, Ratchanok, et al.
(författare)
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Astakine1 forms protein complex in plasma
- 2019
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Ingår i: Fish and Shellfish Immunology. - : Elsevier BV. - 1050-4648 .- 1095-9947. ; 94, s. 66-71
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Tidskriftsartikel (refereegranskat)abstract
- Astakine 1 is a small cytokine-like peptide which is directly involved in hematopoiesis in crustaceans. Astakines are present in many different invertebrate groups primarily in arthropods. In this study we found that astakine1 was present as a high molecular weight (HMW) complex in plasma. It is known that calcium concentration are fluctuating in several crustaceans especially during the molting process. This HMW-complex was formed under low calcium concentrations in plasma and could be partially reversed provided calcium was added. The biological function of the naïve astakine1 and that in the HMW complex was about the same, but if the protein is to be isolated or studied for its function it is important to know about this property of astakine1 which may previously have hampered isolation and functional studies in other animals than freshwater crayfish.
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| 22. |
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| 23. |
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| 24. |
- Wu, Chenglin, et al.
(författare)
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An insect TEP in a crustacean is specific for cuticular tissues and involved in intestinal defense
- 2012
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Ingår i: Insect Biochemistry and Molecular Biology. - : Elsevier BV. - 0965-1748 .- 1879-0240. ; 42:2, s. 71-80
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Tidskriftsartikel (refereegranskat)abstract
- In an attempt to identify genes encoding thioester-containing proteins in the freshwater crayfish, Pacifastacus leniusculus, three different cDNAs were found. A phylogenetic analysis of these proteins indicates that they can be classified into two subfamilies: two alpha-2-macroglobulins (Pl-A2M1, Pl-A2M2) showing a close similarity to shrimp A2M, and one insect TEP-like protein (Pl-TEP). This is the first report of an insect TEP-like protein in a crustacean. Crayfish Pl-A2M1, Pl-A2M2 and Pl-TEP cDNAs encode proteins with 1480, 1586 or 1507 amino acids, respectively. Pl-A2M1, Pl-A2M2 and Pl-TEP have the basic domain structure and functionally important residues for each molecule, and their mRNA was detected in different parts of the body, suggesting that they may have different functions. Pl-A2M1 was mainly expressed in hemocytes and Pl-A2M2 was highly expressed in heart and nerve, while Pl-TEP was exclusively expressed in cuticular tissues such as gill and intestine. RNA interference of Pl-TEP in vivo resulted in that these animals were slightly less resistant when fed with the bacterium, Pseudomonas libanensis/gessardii. Furthermore, when TEP activity was blocked using methylamine followed by bacterial feeding, the animals were killed to a higher extent compared to a control group. Taken together, this indicates that Pl-TEP and/or Pl-A2M1, Pl-A2M2 may be important for the immune defense in crayfish intestine and function as a pattern recognition protein in crayfish cuticular tissues.
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| 25. |
- Zečić, Aleksandra, et al.
(författare)
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Whole-mount in situ hybridization : minimizing the folding problem of thin-sheet tissue-like crayfish haematopoietic tissue
- 2018
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Ingår i: Crustaceana. - : Brill. - 0011-216X .- 1568-5403. ; 91:1, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Crayfish haematopoietic tissue (HPT) has a thin-sheet-like structure with a thickness of 100-160 μm and a width of approximately 1-2 cm. This structure makes HPT extremely easy to fold after removal from the animal. Therefore, it is difficult to handle the tissue without folding when processing for sectioning and histological study. The degree of tissue folding reflects the size of the tissue sections obtained, how complicated it is to interpret the location of each tissue section, and the accuracy of the interpretation of the location of a specific transcript. To facilitate the interpretation of a specific transcript location in the HPT, we optimized a whole-mount in situ hybridization technique to minimize tissue folding. This optimized protocol effectively reduced the tissue folding. Therefore, the location of a specific transcript in the HPT was easily and accurately defined. This protocol will be useful for whole-mount staining of other tissues with similar structure.
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