RESUMO
Transforming growth factor-ß type III receptor (TßR3), as a co-receptor of TGF-ß superfamily, plays critical roles in development and growth as well as some disease pathogeneses by presenting ligands to other receptors in vertebrates. However, the identification and functional characterization of TßR3 had not been reported yet in invertebrates. In the present study, TßR3 was first identified and characterized in mud crab Scylla paramamosain. The obtained cDNA length of SpTßR3 was 2, 424 bp with a 1, 854 bp open reading frame, which encoded a putative peptide of 617 amino acids containing a typical transmembrane region and a Zona pellucida (ZP) domain. Real-time PCR results showed that SpTßR3 was predominantly expressed at early embryonic development stage and early postmolt stage, suggesting its participation in development and growth. We report, for the first time in invertebrates, the challenge of both Vibro alginolyticus and Poly (I:C) could alter the expression patterns of SpTßR3. Notably, the expression levels of SpIKK, two NF-κB members (SpRelish and SpDorsal), and five antimicrobial peptide genes (SpCrustin and SpALF1-4) were significantly suppressed when SpTßR3 was interfered in vivo. Secondly, the overexpression of SpTßR3 in vitro could activate NF-κB signaling through the dual-luciferase reporter assays. Furthermore, the bacterial clearance assay after SpTßR3 was silenced in vivo highlighted the potential of SpTßR3 in activating the innate immune responses. These results implied the involvement of SpTßR3 in the innate immune responses by regulating the NF-κB pathway. This study first indicated that TßR3 was present in invertebrate, and it participated in not only the development and growth but also the innate immunity of S. paramamosain. It also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.
Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Poli I-C/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/química , Alinhamento de Sequência , Vibrio alginolyticus/fisiologiaRESUMO
Transforming growth factor-ß-activating kinase 1 (TAK1) is essential for diverse important biological functions, such as innate immunity, development and cell survival. In the present study, the homologs of TAK1 and TAK1-binding protein 1 (TAB1) were identified and characterized from mud crab Scylla paramamosain for the first time. The full-length cDNAs of SpTAK1 and SpTAB1 were 2, 226 bp and 2, 433 bp with 1, 782 bp and 1, 533 bp open reading frame (ORF), respectively. The deduced SpTAK1 protein contained a conserved S_TKc (Serine/threonine protein kinases, catalytic) domain, and the putative SpTAB1 protein possessed a typical PP2Cc (Serine/threonine phosphatases, family 2C, catalytic) domain and a potential TAK1 docking motif. Real-time PCR analysis showed that SpTAK1 and SpTAB1 were highly expressed at early development stages, suggesting their participation in crab's development process. Moreover, the expression levels of SpTAK1 and SpTAB1 in hepatopancreas were positively stimulated after challenge with Vibro alginolyticus and Poly (I:C), implying the involvement of SpTAK1 and SpTAB1 in innate immune responses against both bacterial and viral infections. When SpTAK1 or SpTAB1 were silenced in vivo, the expression levels of two IMDNFκB signaling components (SpIKKß and SpRelish) and six antimicrobial peptide (AMP) genes (SpALF1-5 and SpCrustin) were significantly reduced, and the bacteria clearance capacity of crabs was also markedly impaired in SpTAK1 or SpTAB1 silenced crabs. Additionally, overexpression of SpTAK1 and SpTAB1 in HEK293T cells could markedly activate the mammalian NF-κB signaling. Collectively, our results suggested that TAK1 and TAB1 regulated crab's innate immunity via modulating the IMDNFκB signaling. These findings may provide new insights into the TAK1/TAB1-mediated signaling cascades in crustaceans and pave the way for a better understanding of crustacean innate immune system.
Assuntos
Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Perfilação da Expressão Gênica , MAP Quinase Quinase Quinases/química , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , Filogenia , Alinhamento de Sequência , Transdução de Sinais/genéticaRESUMO
Various functional 3-acyl-1,5-benzodiazepines containing carboxyl, ester and acyl groups at the 2-position were synthesized via an efficient, sustainable and catalyst-free domino reaction. During the synthesis process, one new cycle and four new bonds (one C-C, two C-N and one C[double bond, length as m-dash]C) were constructed by the nucleophilic substitution, nucleophilic addition, dehydration and cyclization reaction by the H+ shift. Furthermore, a total of 26 examples were examined by reacting inexpensive starting materials of N,N-dimethylformamide dimethyl acetal, aromatic ketones, 1,2-phenylenediamine compounds and aldehyde derivatives. Therefore, it displayed a broad substrate scope, good functional group tolerance, high yields (77-97%) and the ease of obtaining target compounds without the involvement of toxic solvents and column chromatography, which provided a novel method for the synthesis of a wide variety of biologically relevant 1,5-benzodiazepines.
