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1.
Artigo em Inglês | MEDLINE | ID: mdl-31525458

RESUMO

Cladocera are small freshwater crustaceans that have attracted considerable attention in recent years. They are commonly used for studying senescence. In this study, we used LC-MS/MS with eight-plex iTRAQ to perform a comparative proteomic analysis of senescence in Daphnia pulex. Of 3076 primordial proteins, 2325 were credible (the remaining were low-confidence proteins) and 247 significantly differentially expressed proteins (DEPs). Of the latter, 87, 91, and 69 DEPs were identified in the Day 15 vs. Day 5, Day 20 vs. Day 5, and Day 25 vs. Day 5 groups, respectively. Gene ontology enrichment analysis showed that oxidative damage may be the main cause of senescence in D. pulex. Using Kyoto Encyclopedia of Genes and Genomes pathway analysis, we found that the peroxisome pathway played an important role in aging. Our results suggest that D. pulex alleviates excessive oxidative damage by altering key enzymes involved in carbohydrate and protein metabolism.


Assuntos
Envelhecimento/metabolismo , Proteínas de Artrópodes/biossíntese , Daphnia/metabolismo , Proteômica , Animais
2.
Molecules ; 24(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340554

RESUMO

Scorpions, a characteristic group of arthropods, are among the earliest diverging arachnids, dating back almost 440 million years. One of the many interesting aspects of scorpions is that they have venom arsenals for capturing prey and defending against predators, which may play a critical role in their evolutionary success. Unfortunately, however, scorpion envenomation represents a serious health problem in several countries, including Iran. Iran is acknowledged as an area with a high richness of scorpion species and families. The diversity of the scorpion fauna in Iran is the subject of this review, in which we report a total of 78 species and subspecies in 19 genera and four families. We also list some of the toxins or genes studied from five species, including Androctonus crassicauda, Hottentotta zagrosensis, Mesobuthus phillipsi, Odontobuthus doriae, and Hemiscorpius lepturus, in the Buthidae and Hemiscorpiidae families. Lastly, we review the diverse functions of typical toxins from the Iranian scorpion species, including their medical applications.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Antineoplásicos/química , Proteínas de Artrópodes/química , Venenos de Escorpião/química , Escorpiões/química , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/uso terapêutico , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/uso terapêutico , Descoberta de Drogas/métodos , Expressão Gênica , Humanos , Canais Iônicos/agonistas , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/metabolismo , Irã (Geográfico) , Metaloproteases/biossíntese , Metaloproteases/isolamento & purificação , Metaloproteases/toxicidade , Fosfolipases A2/biossíntese , Fosfolipases A2/isolamento & purificação , Fosfolipases A2/toxicidade , Filogenia , Picadas de Escorpião/fisiopatologia , Venenos de Escorpião/biossíntese , Venenos de Escorpião/isolamento & purificação , Escorpiões/classificação , Escorpiões/patogenicidade , Escorpiões/fisiologia , Inibidores de Serino Proteinase/biossíntese , Inibidores de Serino Proteinase/isolamento & purificação , Inibidores de Serino Proteinase/toxicidade , Especificidade da Espécie
3.
Biochem J ; 476(12): 1753-1769, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31189566

RESUMO

To cope with harsh environments, the Artemia shrimp produces gastrula embryos in diapause, a state of obligate dormancy, having cellular quiescence and suppressed metabolism. The mechanism behind these cellular events remains largely unknown. Here, we study the regulation of cell quiescence using diapause embryos of Artemia We found that Artemia DEK (Ar-DEK), a nuclear factor protein, was down-regulated in the quiescent cells of diapause embryos and enriched in the activated cells of post-diapause embryos. Knockdown of Ar-DEK induced the production of diapause embryos whereas the control Artemia released free-swimming nuaplii. Our results indicate that Ar-DEK correlated with the termination of cellular quiescence via the increase in euchromatin and decrease in heterochromatin. The phenomena of quiescence have many implications beyond shrimp ecology. In cancer cells, for example, knockdown of DEK also induced a short period of cellular quiescence and increased resistance to environmental stress in MCF-7 and MKN45 cancer cell lines. Analysis of RNA sequences in Artemia and in MCF-7 revealed that the Wnt and AURKA signaling pathways were all down-regulated and the p53 signaling pathway was up-regulated upon inhibition of DEK expression. Our results provide insight into the functions of Ar-DEK in the activation of cellular quiescence during diapause formation in Artemia.


