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1.
Commun Biol ; 4(1): 959, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381146

RESUMO

The association between kidney stone disease and renal fibrosis has been widely explored in recent years but its underlying mechanisms remain far from complete understanding. Using label-free quantitative proteomics (nanoLC-ESI-LTQ-Orbitrap MS/MS), this study identified 23 significantly altered secreted proteins from calcium oxalate monohydrate (COM)-exposed macrophages (COM-MP) compared with control macrophages (Ctrl-MP) secretome. Functional annotation and protein-protein interactions network analysis revealed that these altered secreted proteins were involved mainly in inflammatory response and fibroblast activation. BHK-21 renal fibroblasts treated with COM-MP secretome had more spindle-shaped morphology with greater spindle index. Immunofluorescence study and gelatin zymography revealed increased levels of fibroblast activation markers (α-smooth muscle actin and F-actin) and fibrotic factors (fibronectin and matrix metalloproteinase-9 and -2) in the COM-MP secretome-treated fibroblasts. Our findings indicate that proteins secreted from macrophages exposed to COM crystals induce renal fibroblast activation and may play important roles in renal fibrogenesis in kidney stone disease.


Assuntos
Oxalato de Cálcio/metabolismo , Fibroblastos/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Animais , Oxalato de Cálcio/química , Cricetinae , Humanos , Mapas de Interação de Proteínas , Células U937
2.
Anal Methods ; 13(30): 3359-3367, 2021 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-34296239

RESUMO

Tamm-Horsfall protein (THP) is a high-abundance urinary protein. Although its functions have been studied for years, several aspects of these remain unclear. To achieve more knowledge on THP functions, an effective isolation/purification method providing a high yield and high purity is required. This is the first report that applied tandem fast protein liquid chromatography (FPLC) (by combining Mono Q anion-exchange with Superdex 200 size-exclusion columns in a tandem manner) to isolate intact THP from human urine. Its efficiency was then systematically compared with that of two conventional methods, diatomaceous earth (DE) adsorption and salt precipitation. The first ever systematic comparisons among the three methods revealed that, while Mono Q-Superdex 200 tandem FPLC offered the lowest %yield and was most time-consuming, it provided substantially high %purity and could selectively purify the monomeric and aggregated forms of urinary THP. On the other hand, DE adsorption provided the highest %yield and %purity, whereas salt precipitation offered the lowest %purity. In summary, the tandem FPLC system is most useful for selective purification of the monomeric and aggregated forms of urinary THP for further functional study, whereas DE adsorption remains the method of choice for general purification of THP from human urine.


Assuntos
Terra de Diatomáceas , Cloreto de Sódio , Adsorção , Cromatografia Líquida de Alta Pressão , Humanos , Uromodulina
3.
Theranostics ; 11(9): 4436-4451, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33754070

RESUMO

Inflammasome is a complex of multiple proteins found in cytoplasm of the cells activated by infectious and/or non-infectious stimuli. This complex involves caspase-1 activation, leading to unconventional secretion of interleukin-1ß (IL-1ß) and IL-18 and inflammatory cascade. Exosome is the nanoscale membrane-bound extracellular vesicle that plays significant roles in intercellular communications by carrying bioactive molecules, e.g., proteins, RNAs, microRNAs (miRNAs), DNAs, from one cell to the others. In this review, we provide the update information on the crosstalk between exosome and inflammasome and their roles in inflammatory responses. The effects of inflammasome activation on exosomal secretion are summarized. On the other hand, the (dual) effects of exosomes on inhibiting and promoting inflammasome activation are discussed. Finally, perspectives on therapeutic roles of exosomes in human diseases and future direction of the research on exosome-inflammasome crosstalk are provided.


Assuntos
Exossomos/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Animais , Comunicação Celular/fisiologia , Humanos
4.
Fish Shellfish Immunol ; 102: 177-184, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32311459

RESUMO

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 µg/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 µg/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 µg/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 µg/L could marginally and for a few days have an immunostimulatory effect.


Assuntos
Proteínas de Artrópodes/imunologia , Astacoidea/efeitos dos fármacos , Sulfametoxazol/efeitos adversos , Poluentes Químicos da Água/efeitos adversos , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Anti-Infecciosos/efeitos adversos , Proteínas de Artrópodes/genética , Astacoidea/virologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
5.
Fish Shellfish Immunol ; 94: 66-71, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31465872

RESUMO

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.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/genética , Astacoidea/imunologia , Cálcio/metabolismo , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/imunologia , Animais , Proteínas de Artrópodes/imunologia , Plasma/química
6.
J Invertebr Pathol ; 157: 67-73, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30077692

RESUMO

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.


Assuntos
Astacoidea/imunologia , Astacoidea/parasitologia , Infecções por Bactérias Gram-Negativas/veterinária , Animais , Interações Hospedeiro-Parasita , Temperatura
7.
Dev Comp Immunol ; 89: 7-13, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30071208

RESUMO

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 at 6 °C. 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.