RESUMO
Ambient temperature-associated stress can affect normal physiological functions in ectotherms. To assess the effects of cold or heat stress on amphibians, giant spiny frogs (Quasipaa spinosa) were acclimated at 22°C followed by exposure to 5°C or 30°C for 0, 3, 6, 12, 24 and 48â h, respectively. Histological alterations, apoptotic index, generation of mitochondrial reactive oxygen species (ROS), antioxidant activity indices and stress-response gene expression in frog livers were subsequently determined. Results showed that many fat droplets appeared after 12â h of heat stress and the percentage of melanomacrophage centres significantly changed after 48â h at both stress conditions. Furthermore, the mitochondrial ROS levels were elevated in a time-dependent manner up to 6â h and 12â h in the cold and heat stress groups, respectively. The activities of superoxide dismutase, glutathione peroxidase and catalase were successively increased with increasing periods of cold or heat exposure, and their gene expression levels showed similar changes in both stress conditions. Most tested heat shock protein (HSP) genes were sensitive to temperature exposure, and the expression profiles of most apoptosis-related genes was significantly upregulated at 3 and 48â h under cold and heat stress, respectively. Apoptotic index at 48â h under cold stress was significantly higher than that under heat stress. Notably, lipid droplets, HSP30, HSP70 and HSP110 might be suitable bioindicators of heat stress. The results of these alterations at physiological, biochemical and molecular levels might contribute to a better understanding of the stress response of Q. spinosa, and perhaps amphibians more generally, under thermal stress.
Assuntos
Anuros/fisiologia , Resposta ao Choque Frio/fisiologia , Resposta ao Choque Térmico/fisiologia , Fígado/fisiologia , Mitocôndrias/metabolismo , Transcriptoma , Animais , Antioxidantes/metabolismo , Anuros/genética , Apoptose/fisiologia , Resposta ao Choque Frio/genética , Resposta ao Choque Térmico/genética , Fígado/citologia , Fígado/ultraestrutura , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismoRESUMO
Immune deficiency (IMD) pathway, one of the most essential pattern recognition receptor signaling pathways, plays vital roles in innate immune responses to eliminate pathogen infection in invertebrates. In the present study, an immune deficiency (IMD) gene and two NF-κB family members, Relish and Dorsal, were identified and characterized in mud crab Scylla paramamosain for the first time. The deduced SpIMD, SpRelish and SpDorsal protein contained conserved death domain and classical NF-κB domains, respectively. Phylogenetic analysis suggested that SpIMD was classified into the invertebrate IMD branch, and SpRelish could be classified into the type I NF-κB class while SpDorsal could be grouped into the type II NF-κB class. Tissue distribution results showed these three genes were ubiquitously expressed in all tested tissues. The expression patterns of IMD signaling pathway and NF-κB genes, including SpIMD, SpIKKß, SpIKKε, SpRelish and SpDorsal, were distinct when crabs were stimulated with Vibro alginolyticus, indicating that they might be involved in responding to bacterial infection. When SpIMD was silenced by in vivo RNA interference assay, the expression levels of IMD pathway and antimicrobial peptides (AMPs) genes, including SpIKKß, SpRelish, SpALF1-6 and SpCrustin, were significantly down-regulated (pâ¯<â¯0.05). Correspondingly, the bacteria clearance ability of hemolymph was extremely impaired in IMD silenced crabs. Overall, the IMD played vital roles in innate immune response by regulating the expressions of its down-stream signaling genes and AMPs in S. paramamosain. These findings might pave the way for a better understanding of innate immune system and establish a fundamental network for the IMD signaling pathway in crustaceans.