Assuntos
Artemia/embriologia , Proteínas de Artrópodes/biossíntese , Diapausa/fisiologia , Embrião não Mamífero/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Receptores da Família Eph/biossíntese , Via de Sinalização Wnt/fisiologia , Animais , Artemia/genética , Proteínas de Artrópodes/genética , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Humanos , Células MCF-7 , Receptores da Família Eph/genética
4.
Artigo em Inglês | MEDLINE | ID: mdl-31075502

RESUMO

Cherax quadricarinatus, as one of the world's most valuable freshwater shrimp species, has received extensive attention in recent years. As males grow larger and faster than females, development of the sex control breeding techniques is of great interest, but knowledge on sex determination and differentiation in C. quadricarinatus remains poorly unknown. Sxl (Sex-lethal) is an important gene in the sexual differentiation regulatory hierarchy. It reflects the ratio of sex chromosomes to autosomes into molecule changes and directs sex-specific splicing forms of precursor mRNA. In the present study, the full-length cDNA sequences of four Sxl splice variants were identified from C. quadricarinatus, designated as CqSxl1, CqSxl2, CqSxl3 and CqSxl4, respectively. Sequence analysis determined different splicing sites near the translation termination region of four Sxl transcript isoforms. Two highly conserved classical RRM domains were found according to predicted secondary structures of Sxl proteins. mRNA expression of CqSxl in different tissues, developmental stage of embryos and testes were investigated by real-time quantitative PCR. Among four isoforms, CqSxl3 showed tissue specificity with higher expression levels in testis than in ovary. CqSxl1 and CqSxl4 were found widely expressed in various tissues and CqSxl2 was almost undetectable. In early developmental stages, the expression levels of CqSxl1/3/4 gradually increased along with embyonic development. In addition, CqSxl genes presented the higher transcript levels in the early stage of testis development. Furthermore, CqSxl3 silencing induced a significant decrease of the transcript of Cq-IAG, an androgenic hormone-encoding gene responsible for masculine development. These data indicate that CqSxl3 might be involved in male sex determination in C. quadricarinatus. Our study will contribute to understanding the mechanism of sex determination in C. quadricarinatus, and also can provide theoretical guidance for establishing a sex control technology.


Assuntos
Proteínas de Artrópodes , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Letais , Processamento de RNA/fisiologia , RNA Mensageiro , Diferenciação Sexual/fisiologia , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , /genética , Feminino , Perfilação da Expressão Gênica , Masculino , RNA Mensageiro/biossíntese , RNA Mensageiro/genética
5.
Artigo em Inglês | MEDLINE | ID: mdl-30831207

RESUMO

The capacity of crustaceans to biosynthesise long-chain polyunsaturated fatty acids has yet to be fully defined, due to the lack of evidence on the functional activities of enzymes involved in desaturation or elongation of fatty acid substrates. We report here the cloning and in vitro functional analysis of an elongase from the orange mud crab, Scylla olivacea. Sequence and phylogenetic analysis placed the elovl close to the vertebrate Elovl1 and Elovl7 clade, which is distinct from the other remaining five Elovl families. The elongase was also clustered together with several elongases from crustaceans and insects. This elongase showed activities towards 16:1n-7, and at lower rate, linoleic acid (18:2n-6) and linolenic acid (18:3n-3). To our knowledge this is the first description of a functional enzyme involved in biosynthesis of long-chained polyunsaturated fatty acids in a crustacean species. Expression of the S. olivacea elovl7-like mRNA was prominent in stomach, intestine and gill tissues, due to the need to regulate the permeability of epithelial tissue through modification of fatty acid compositions. The implication of our findings, in terms of ability of Crustacea phylum to biosynthesise polyunsaturated fatty acids is discussed.