Assuntos
Astacoidea/virologia , Vírus da Síndrome da Mancha Branca 1/patogenicidade , Animais , Astacoidea/imunologia , Astacoidea/fisiologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Infecções por Vírus de DNA/etiologia , Infecções por Vírus de DNA/veterinária , Demecolcina/farmacologia , Progressão da Doença , Água Doce , Expressão Gênica , Genes Virais , Hemócitos/efeitos dos fármacos , Hemócitos/imunologia , Hemócitos/virologia , Interações Hospedeiro-Patógeno/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Serina Proteases/genética , Temperatura , Proteínas do Envelope Viral/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia , Vírus da Síndrome da Mancha Branca 1/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia
8.
Dev Comp Immunol ; 86: 189-195, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29778989

RESUMO

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.


Assuntos
Astacoidea/metabolismo , Hematopoese/fisiologia , Serotonina/metabolismo , Animais , Proliferação de Células/fisiologia , Fatores de Crescimento de Células Hematopoéticas/metabolismo , Hemócitos/metabolismo
9.
Stem Cells Dev ; 26(20): 1449-1459, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28805145

RESUMO

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 ß-tubulin and elongation of ß-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.


Assuntos
Astacoidea/citologia , Astacoidea/metabolismo , Movimento Celular , Hematopoese , Receptores Proteína Tirosina Quinases/metabolismo , Transglutaminases/metabolismo , Animais , Astacoidea/enzimologia , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Evolução Molecular , Hematopoese/efeitos dos fármacos , Indóis/farmacologia , Pirróis/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/efeitos dos fármacos , Sunitinibe , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo
10.
Fish Shellfish Immunol ; 58: 429-435, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27663854

RESUMO

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 PlHHAP 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 PlHHAP knockdown in vivo, in contrast to the results found in shrimp. Moreover, suppression of PlHHAP 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 PlHHAP in intestine by RNAi resulted in higher number of bacteria in the crayfish intestine. Together, these results suggest that PlHHAP 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 PlHHAP has different functions from PmHHAP, we therefore named it HHAP-like protein.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/fisiologia , Homeostase , Imunidade Inata/genética , Animais , Proteínas de Artrópodes/metabolismo , Astacoidea/genética , Astacoidea/imunologia , Astacoidea/microbiologia , Microbioma Gastrointestinal , Hematopoese , Hemócitos/citologia , Hemócitos/imunologia , Intestinos/imunologia , Intestinos/microbiologia , Análise de Sequência de DNA
11.
J Biol Chem ; 291(34): 17593-601, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27339892

RESUMO

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.


Assuntos
Proteínas de Artrópodes/metabolismo , Astacoidea/metabolismo , Hematopoese/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Transglutaminases/metabolismo , Animais
13.
Pharm Biol ; 54(5): 853-62, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26455646

RESUMO

CONTEXT: Curcuma comosa Roxb. (Zingiberaceae) has traditionally been used as an anti-inflammatory agent in liver, and recent study has shown its hepatoprotective effect against CCl4-induced liver injury in vivo. OBJECTIVE: This study further assesses the protective effect of C. comosa extracts and its isolated compounds against tert-butyl hydroperoxide (t-BHP)-induced hepatotoxicity in isolated primary rat hepatocytes. MATERIALS AND METHODS: Isolated primary hepatocytes were pretreated with either ethanol (5-50 µg/ml) or hexane extract (1-50 µg/ml), or two diarylheptanoids (4-35 µM): compound D-91 [1-(4-hydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol] and compound D-92 [(3S)-1-(3,4-dihydroxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol], from C. comosa for 2 h prior to exposure to 1.5 mM t-BHP for 15 and 30 min. Their hepatoprotective activities were then determined. RESULTS: t-BHP markedly caused the formation of MDA and ALT leakage from the hepatocytes. Pretreatment with the C. comosa ethanol extract showed greater protective effect than the hexane extract, and the effect was concentration related. Treating the hepatocytes with compound D-92 provided greater protective effect than compound D-91. IC50 values of compounds D-91, D-92, and silymarin for the protection of ALT leakage at 30 min were 32.7 ± 1.1, 9.8 ± 0.7, and 160 ± 8 µM, respectively. Further investigation showed that compound D-92 was more effective in maintaining the intracellular glutathione content in the t-BHP treated group, whereas the reduction in antioxidant enzymes, glutathione peroxidase and glutathione-S-transferase activities, were not improved. DISCUSSION AND CONCLUSION: Results suggest that diarylheptanoids are the active principles that provide protection against t-BHP-induced injury. Their ability to maintain intracellular glutathione content is the main mechanisms underlying the protective action.


Assuntos
Curcuma , Diarileptanoides/toxicidade , Hepatócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , terc-Butil Hidroperóxido/toxicidade , Animais , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidação de Lipídeos/fisiologia , Masculino , Extratos Vegetais/isolamento & purificação , Substâncias Protetoras/isolamento & purificação , Substâncias Protetoras/farmacologia , Ratos , Ratos Wistar
14.
Dev Cell ; 30(3): 322-33, 2014 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-25117683

RESUMO

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.