Assuntos
Infecções Bacterianas/imunologia , Braquiúros/imunologia , Imunidade Inata , Transdução de Sinais/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Braquiúros/genética , Perfilação da Expressão Gênica , Hemolinfa , NF-kappa B/genética , Filogenia , RNA , Vibrioses/imunologia , Vibrio alginolyticusRESUMO
Nuclear factor E2-related factor 2 (Nrf2) is a crucial transcription factor that regulates the basal and inducible expression of many antioxidant-relevant genes, and the Nrf2-mediated antioxidant pathway has been regarded as a critical switch in the initiation of cellular defence systems against oxidative damages. In this study, Nrf2 was first identified and characterized in the Chinese giant salamander (Andrias davidianus). A. davidianus was exposed to a high ambient temperature of 30⯰C for various periods of time (0, 3, 6, 12, 24, 48 and 72â¯h). We investigated the effects of heat stress on alterations of the hepatic malondialdehyde (MDA) concentration, the activities of lactic acid dehydrogenase (LDH), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), the histology of the liver, and the mRNA expression patterns of 11 genes involved in the Nrf2-mediated antioxidant pathway in A. davidianus. The results showed that both the hepatic LDH activity and MDA content significantly increased after heat exposure, indicating that heat stress could induce cell injury and oxidative damage. Histological analysis of the liver showed that heat stress caused hepatocyte abnormalities, fat accumulation and ultrastructural alterations of the hepatocytes, endoplasmic reticulum and nuclei. The expression patterns of genes involved in the Nrf2-mediated antioxidant pathway in the liver were distinct when A. davidianus was exposed to heat stress. To the best of our knowledge, this study is the first on the characterization of Nrf2 in A. davidianus and even in amphibians. The results indicated that heat stress could induce oxidative damage, and the Nrf2 antioxidant pathway might play a critical role in the resistance against heat stress in A. davidianus. These findings will deepen and enrich the current knowledge on the evolutionary conserved antioxidant roles and mechanisms of Nrf2 in A. davidianus, or even in amphibians, in the antioxidant defence against heat stress.
Assuntos
Antioxidantes/metabolismo , Resposta ao Choque Térmico , Fígado/metabolismo , Fígado/patologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Fígado/ultraestrutura , Fator 2 Relacionado a NF-E2/isolamento & purificação , Transdução de Sinais , Temperatura , UrodelosRESUMO
36 Novel 1,5-benzodiazepine derivatives were rationally designed and synthesized according to the principle of superposition of bioactive substructures by the combination of 1,5-benzodiazepines, thiophene or thiazole and ester group. The structures of the target compounds have been characterized by IR, (1)H NMR, (13)C NMR, MS and elemental analysis. The structure of 1v was further determined using X-ray single crystal diffraction. All synthesized 1,5-benzodiazepine derivatives were evaluated for their in vitro antimicrobial activity against C. neoformans, C. neoformans clinical isolates, C. albicans, E. coli and S. aureus. The bioactive assay results revealed that most of the 1,5-benzodiazepine derivatives exhibited considerable potency against all of the tested strains. In particular, compounds 1v and 1w (MIC: 2-6 µg mL(-1), MFC: 10-14 µg mL(-1)) exhibited excellent antifungal activity and were found to be 32-64 and 9-12.8 times more potent than the reference drugs against C. neoformans, respectively. Moreover, compound (MIC: 40 µg mL(-1)) displayed equipotent antibacterial activity against E. coli and S. aureus compared to the reference drugs. The most potent of the synthesized compounds 1v and 1w were further studied by evaluating their cytotoxicities, and the results showed that they had relatively low level cytotoxicity for BV2 cell. A preliminary study of the structure-activity relationship revealed that substituents in the phenyl ring and the thiophene ring had a great effect on the antimicrobial activity of these compounds. In addition, the thiazole ring at C2 may be a pharmacophore of these compounds and COOC2H5 group at C3 is the best substituent for the maintenance of antimicrobial activities at low concentrations (1.5625 µg per disc).