Assuntos
Acetiltransferases , Proteínas de Artrópodes , Braquiúros , Ácidos Graxos Insaturados , Regulação Enzimológica da Expressão Gênica/fisiologia , Filogenia , Acetiltransferases/biossíntese , Acetiltransferases/química , Acetiltransferases/genética , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Braquiúros/enzimologia , Braquiúros/genética , Clonagem Molecular , Ácidos Graxos Insaturados/biossíntese , Ácidos Graxos Insaturados/genética , Especificidade de Órgãos/fisiologia
6.
Artigo em Inglês | MEDLINE | ID: mdl-30880278

RESUMO

Methyl farnesoate (MF), a sesquiterpenoid synthesized in the mandibular organ, regulates many physiological processes in crustaceans including growth and reproduction. In the present study, farnesoic acid O-methyltransferase (FAMeT), the key enzyme responsible for final step conversion of farnesoic acid (FA) to methyl farnesoate (MF), was cloned and characterized from the nervous tissues of Penaeus indicus. Multiple sequence alignment, prediction of conserved domain regions, phosphorylation sites identification and phylogenetic analysis indicated that putative FAMeT fragment from P. indicus (PiFAMeT), shares a high degree of sequence identity to FAMeT proteins isolated from other crustaceans species. Quantitative real-time PCR analysis revealed ubiquitous expression of PiFAMeT in all the tissues examined, with comparative higher mRNA levels in nervous tissue and ovary. Additionally, the levels of PiFAMeT also showed gradual increase of expression correlating with the advancement in ovarian maturation. Further to support their role in promoting ovarian development, serotonin treatment (5HT, 50 µg/g body weight) was given to eyestalk intact and unilaterally eyestalk ablated females which resulted in significant increase in PiFAMeT transcript levels at day 7 and day 14. The relatively higher levels of PiFAMeT, reflecting higher levels of MF, suggest a role during secondary vitellogenesis thereby regulating ovarian development in P. indicus. Further research is required to understand the synergistic interaction of MF pathways with serotonergic and other regulatory pathways in regulating ovarian maturation in penaeid shrimps.


Assuntos
Proteínas de Artrópodes , Regulação Enzimológica da Expressão Gênica/fisiologia , Metiltransferases , Ovário/enzimologia , Penaeidae , Vitelogênese/fisiologia , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Clonagem Molecular , Feminino , Metiltransferases/biossíntese , Metiltransferases/genética , Ovário/citologia , Penaeidae/enzimologia , Penaeidae/genética
7.
Artigo em Inglês | MEDLINE | ID: mdl-30472332

RESUMO

In addition to the typical electron transport system (ETS) in animal mitochondria responsible for oxidative phosphorylation, in some species there exists an alternative oxidase (AOX) pathway capable of catalyzing the oxidation of ubiquinol and the reduction of oxygen to water. The discovery of AOX in animals is recent and further investigations into its expression, regulation, and physiological role have been hampered by the lack of a tractable experimental model organism. Our recent DNA database searches using bioinformatics revealed an AOX sequence in several marine copepods including Tigriopus californicus. This species lives in tidepools along the west coast of North America and is subject to a wide variety of daily environmental stresses. Here we verify the presence of the AOX gene in T. californicus and the expression of AOX mRNA and AOX protein in various life stages of the animal. We demonstrate that levels of the AOX protein increase in T. californicus in response to cold and heat stress compared to normal rearing temperature. We predict that a functional AOX pathway is present in T. californicus, propose that this species will be a useful model organism for the study of AOX in animals, and discuss future directions for animal AOX research.


Assuntos
Proteínas de Artrópodes , Copépodes , Regulação Enzimológica da Expressão Gênica/fisiologia , Resposta ao Choque Térmico/fisiologia , Oxirredutases , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Temperatura Baixa , Copépodes/enzimologia , Copépodes/genética , Oxirredutases/biossíntese , Oxirredutases/genética
8.
Mol Reprod Dev ; 86(2): 122-131, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30286264

RESUMO

Mud crab Scylla paramamosain is a commercially important species widely cultured in China. It is well known that the eyestalk regulates reproductive activities in crustaceans. In our previous research, we found that the miR-34 expression level in male eyestalk was significantly higher than that in females. Thus, we assumed that it may play an important role in regulating reproduction. In this study, we used bioinformatic tools to identify the target genes of miR-34 in eyestalk. Six reproduction-related genes with an intact 3'-untranslated region (UTR), including molt-inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), vitellogenesis-inhibiting hormone, red pigment concentrating hormone, ecdysone receptor (EcR), and farnesoic acid methyltransferase (FAMeT) were identified. When the 3'-UTR plasmid vectors of the six genes were cotransfected with miR-34 mimics into 293FT cells, respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly decreased compared with that in the control group; on the contrary, when the six plasmid vectors were cotransfected with the miR-34 inhibitor respectively, the luciferase activities of four genes (MIH, CHH, EcR, and FAMeT) were significantly higher than that in the control group. When agomiR-34 and antagomiR-34 were injected into the eyestalk respectively in vivo, the expression levels of the MIH, CHH, EcR, and FAMeT genes were detected by a quantitative real-time polymerase chain reaction. The results showed that agomiR-34 suppressed the expression of the four genes, whereas antagomiR-34 enhanced their expression. These experimental results confirmed our hypothesis that miR-34 may indirectly regulate reproduction via binding to the 3'-UTRs of MIH, CHH, EcR, and FAMeT genes and suppressing their expression.