Assuntos
Envelhecimento/imunologia , Astacoidea/citologia , Sistema Imunitário/citologia , Células-Tronco Neurais/citologia , Neurogênese/imunologia , Neurônios/citologia , Animais , Encéfalo/citologia , Encéfalo/imunologia , Movimento Celular/fisiologia , Proliferação de Células , Nicho de Células-Tronco/imunologia
15.
PLoS Pathog ; 10(4): e1004059, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24722332

RESUMO

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.


Assuntos
Proteínas de Artrópodes/imunologia , Astacoidea/imunologia , Caspase 1/imunologia , Catecol Oxidase/imunologia , Precursores Enzimáticos/imunologia , Imunidade Inata/fisiologia , Peptídeos/imunologia , Proteólise , Animais , Proteínas de Artrópodes/metabolismo , Astacoidea/enzimologia , Caspase 1/metabolismo , Catecol Oxidase/metabolismo , Precursores Enzimáticos/metabolismo , Peptídeos/metabolismo
16.
PLoS One ; 8(4): e60974, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23565293

RESUMO

Thymosin proteins are well known for their actin-binding activity. Thymosin beta 4 (Tß4) 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 ß-subunit of ATP synthase. Here, we identified five different ß-thymosins from Pacifastacus leniusculus, designated Pl-ß-thymosin1-5. The two dominant isoforms in brain, HPT and hemocytes, Pl-ß-thymosin1 and 2, were chosen for functional studies. Both isoforms could bind to the ß-subunit of ATP-synthase, and Pl-ß-thymosin1, but not Pl-ß-thymosin2, significantly increased extracellular ATP formation. Moreover, Pl-ß-thymosin1 stimulated HPT cell migration in vitro and Ast1 blocked this effect. Pl-ß-thymosin2 increased the circulating hemocyte number at an early stage after injection. Additionally, in vivo injection of Pl-ß-thymosin1 resulted in significant reduction of reactive oxygen species (ROS) production in crayfish HPT whereas Pl-ß-thymosin2 had a similar but transient effect. Both Pl-ß-thymosins induced the expression of Ast1 and superoxide dismutase (SOD) transcripts, while silencing of endogenous Pl-ß-thymosin 1 and 2 by RNAi resulted in significant reduction of the Ast1 and SOD transcripts. The diverse effects exhibited by Pl-ß-thymosin1 and Pl-ß-thymosin2 indicates that these proteins are involved in a complex interaction that regulates the hematopoietic stem cell proliferation and differentiation.


Assuntos
Crustáceos/enzimologia , Hemócitos/metabolismo , Timosina/metabolismo , Animais , Astacoidea , Homeostase , Espécies Reativas de Oxigênio/metabolismo
17.
Dev Comp Immunol ; 40(2): 218-26, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23500514

RESUMO

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.


Assuntos
Astacoidea/metabolismo , Encéfalo/enzimologia , Monofenol Mono-Oxigenase/metabolismo , Neuropeptídeos/metabolismo , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Encéfalo/citologia , Ritmo Circadiano , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hemócitos/metabolismo , Melaninas/biossíntese , Monofenol Mono-Oxigenase/genética , Neuropeptídeos/genética , Homologia de Sequência de Aminoácidos , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética
18.
Stem Cells Dev ; 21(17): 3173-86, 2012 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-22564088

RESUMO

During evolution, the innate and adaptive immune systems were developed to protect organisms from non-self 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 for studying 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 for studying 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 center (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, thereby obtaining new information about the evolution of different blood and nerve cell lineages.


Assuntos
Astacoidea/citologia , Encéfalo/citologia , Proliferação de Células , Hematopoese , Hemócitos/citologia , Animais , Astacoidea/metabolismo , Astacoidea/fisiologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Bromodesoxiuridina/metabolismo , Contagem de Células , Diferenciação Celular , Núcleo Celular/metabolismo , Células Cultivadas , Mucosa Gástrica/metabolismo , Sistema Hematopoético/citologia , Sistema Hematopoético/metabolismo , Hemócitos/metabolismo , Lipopolissacarídeos , Microscopia Eletrônica de Transmissão , Mitose , Espécies Reativas de Oxigênio/metabolismo , Coloração e Rotulagem , Estômago/citologia , Estômago/fisiologia
19.
Insect Biochem Mol Biol ; 42(2): 71-80, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22193393

RESUMO

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.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/genética , alfa-Macroglobulinas/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/metabolismo , Astacoidea/imunologia , Astacoidea/metabolismo , Clonagem Molecular , Trato Gastrointestinal/imunologia , Dados de Sequência Molecular , Filogenia , Interferência de RNA , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , alfa-Macroglobulinas/metabolismo
20.
PLoS One ; 5(12): e15728, 2010 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-21206752

RESUMO

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.


Assuntos
Aeromonas hydrophila/metabolismo , Aeromonas hydrophila/patogenicidade , Crustáceos/metabolismo , Crustáceos/microbiologia , Melaninas/metabolismo , Tenebrio/metabolismo , Tenebrio/microbiologia , Animais , Catecol Oxidase/química , Precursores Enzimáticos/química , Regulação da Expressão Gênica , Insetos , Melaninas/química , Modelos Genéticos , Mutação , Antígenos O/metabolismo , Proteínas/metabolismo , Células-Tronco , Virulência , Fatores de Virulência
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