Assuntos
Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Benzodiazepinas/síntese química , Benzodiazepinas/farmacologia , Desenho de Fármacos , Benzodiazepinas/química , Morte Celular/efeitos dos fármacos , Cristalografia por Raios X , Fluconazol/farmacologia , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Relação Estrutura-AtividadeRESUMO
Chemokines are a superfamily of structurally related chemotactic cytokines exerting significant roles in acting as a bridge between the innate and adaptive immune responses. In this study, we identified three CXC motif chemokine 10 (CXCL10) homologues (QsCXCL10-1, QsCXCL10-2 and QsCXCL10-3) from giant spiny frog Quasipaa spinosa. All three deduced QsCXCL10 proteins contained four conserved cysteine residues as found in other known CXC chemokines. Phylogenetic analysis showed that QsCXCL10-1, 2, 3 and other CXCL10s in amphibian were grouped together to form a separate clade. These three QsCXCL10s were highly expressed in spleen and blood. Upon infection with Staphylococcus aureus or Aeromonas hydrophila, the expressions of QsCXCL10s were markedly increased in spleen and blood during biotic stresses. Meanwhile, the QsCXCL10s transcription in liver could also be up-regulated under abiotic stresses such as cold and heat stresses. The recombinant proteins of frog CXCL10 homologues were produced and purified in E. coli and possessed similar but differential bioactivities. Both rCXCL10-1 and rCXCL10-2 had strong effects on the up-regulation of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-8) in vivo, whereas rCXCL10-3 induced a weak expression of these cytokines. Moreover, the rCXCL10-1 and rCXCL10-2 could strongly promote splenocyte proliferation and induce lymphocytes migration, while rCXCL10-3 had limited effects on these biological processes. All three frog chemokines triggered their functional activities by engaging CXC motif chemokine receptor 3 (CXCR3). Taken together, these results revealed that the three QsCXCL10s had similar but differential functional activities in mediating immune responses and host defenses, which might contribute to a better understanding of the functional evolution of CXCL10 in vertebrates.
Assuntos
Anuros/genética , Proliferação de Células/genética , Quimiocina CXCL10/genética , Expressão Gênica , Baço/metabolismo , Aeromonas hydrophila/fisiologia , Sequência de Aminoácidos , Animais , Anuros/metabolismo , Quimiocina CXCL10/classificação , Quimiocina CXCL10/metabolismo , Interações Hospedeiro-Patógeno , Fígado/metabolismo , Filogenia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Baço/citologia , Baço/microbiologia , Staphylococcus aureus/fisiologia , TemperaturaRESUMO
Low-valent aluminum Al(i) chemistry has attracted extensive research interest due to its unique chemical and catalytic properties but is limited by its low stability. Herein, a hourglass phosphomolybdate cluster with a metal-center sandwiched by two benzene-like planar subunits and large steric-hindrance is used as a scaffold to stabilize low-valent Al(i) species. Two hybrid structures, (H3O)2(H2bpe)11[AlIII(H2O)2]3{[AlI(P4MoV 6O31H6)2]3·7H2O (abbr. Al6{P4Mo6}6) and (H3O)3(H2bpe)3[AlI(P4MoV 6O31H7)2]·3.5H2O (abbr. Al{P4Mo6}2) (bpe = trans-1,2-di-(4-pyridyl)-ethylene) were successfully synthesized with Al(i)-sandwiched polyoxoanionic clusters as the first inorganic-ferrocene analogues of a monovalent group 13 element with dual Lewis and Brønsted acid sites. As dual-acid catalysts, these hourglass structures efficiently catalyze a solvent-free four-component domino reaction to synthesize 1,5-benzodiazepines. This work provides a new strategy to stabilize low-valent Al(i) species using a polyoxometalate scaffold.
RESUMO
Interferon regulatory factor 3 (IRF3), a crucial member of interferon regulatory factor (IRF) family, plays an important role in innate immunity in vertebrates. However, there are no reports on the characterization and especially their respective functional analysis of two IRF3 genes in some species. In this study, two IRF3 genes as well as their roles in the immune response were identified and investigated in Chinese giant salamander, Andrias davidianus. The complete open reading frames of AdIRF3A and AdIRF3B were 1, 113 bp and 1, 380 bp in length, encoding 370 and 459 amino acids, respectively. Both AdIRF3A and AdIRF3B protein contain an IRF and an IRF3 domain. Phylogenetic analysis indicated that AdIRF3s clustered together with other IRF3 proteins. Tissue distribution analysis showed that AdIRF3s were expressed in all tissues tested, with highest expression levels in blood. Both AdIRF3s actively responded to Chinese giant salamander iridovirus (GSIV) and poly (I:C) challenge in A. davidianus. AdIRF3A/B silencing significantly suppressed the DNA virus and viral RNA analog-induced expression of IFN-inducible genes. Luciferase reporter assay further confirmed the regulatory role of AdIRF3s in IFN signaling. These results provide new insights into the origin or evolution of IRF3 in amphibians and even in vertebrates.