Assuntos
Proteínas de Artrópodes/biossíntese , Braquiúros/metabolismo , Regulação da Expressão Gênica/fisiologia , MicroRNAs/metabolismo , Animais , Proteínas de Artrópodes/genética , Braquiúros/genética , Feminino , Masculino , MicroRNAs/genética , Reprodução/fisiologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-30448604

RESUMO

An 8-week feeding trial was conducted to evaluate the effect of fish-meal replacement on growth performance, protein synthesis and immune response of juvenile Pacific white shrimp, Litopenaeus vannamei reared at low salinity (7‰). Five isonitrogenous and isolipidic diets were formulated to contain graded levels (25, 20, 15, 10 and 5%) of fish-meal. High quality alternative solutions were performed, crystalline amino acids, phytase, mannan oligosaccharides and some micro-nutrients were supplemented in the low fish-meal diets. Each diet was randomly assigned to triplicate tanks, each tank with 30 shrimp (mean weight 0.3 g), the shrimp were fed 3 times a day. Weight gain and survival were decreased with the decreasing dietary fish meal levels. When dietary fish-meal decreased, the gene expression of TOR, Raptor and eIF4E2 in hepatopancreas were decreased with the decreasing fish meal levels, eIF4E2 in intestine was decreased while 4E-BP was increased with the decreasing fish meal levels. The mRNA level of SOD in hepatopancreas decreased, and the expression of GPx and CAT increased with the decreasing FM levels. The Toll pathway was affected by dietary FM levels, the expression of Toll2, TNFSF, MyD88, Rho and p38 in intestine were increased with the decreasing FM levels. The results indicated that at low salinity condition, fish meal level lower than 15% would inhibit the protein synthesis and harm to the health of shrimp.


Assuntos
Ração Animal , Proteínas de Artrópodes , Regulação da Expressão Gênica/imunologia , Penaeidae , Salinidade , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Penaeidae/imunologia , Penaeidae/metabolismo
10.
Sheng Wu Gong Cheng Xue Bao ; 34(10): 1668-1678, 2018 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-30394034

RESUMO

To establish a simple, quick and effective method to get a large amount of spider toxin JZTX-26 (35 aa) and JZTX-51 (27 aa) with 3 disulfide bonds each, the mature peptides coding gene fragments were constructed and fused with maltose-binding protein (MBP) tag in an Escherichia coli expression vector pMAL-p2x. The recombinant constructs pMAL-jz26 and pMAL-jz51 were transformed and cultured in E. coli TB1 and BL21 (DE3). After being induced by isopropyl-ß-d-thiogalactoside (IPTG), the periplasmic proteins were purified by amylose affinity chromatography and analyzed by SDS-PAGE. The fusion proteins were digested with factor X, and purified by Sizes-Exclusion chromatography and Reversed Phase HPLC. Molecular weights of the purified peptides were obtained by using a MALDI-TOF-TOF mass spectrometer, which were consistent with the theoretical molecular weights. Five milligram of target protein could be purified from 1 L of culture medium. The results indicate that the peptides with three disulfide bonds can be expressed by using the prokaryotic expression system with MBP tag. Our findings suggest the possibility of genetic engineering to obtain large amount of spider peptide toxins.


