| 1. |
- Junkunlo, Kingkamon, et al.
(författare)
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A transcription factor glial cell missing (Gcm) in the freshwater crayfish Pacifastacus leniusculus
- 2020
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 113
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Tidskriftsartikel (refereegranskat)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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| 2. |
- Junkunlo, Kingkamon, et al.
(författare)
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Clotting protein : An extracellular matrix (ECM) protein involved in crustacean hematopoiesis
- 2018
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 78, s. 132-140
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Tidskriftsartikel (refereegranskat)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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| 3. |
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| 4. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
- Junkunlo, Kingkamon, et al.
(författare)
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Transglutaminase 1 and 2 are localized in different blood cells in the freshwater crayfish Pacifastacus leniusculus
- 2020
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Ingår i: Fish and Shellfish Immunology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 1050-4648 .- 1095-9947. ; 104, s. 83-91
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Tidskriftsartikel (refereegranskat)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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| 8. |
- Junkunlo, Kingkamon, et al.
(författare)
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Transglutaminase inhibition stimulates hematopoiesis and reduces aggressive behavior of crayfish, Pacifastacus leniusculus
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; :2, s. 708-715
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Tidskriftsartikel (refereegranskat)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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| 9. |
- 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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| 10. |
- 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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| 11. |
- Sirikharin, Ratchanok, et al.
(författare)
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Role of astakine1 in regulating transglutaminase activity
- 2017
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Ingår i: Developmental and Comparative Immunology. - : Elsevier. - 0145-305X .- 1879-0089. ; 76, s. 77-82
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Tidskriftsartikel (refereegranskat)abstract
- Transglutaminase (TGase) has been implicated in maintaining the undifferentiated stage of hematopoietic stem cells (HSC) in the crayfish Pacifastacus leniusculus. TGase activity has been reported to be regulated by astakine1, an essential crayfish cytokine for inducing new hemocyte synthesis in hematopoietic tissue (HPT). Here, the role of astakine1 in TGase activity regulation and clotting protein (CP) cross-linking was characterized. A reduction in TGase activity was observed by the addition of purified astakine1 in vitro for both endogenous crayfish TGase and a commercial purified guinea pig liver TGase. As a result, we observed that astakine1 inhibits TGase enzyme activity and acts as a non-competitive inhibitor for the TGase enzyme. Additionally, the clotting reaction was impaired in the presence of astakine1. A decrease in TGase-mediated crosslinking of ε(γ-glutamyl)-lysine bonds was also observed in the presence of astakine1. In conclusion, this study shows that astakine1 acts as an inhibitor of TGase activity and that it also affects CP cross-linking during crayfish hematopoiesis.
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| 12. |
- Söderhäll, Irene, et al.
(författare)
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A comparative global proteomic analysis of the hematopoietic lineages in the crustacean Pacifastacus leniusculus
- 2019
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 92, s. 170-178
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Tidskriftsartikel (refereegranskat)abstract
- In crustaceans as in other arthropods, the circulating hemocytes are vital for protecting the animal against attacking microorganisms. As many hemocytes are destroyed early during an infection, new hemocytes must fast get in place to prevent disperse of a pathogenic microbe, In order to understand the hematopoietic process in more detail we here report a complete proteomic analysis from purified cell types from the APC of the hematopoietic tissue, via the remaining parts of the HPT to the mature semigranular and granular hemocytes. Several possible cell type specific proteins are detected and new putative biomarkers within the crayfish hematopoietic lineage that can be used to increase the understanding of how the differentiation process is regulated is described.
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| 13. |
- Udompetcharaporn, Attasit, et al.
(författare)
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Identification and characterization of a QM protein as a possible peptidoglycan recognition protein (PGRP) from the giant tiger shrimp Penaeus monodon
- 2014
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Ingår i: Developmental and Comparative Immunology. - : Elsevier BV. - 0145-305X .- 1879-0089. ; 46:2, s. 146-154
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Tidskriftsartikel (refereegranskat)abstract
- In an attempt to identify a peptidoglycan recognition protein (PGRP) in Penaeus (Penaeus) monodon, in vitro pull-down binding assays were used between shrimp proteins and purified peptidoglycan (PG). By gel electrophoresis and mass spectrometry followed by Mascot program analysis, proteins from shrimp hemocyte peripheral membrane proteins showed significant homology to records for a QM protein, actin and prophenoloxidase 2 precursor (proPO2), while proteins from cell-free plasma showed significant homology to records for a vitellogenin, a fibrinogen related protein (FREP) and a C-type lectin. Due to time and resource limitations, specific binding to PG was examined only for recombinant PmQM protein and PmLec that were synthesized based on sequences reported in the Genbank database (accession numbers FJ766846 and DQ078266, respectively). An in vitro assay revealed that hemocytes would bind with and encapsulate agarose beads coated with recombinant PmQM (rPmQM) or rPmLec and that melanization followed 2 h post-encapsulation. ELISA tests confirmed specific binding of rPmQM protein to PG. This is the first time that PmQM has been reported as a potential PGRP in shrimp or any other crustacean. The two other potential PGRP identified (FREP and the vitellin-like protein present in male P. monodon, unlike other vitellin subunits) should also be expressed heterologously and tested for their ability to activate shrimp hemocytes.
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| 14. |
- Österroos, Albin, et al.
(författare)
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Integrated multi-omic profiling of azacitidine-venetoclax in AML reveals additional targetable pathways to improve the treatment
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The combination of venetoclax and azacitidine constitutes the first-line option for patients with acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy. Azacitidine-venetoclax has shown pronounced clinical and preclinical ecacy but the molecular mechanisms causing this synergy remain to be clarified. We applied an integrative multi-omic approach with focus in epigenetics on two AML cell lines to characterize the effects of azacitidine-venetoclax in vitro on chromatin accessibility, DNA methylation and gene expression patterns.We report distinct epigenetic and transcriptomic eects when combining azacitidine and venetoclax as compared to either substance alone. With the application of ATAC-seq, we delineate combination-unique gained pathways including the activation of mRNA splicing and NOTCH-HSF1 signaling but also more widespread heterochromatin compared to azacitidine or venetoclax alone. When assessing methylation status, we observed combination-unique hypermethylation of Rac1- and Rho-associated signaling. We integrate the epigenetic alterations of azacitidine-venetoclax with RNA-seq data and report activation of genes involved in the serine synthesis pathway and NTRK signaling that represent potentially upregulated survival pathways upon azacitidine-venetoclax exposure.We also propose potential synergistic triplet therapies based on in silico drug predictions using condition-wise weighted co-expression networks including inhibitors of proteaseomes, MCL-1 and histone deacetylase. Future studies are needed to investigate whether the inhibition of the mentioned pro-survival pathways or proposed triplets may enhance the effects of azacitidine-venetoclax in AML.
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