Assuntos
Proteínas de Anfíbios/genética , Infecções por Vírus de DNA/imunologia , Fator Regulador 3 de Interferon/genética , Iridoviridae/fisiologia , Urodelos/imunologia , Proteínas de Anfíbios/metabolismo , Animais , Células Cultivadas , Clonagem Molecular , Inativação Gênica , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Interferons/metabolismo , Filogenia , Poli I-C/imunologia , Transdução de Sinais , Transcriptoma , Urodelos/genéticaRESUMO
Interleukin 18 (IL-18), a member of IL-1 cytokine superfamily, is an important proinflammatory cytokine with multiple functions in both innate immunity and acquired immunity. However, the characteristics and functional roles of IL-18 remain largely unknown in amphibians, which were classed as major group of vertebrates. In the present study, two IL-18 genes (AdIL-18A and AdIL-18B) and four transcripts (AdIL-18A1, AdIL-18A2, AdIL-18B1 and AdIL-18B2) were firstly identified and characterized from Chinese giant salamander (Andrias davidianus). To the best of our knowledge, this is the first report on the presence of more than one gene copy or two transcripts of IL-18 in one species. The complete open reading frames of AdIL-18A1, AdIL-18A2, AdIL-18B1 and AdIL-18B2 were 588 bp, 603 bp, 591 bp and 606 bp, respectively. The putative AdIL-18 proteins possessed the typical IL-1 domains and phylogenetic analysis indicated that AdIL-18s grouped together with other vertebrate IL-18 proteins. The expression profiles of AdIL-18s were investigated under the challenges of Aeromonas hydrophila, Staphylococcus ureae and Poly (I:C) respectively, and the results suggested that AdIL-18s were involved in the immune responses against both bacterial and viral infections. Moreover, the expression levels of two NF-κBs (P100 and P105) and four proinflammatory cytokines (IL-1ß, IL-6, TNF-α and IFN-γ) were inhibited in AdIL-18A1/A2-silenced cells when treated with bacteria and viral RNA analog. Additionally, the transcription levels of these immune-related cytokine genes were markedly induced when the lymphocytes were treated with recombinant AdIL-18A1 or AdIL-18A2 proteins, implying the involvement of AdIL-18s in triggering NF-κB signaling and proinflammatory responses. These results might provide new insights into the origin or evolution of IL-18 in amphibians and even in vertebrates.
Assuntos
Aeromonas hydrophila/imunologia , Aeromonas hydrophila/fisiologia , Proteínas de Anfíbios/genética , Anfíbios/imunologia , Interleucina-18/genética , Infecções Estafilocócicas/imunologia , Staphylococcus/fisiologia , Proteínas de Anfíbios/metabolismo , Animais , Clonagem Molecular , Citocinas/metabolismo , Dosagem de Genes , Imunidade , Mediadores da Inflamação/metabolismo , Interleucina-18/metabolismo , NF-kappa B/metabolismo , Filogenia , Poli I-C/imunologia , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
In the present paper, two series of substituted tetraphenylporphyrin iron compounds RTPPFe(II) and RTPPFeCI (III) were synthesized by two-step synthetic method. Raman and IR spectra of tetraphenylporphyrin iron complexes were investigated. By analyzing the IR and Raman spectra of RTPPFe(II) and RTPPFe(III)Cl, it was confirmed that the RTPPFe(II) complexes were usually unstable at normal temperature and existed via the coordination of dioxygen with the iron (II) center, i.e. they formed into TPPFe-O2, and the stable configuration of dioxygen with the iron (II) center was "end-on". In addition, the geometric structure optimization of substituted iron tetraphenylporphyrin complexes was performed by PM3 semi-empirical and MM+ method quantum calculation. The structure parameters were obtained, including E(HOMO), E(LUMO) and total energy, bond length of Fe-Cl etc. The effect of molecular structure on vibrational frequency shift was studied by combing the IR and Raman spectra. The results showed that for iron tetraphenylporphyrins (RTPPFe-O2) with different structure, the vibrational frequencies of the Fe-O bond were associated with their twist degree of molecular structures, i.e. the Raman and IR characterization vibrational frequencies of the Fe-O2 were up shifted with the increase in the Fe-O-O angle of iron tetraphenylporphyrins (RTPPFe-O2) molecule.