Assuntos
Proteínas de Artrópodes/biossíntese , Peptídeos , Venenos de Aranha/química , Animais , Escherichia coli , Engenharia Genética , Isopropiltiogalactosídeo , Proteínas Ligantes de Maltose , Proteínas Recombinantes de Fusão/biossíntese , Aranhas
11.
G3 (Bethesda) ; 8(12): 3865-3879, 2018 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-30333191

RESUMO

The two-spotted spider mite Tetranychus urticae is an important pest with an exceptionally broad host plant range. This generalist rapidly acclimatizes and adapts to a new host, hereby overcoming nutritional challenges and a novel pallet of constitutive and induced plant defenses. Although recent studies reveal that a broad transcriptomic response upon host plant transfer is associated with a generalist life style in arthropod herbivores, it remains uncertain to what extent these transcriptional changes are general stress responses or host-specific. In the present study, we analyzed and compared the transcriptomic changes that occur in a single T. urticae population upon long-term transfer from Phaseolus vulgaris to a similar, but chemically defended, host (cyanogenic Phaseolus lunatus) and to multiple economically important crops (Glycine max, Gossypium hirsutum, Solanum lycopersicum and Zea mays). These long-term host plant transfers were associated with distinct transcriptomic responses with only a limited overlap in both specificity and directionality, suggestive of a fine-tuned transcriptional plasticity. Nonetheless, analysis at the gene family level uncovered overlapping functional processes, recruiting genes from both well-known and newly discovered detoxification families. Of note, our analyses highlighted a possible detoxification role for Tetranychus-specific short-chain dehydrogenases and single PLAT domain proteins, and manual genome annotation showed that both families are expanded in T. urticae Our results shed new light on the molecular mechanisms underlying the remarkable adaptive potential for host plant use of generalist arthropods and set the stage for functional validation of important players in T. urticae detoxification of plant secondary metabolites.


Assuntos
Proteínas de Artrópodes/biossíntese , Produtos Agrícolas/parasitologia , Regulação da Expressão Gênica/fisiologia , Interações Hospedeiro-Parasita/fisiologia , Tetranychidae/fisiologia , Transcriptoma/fisiologia , Animais
12.
Int J Mol Sci ; 19(8)2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-30071669

RESUMO

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), ß-act (ß-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and ß-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.


Assuntos
Proteínas de Artrópodes , Palaemonidae , Reação em Cadeia da Polimerase em Tempo Real/estatística & dados numéricos , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Palaemonidae/genética , Palaemonidae/metabolismo
13.
Gene ; 665: 111-118, 2018 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-29730424

RESUMO

As an essential mediator in the Gonadotropin-releasing hormone (GnRH) signaling pathway, GnRH receptor (GnRHR) coupled to GnRH, plays an important role in activating the downstream pathway after stimulating a series of cascades to regulate reproduction. To detect the existence of GnRHR and potential GnRH signaling pathway, we cloned and characterized GnRHR in the Chinese mitten crab, Eriocheir sinensis (named EsGnRHR). The full-length EsGnRHR cDNA is 2038 bp in length, including an open reading frame (ORF) of 1566 bp, a 57 bp 5'-untranslated region (5'-UTR) and a 415 bp 3'-UTR. Prediction of transmembrane domains in protein sequence revealed that the EsGnRHR protein contained seven hydrophobic transmembrane regions (TMs). Reverse transcription PCR revealed that EsGnRHR was mainly expressed in the thoracic nerve group and ovary, and weakly distributed in the testis and brain. In situ hybridization further demonstrated that EsGnRHR mRNA was localized at the protocerebrum and deutocerebrum. In the ovary and testis, the hybridization signal was dominantly at the earlier developmental stages. The signal was mainly localized in the cytoplasm cell in the ovary, and in the epithelium cell in the testis. During the different stages of gonadal development, EsGnRHR displayed increasing trends in both female and male when analyzed by quantitative real-time PCR, suggesting that EsGnRHR was involved in controlling gonadal development. Our study provides important information for further research on the molecular mechanisms underlying crab development.


Assuntos
Proteínas de Artrópodes , Braquiúros , Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Receptores LHRH , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Braquiúros/genética , Braquiúros/metabolismo , Feminino , Masculino , Ovário/metabolismo , Receptores LHRH/biossíntese , Receptores LHRH/genética , Testículo/metabolismo
14.
Comp Biochem Physiol B Biochem Mol Biol ; 221-222: 18-28, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29649577