RESUMO
NFIL3 is a transcriptional activator of the IL-3 promoter in T cells. In vertebrates, it has been characterized as an essential regulator of several cellular processes such as immunity response, apoptosis and NK cells maturation. However, the identification and functional characterization of NFIL3 still remains unclear in arthropods. In this study, the NFIL3 homologue was firstly cloned and characterized in mud crab Scylla paramamosain. The full-length of SpNFIL3 was 2, 041 bp in length with an open reading frame of 1, 509 bp, containing a conserved basic region of leucin zipper domain. The qRT-PCR analysis indicated that SpNFIL3 was significantly highly expressed in hepatopancreas and in hemocytes. Moreover, the SpNFIL3 transcription could be up-regulated after the challenge of Vibrio alginolyticus or virus-analog Poly (I:C). The dual-luciferase reporter assays revealed that SpNFIL3 could activate NF-κB pathway. The immunofluorescence assay indicated SpNFIL3 was located in nucleus. After NFIL3 was interfered in vivo and in vitro, the expressions of two NF-κB members (SpRelish and SpDorsal), six antimicrobial peptide genes (SpCrustin and SpALF2-6) and pro-inflammatory cytokine SpIL-16 were suppressed, and the bacteria clearance capacity of crabs was also markedly impaired in NFIL3 silenced crabs. These results indicated that SpNFIL3 played crucial role in the innate immunity of S. paramamosain and it also brought new insight into the origin and evolution of NFIL3 in arthropods and even in invertebrates.
Assuntos
Proteínas de Artrópodes/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Braquiúros/imunologia , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Braquiúros/metabolismo , NF-kappa B/metabolismo , Filogenia , Alinhamento de SequênciaRESUMO
Activins, members of transforming growth factor ß (TGF-ß) superfamily, are pleiotropic cytokines with critical roles in mediating cell proliferation, differentiation, homeostasis, apoptosis and immune response. However, the structural characteristics and specific functions of Activins remain largely unknown in invertebrates. In the present study, an Activin-like ligand Dawdle (Daw) was firstly identified and characterized from mud crab Scylla paramamosain. The obtained cDNA sequence of SpDaw was 2, 196 bp long with a 1, 149 bp open reading fame, which encoded a putative protein of 382 amino acids. The putative SpDaw protein contained a signal peptide, a TGF-ß propeptide region and a TGF-ß domain. Real-time PCR analysis demonstrated that SpDaw was predominantly expressed at early embryonic development stage and premolt stages, implying its participation in development and growth. Furthermore, SpDaw responded to both Vibro alginolyticus and Poly (I:C) challenges, suggesting the involvement of SpDaw in innate immune responses. Knockdown of SpDaw in vivo dramatically increased the expressions of NF-κB signaling genes and anti-lipopolysaccharide factor (ALF) genes, and the bacteria clearance efficiency was also markedly enhanced in SpDaw-silenced crabs. Moreover, the in vitro experiment further demonstrated that recombinant SpDaw protein could block the increased transcription of IKKs, NF-κBs and ALFs induced by pathogen challenges. Taken together, these results indicated that SpDaw not only participated in development and growth processes but also played an immune-regulatory role in crabs' innate immunity, which may pave the way for a better understanding of TGF-ß superfamily members in crustacean species.
Assuntos
Proteínas de Artrópodes/fisiologia , Braquiúros/imunologia , Imunidade Inata/imunologia , NF-kappa B/metabolismo , Transdução de Sinais/imunologia , Ativinas/imunologia , Sequência de Aminoácidos , Animais , Braquiúros/genética , Braquiúros/crescimento & desenvolvimento , Proteínas de Transporte/fisiologia , Ligantes , Filogenia , Alinhamento de SequênciaRESUMO
The six-membered heterocyclic ring in the title compound, C(16)H(12)ClNO(2)S, exists in a conformation intermediate between twist-boat and chair. A one-dimensional chain structure is formed as a result of inter-molecular N-Hâ¯O and C-Hâ¯O hydrogen bonds via crystallographic inversion symmetry and translation along the a axis.
RESUMO
A full IR, UV-Vis, FIR, spectroscopic analysis on substituted porphyrin iron, manganese, cobalt, copper and Zinc complexes was performed, and the spectroscopic patterns were found for the metalloporphyrin compounds with various structures. Base on molecular structures theory, the reasons for the spectroscopic patterns were discussed deeply. Considering the inconsistency of the far-infrared spectra of the Fe-Cl axial bonds in chloro-iron tetraphenylporphyrins reported in the literature. Their far-infrared absorptions were investigated by using both theoretical analysis and experimental determination, and the results showing that their vibrational frequencies of the Fe--Cl bonds in different substitution chloro-iron tetraphenylporphyrins were associated with their structures, and were linearly related to the length of Fe--Cl bonds.