RESUMO

Cathepsin B is a lysosomal proteolytic enzyme that has been suggested to play a role in pathological processes of immune system. In this study, the full-length cDNA sequence of cathepsin B transcript in the giant river prawn Macrobrachium rosenbergii (MrCTSB) was obtained from 454 pyrosequencing of cDNAs from hepatopancreas and muscle. It was 1158 bp in length, containing an open reading frame (ORF) of 987 bp corresponding to 328 amino acids. The predicted molecular mass and pI of MrCTSB protein was 36.04 kDa and 4.73. The major characteristics of MrCTSB protein consisted of a propeptide of C1 peptidase family at the N-terminus and a cysteine protease (Pept_C1) domain at the C-terminus. The 3-dimentional structure of MrCTSB was constructed by computer-assisted homology modeling. The folding of MrCTSB was highly conserved to human CTSB structure and the modeled MrCTSB displayed characteristics of cysteine proteinases superfamily. The docking study was performed to investigate binding interactions between known inhibitors against MrCTSB. Known inhibitors were oriented in the groove of catalytic site cleft. They bound to subsites from S2, S1, S1', and S2', respectively, with key residues in each subsite. Challenge of juvenile prawns with Aeromonas hydrophila revealed that the MrCTSB transcript in hepatopancreas significantly increased at 60-96 h post injection (hpi). This suggested that MrCTSB may play roles in innate immunity of M. rosenbergii. Our results provide useful information for a more comprehensive study in immune-related functions of MrCTSB.


Assuntos
Aeromonas hydrophila , Proteínas de Artrópodes , Catepsina B , Regulação Enzimológica da Expressão Gênica , Palaemonidae , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Catepsina B/biossíntese , Catepsina B/genética , Biologia Computacional , Palaemonidae/enzimologia , Palaemonidae/genética , Palaemonidae/microbiologia
15.
PLoS One ; 13(3): e0194459, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29590153

RESUMO

Vitellogenesis is the process of yolk formation via accumulating vitellin (Vn) with nutrients in the oocytes. Expression of vitellogenin (Vg), the precursor of Vn, is one of the indicators for the start of vitellogenesis. In Pacific white shrimp (Litopenaeus vannamei), the type-II vitellogenesis-inhibiting hormone (VIH-2) effectively suppresses hepatopancreatic Vg mRNA expression. In this study, we demonstrate the increasing transcript levels of hepatopancreatic Vg during L. vannamei ovarian development, suggesting that the hepatopancreas-derived Vg/Vn may also contribute to vitellogenesis in this species. Using a combination of in vivo injections and in vitro primary cell cultures, we provide evidences that the inhibition of VIH-2 on hepatopancreatic Vg gene expression is mediated through a functional coupling of the GC/cGMP pathway with different MAPK-dependent cascades in female shrimp. In VIH-2 signaling, the NO-independent GC/cGMP/PKG cascades were upstream of the MAPKs. Activations of the MAPK signal by VIH-2 include the phosphorylation of JNK and the mRNA/protein expression of P38MAPK. Additionally, the cAMP/PKA pathway is another positive intracellular signal for hepatopancreatic Vg mRNA expression but is independent of its VIH-2 regulation. Our findings establish a model for the signal transduction mechanism of Vg regulation by VIH and shed light on the biological functions and signaling of the CHH family in crustaceans.


Assuntos
Proteínas de Artrópodes/biossíntese , Proteínas de Transporte/metabolismo , Hepatopâncreas/metabolismo , Hormônios de Invertebrado/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Penaeidae/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transcrição Genética/fisiologia , Vitelogeninas/biossíntese , Animais , GMP Cíclico/metabolismo , Feminino
16.
Insect Biochem Mol Biol ; 95: 44-54, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29526768

RESUMO

Blood-feeding parasites are inadvertently exposed to high doses of potentially cytotoxic haem liberated upon host blood digestion. Detoxification of free haem is a special challenge for ticks, which digest haemoglobin intracellularly. Ticks lack a haem catabolic mechanism, mediated by haem oxygenase, and need to dispose of vast majority of acquired haem via its accumulation in haemosomes. The knowledge of individual molecules involved in the maintenance of haem homeostasis in ticks is still rather limited. RNA-seq analyses of the Ixodes ricinus midguts from blood- and serum-fed females identified an abundant transcript of glutathione S-transferase (gst) to be substantially up-regulated in the presence of red blood cells in the diet. Here, we have determined the full sequence of this encoding gene, ir-gst1, and found that it is homologous to the delta-/epsilon-class of GSTs. Phylogenetic analyses across related chelicerates revealed that only one clear IrGST1 orthologue could be found in each available transcriptome from hard and soft ticks. These orthologues create a well-supported clade clearly separated from other ticks' or mites' delta-/epsilon-class GSTs and most likely evolved as an adaptation to tick blood-feeding life style. We have confirmed that IrGST1 expression is induced by dietary haem(oglobin), and not by iron or other components of host blood. Kinetic properties of recombinant IrGST1 were evaluated by model and natural GST substrates. The enzyme was also shown to bind haemin in vitro as evidenced by inhibition assay, VIS spectrophotometry, gel filtration, and affinity chromatography. In the native state, IrGST1 forms a dimer which further polymerises upon binding of excessive amount of haemin molecules. Due to susceptibility of ticks to haem as a signalling molecule, we speculate that the expression of IrGST1 in tick midgut functions as intracellular buffer of labile haem pool to ameliorate its cytotoxic effects upon haemoglobin intracellular hydrolysis.


Assuntos
Proteínas de Artrópodes , Regulação Enzimológica da Expressão Gênica/fisiologia , Glutationa Transferase , Ixodes , Filogenia , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Glutationa Transferase/biossíntese , Glutationa Transferase/química , Glutationa Transferase/genética , Hemoglobinas/química , Hemoglobinas/metabolismo , Ixodes/enzimologia , Ixodes/genética , Especificidade por Substrato
17.
Colloids Surf B Biointerfaces ; 163: 19-28, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29268210

RESUMO

Recombinant spider silk protein (pNSR32) and gelatin (Gt) were demonstrated to enhance cytocompatibility of electrospun pNSR32/PCL/Gt scaffold. However, its potential pro-inflammatory effects and interactions with tissue and blood are unknown. In this study, the physicochemical properties and in vitro and in vivo biocompatibility of such scaffolds were evaluated. The results showed that the pNSR32/PCL/Gt scaffold possessed larger average fiber diameters, wider fiber diameter distribution and faster degradation rate than that of pNSR32/PCL and PCL scaffolds. The addition of pNSR32 and Gt had little influence on the hemolysis and plasma re-calcification time, but prolonged kinetic clotting time and reduced the platelet adhesion. The Il-6 and Tnf-α mRNA expression levels were up-regulated in macrophages seeded on the PCL and pNSR32/PCL scaffolds. The lowest release of IL-6 and TNF-α appeared in the pNSR32/PCL/Gt scaffold. Histological results revealed that the PCL and pNSR32/PCL scaffolds elicited severe host tissue responses after implantation, while prominent ingrowth of host cells were observed in the pNSR32/PCL and pNSR32/PCL/Gt scaffolds. The comet assay and bone marrow micronucleus test demonstrated that the pNSR32/PCL/Gt scaffold did not increase the frequency of DNA damage or bone marrow micronucleus. In short, this study confirmed that the pNSR32/PCL/Gt scaffold exhibited better blood and tissue compatibility than pNSR32/PCL and PCL scaffolds. No induction of genotoxicity and inflammatory factor releases makes the pNSR32/PCL/Gt scaffold a good candidate for engineering small diameter vascular tissue.


Assuntos
Proteínas de Artrópodes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Gelatina/farmacologia , Proteínas Recombinantes/farmacologia , Tecidos Suporte , Animais , Proteínas de Artrópodes/biossíntese , Vasos Sanguíneos/citologia , Técnicas Eletroquímicas , Gelatina/química , Expressão Gênica , Interleucina-6/genética , Interleucina-6/imunologia , Camundongos , Adesividade Plaquetária/efeitos dos fármacos , Células RAW 264.7 , Ratos Sprague-Dawley , Proteínas Recombinantes/biossíntese , Seda/química , Seda/farmacologia , Aranhas/química , Aranhas/fisiologia , Engenharia Tecidual/métodos , Fator de Necrose Tumoral alfa/imunologia
18.
Artigo em Inglês | MEDLINE | ID: mdl-29237574