RESUMO
The transforming growth factor-ß (TGF-ß) receptor-mediated TGF-ß signaling cascade plays important roles in diverse cellular processes, including cell proliferation, differentiation, growth, apoptosis and inflammation in vertebrates. In the present study, the type I TGF-ß receptor (TßR1) was firstly identified and characterized in mud crab Scylla paramamosain. The full-length cDNA of SpTßR1 was 1, 986 bp with a 1, 608 bp open reading frame, which encoded a putative protein of 535 amino acids including a typical transmembrane region, a conserved glycine-serine (GS) motif and a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Real-time PCR analysis showed that SpTßR1 was predominantly expressed at early embryonic development stage and was highly expressed at postmolt stages during molt cycle, suggesting its participation in development and growth. Moreover, the expression levels of SpTßR1 in hepatopancreas and hemocytes were positively induced after the challenges of Vibro alginolyticus and Poly (I:C), indicating the involvement of SpTßR1 in responding to both bacterial and viral infections. The in vivo RNA interference assays demonstrated that the expression levels of two NF-κB members (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF2-6) were significantly suppressed when the SpTßR1 was silenced. Additionally, the expression levels of SpTßR1, SpRelish, SpDorsal and AMPs were consistently down-regulated or up-regulated when the primary cultured hemocytes were treated with TßR1 antagonist or agonist for 24â¯h. These results indicated that TßR1 not only contributed to the crabs' development and growth but also played vital role in the innate immunity of S. paramamosain, and it also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.
Assuntos
Proteínas de Artrópodes/fisiologia , Braquiúros/fisiologia , Doenças dos Peixes/imunologia , Imunidade Inata , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Animais , Aquicultura , Proteínas de Artrópodes/agonistas , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/isolamento & purificação , Células Cultivadas , Evolução Molecular , Doenças dos Peixes/virologia , Hemócitos/imunologia , Hemócitos/metabolismo , Hepatopâncreas/imunologia , Hepatopâncreas/metabolismo , Larva/crescimento & desenvolvimento , Larva/imunologia , Filogenia , Poli I-C/imunologia , Cultura Primária de Células , Receptores de Fatores de Crescimento Transformadores beta/agonistas , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/isolamento & purificação , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vibrioses/imunologia , Vibrioses/veterinária , Vibrioses/virologia , Vibrio alginolyticus/imunologiaRESUMO
A series of novel 1,5-benzodiazepine derivatives were rationally designed and synthesized following the principle of the superposition of bioactive substructures by the combination of 1,5-benzodiazepine, pyridine (phenyl), and an ester group. The structures of the target compounds were determined by (1) H NMR, (13) C NMR, MS, IR, and elemental analysis. All the synthesized compounds were evaluated for their antimicrobial activities in vitro against the fungi C. neoformans, C. neoformans clinical isolates (ATCC 32264), C. albicans (ATCC 10231), Gram-negative bacterium E. coli (ATCC 44752), and Gram-positive bacterium S. aureus (ATCC 25923). The results of the bioactive assay demonstrated that most of the tested compounds exhibited variable inhibitory effects on the growth of the tested microorganisms. All the active compounds showed better antifungal activity than antibacterial activity. Notably, compound 2b displayed the highest activity (MIC = 30 µg/mL) against C. neoformans and (MIC = 31 µg/mL) against C. neoformans clinical isolates. In addition, compound 2a also showed excellent activity against C. neoformans and C. neoformans clinical isolates with minimum inhibitory concentration of 35 and 36 µg/mL, respectively. Compounds 2a and 2b were further studied by evaluating their cytotoxicities, and the results showed that they have relatively low level cytotoxicity for BV2 and 293T cell. Preliminary structure-activity relationship study on three diverse sets (C-2, C-3, and C-8 positions) of 1,5-benzodiazepines was performed. The results revealed that the presence of a -CH3 group at the C-8 position had a positive effect on the inhibitory activity of these compounds. Additionally, the 2-pyridyl group at the C-2 position may be a pharmacophore and -COOC2 H5 at C-3 position is the best substituent for the maintenance of antimicrobial activities.