RESUMO

Some Macrobrachium shrimps (Caridea, Palaemonidae) are diadromous; freshwater adults are truly euryhaline, while larvae need saline water for development. Branchial Na+/K+-ATPase (NKA) and carbonic anhydrase (CA) are involved in NaCl absorption in freshwater. This study aimed at verifying the time course of the osmoregulatory response of adult Macrobrachium acanthurus to high salinity brackish water (20‰), from the first 30min to 5days. The goal was to detect possible transition from hyper- to hyporegulation, the putative involvement of branchial NKA and CA, or the induction of muscular HSP70 expression. Hemolymph osmotic and ionic concentrations remained relatively stable and close to control levels until ~9h of exposure, but later increased consistently (~50%). A fast reduction in NKA activity (3-6h) was observed; these shrimps seem to shut off salt absorption already in the first hours. Later on, especially after 24h, hemolymph concentrations rise but HSP70 expression is not induced, possibly because constitutive levels are already sufficient to prevent protein damage. Time-dependent response mechanisms effective in high salinity brackish water, resulting in salt loading avoidance and suggestive of hyporegulation should be further investigated in decapods that evolutionary invaded freshwater.


Assuntos
Proteínas de Artrópodes/biossíntese , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/biossíntese , Hemolinfa/metabolismo , Músculos/metabolismo , Palaemonidae/metabolismo , ATPase Trocadora de Sódio-Potássio/biossíntese , Animais , Concentração Osmolar
19.
Artigo em Inglês | MEDLINE | ID: mdl-29032300

RESUMO

Cathepsin D is an aspartic endopetidase with typical characteristics of lysosomal enzymes. Cathepsin D activity has been reported in the gastric fluid of clawed lobsters where it acts as an extracellular digestive enzyme. Here we investigate whether cathepsin D is unique in clawed lobsters or, instead, common in decapod crustaceans. Eleven species of decapods belonging to six infraorders were tested for cathepsin D activity in the midgut gland, the muscle tissue, the gills, and when technically possible, in the gastric fluid. Cathepsin D activity was present in the midgut gland of all 11 species and in the gastric fluid from the seven species from which samples could be taken. All sampled species showed higher activities in the midgut glands than in non-digestive organs and the activity was highest in the clawed lobster. Cathepsin D mRNA was obtained from tissue samples of midgut gland, muscle, and gills. Analyses of deduced amino acid sequence confirmed molecular features of lysosomal cathepsin D and revealed high similarity between the enzymes from Astacidea and Caridea on one side, and the enzymes from Penaeoidea, Anomura, and Brachyura on the other side. Our results support the presence of cathepsin D activity in the midgut glands and in the gastric fluids of several decapod species suggesting an extracellular function of this lysosomal enzyme. We discuss whether cathepsin D may derive from the lysosomal-like vacuoles of the midgut gland B-cells and is released into the gastric lumen upon secretion by these cells.


Assuntos
Proteínas de Artrópodes , Catepsina D , Regulação Enzimológica da Expressão Gênica/fisiologia , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , Catepsina D/biossíntese , Catepsina D/genética , /genética , Especificidade de Órgãos/fisiologia
20.
Biochimie ; 140: 117-121, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28735872

RESUMO

The Rhipicephalus (Boophilus) microplus is an exclusive bovine ectoparasite responsible for the transmission of pathogens that decrease meat, leather and milk productions. Cattle vaccination is an alternative to control tick infestations, but the discovery of potential antigens is still a challenge for researchers. Recently, our group performed a midgut transcriptome of engorged R. microplus tick, and out of 800 ESTs sequences one cystatin-coding sequence was identified and named Rmcystatin-4. In order to understand the physiological role of Rmcystatin-4, the aim of this work was the expression, purification and functional characterization of a novel type 2 cystatin from the tick R. microplus. Rmcystatin-4 gene expression was identified mostly in tick midgut suggesting its possible role in blood digestion control. Our data showed that rRmcystatin-4 was successfully expressed in active form using Pichia pastoris system and the purified inhibitor presented high selectivity to BmCl-1 (Ki = 0.046 nM). Moreover, rRmcystatin-4 was able to impaired BmCl-1 activity towards bovine hemoglobin.


Assuntos
Proteínas de Artrópodes , Mucosa Intestinal/metabolismo , Rhipicephalus , Cistatinas Salivares , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/isolamento & purificação , Bovinos , Expressão Gênica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Rhipicephalus/química , Rhipicephalus/genética , Rhipicephalus/metabolismo , Cistatinas Salivares/biossíntese , Cistatinas Salivares/química , Cistatinas Salivares/genética , Cistatinas Salivares/isolamento & purificação
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