RESUMO
BACKGROUND: We have identified endogenous p65 to be an SDS-stable dimer protein composed of ~ 37 kDa hnRNPA/B-like subunits. We have investigated molecular properties involved in the stability of dimeric form, and their regulation in the transition between monomeric and dimeric forms of hnRNPA/B-like protein 2. We also investigated a cellular property conserved between squid hnRNPA/B-like protein 2 and human hnRNPA1 protein in a neuronal context. METHODS AND RESULTS: Here we show biochemical properties of a recombinant hnRNPA/B-like protein 2 (rP2) in vitro experiments, as one of p65 subunit. We found that interaction between rP2 and RNA molecules interfered with the dynamics of rP2 dimers formation, involved in disulfide bonds and/or postranslational alterations in distinct stage of SDS-stable dimers formation. In addition, we have performed immunofluorescence in SH-SY5Y cells and observed that the pEGFP-P2 fusion protein was expressed in the nucleus, similar to what is observed for human hnRNPA1 protein. CONCLUSION: Our results reinforce the idea that p65 is an SDS-stable dimer. Thus, a deeper understanding between monomeric and dimeric transition dynamic is critical into evolution of several neurodegenerative disease.
Assuntos
Neuroblastoma , Doenças Neurodegenerativas , Animais , Decapodiformes/genética , Decapodiformes/metabolismo , Humanos , Pressão Osmótica , Proteínas Recombinantes/genéticaRESUMO
ß-chitin was isolated from marine waste, giant Humboldt squid Dosidicus gigas, and further converted to nanofibers by use of a collider machine under acidic conditions (pH 3). The FTIR, TGA, and NMR analysis confirmed the efficient extraction of ß-chitin. The SEM, TEM, and XRD characterization results verified that ß-chitin crystalline structure were maintained after mechanical treatment. The mean particle size of ß-chitin nanofibers was in the range between 10 and 15 nm, according to the TEM analysis. In addition, the ß-chitin nanofibers were converted into films by the simple solvent-casting and drying process at 60 °C. The obtained films had high lightness, which was evidenced by the CIELAB color test. Moreover, the films showed the medium swelling degree (250-290%) in aqueous solutions of different pH and good mechanical resistance in the range between 4 and 17 MPa, depending on film thickness. The results obtained in this work show that marine waste can be efficiently converted to biomaterial by use of mild extractive conditions and simple mechanical treatment, offering great potential for the future development of sustainable multifunctional materials for various industrial applications such as food packaging, agriculture, and/or wound dressing.
Assuntos
Materiais Biocompatíveis , Quitina/isolamento & purificação , Decapodiformes/metabolismo , Nanofibras , Resíduos , Animais , Configuração de Carboidratos , Quitina/química , Tamanho da Partícula , Propriedades de Superfície , ViscosidadeRESUMO
The effect of a hydroxyl radical generating system (HRGS), which contained FeCl3, sodium ascorbate, and different concentrations of H2O2, on the physiochemical properties of myofibrillar protein (MP) from squid mantles, has been investigated. The effect of different exposure times to HRGS was also considered. Compared to non-oxidized MP, a significant (pâ¯<â¯0.05) increase in carbonyl content (more than 50% of its original content) and protein solubility, as well as in surface hydrophobicity, was observed in the oxidative MP. With different treatment times, a sharp decrease (pâ¯<â¯0.05) in sulfhydryl content was detected. In addition, hydroxyl radical treatment significantly reduced the MP gel's texture properties, whiteness and water holding capacity, especially at higher concentrations of H2O2. This observation could be attributed to extensive disorderly and less compact structure of MP gels. The results demonstrate the negative effect of HRGS on the structural and functional properties of MP from squid mantles.
Assuntos
Decapodiformes/metabolismo , Proteínas de Frutos do Mar/química , Animais , Ácido Ascórbico/química , Géis/química , Interações Hidrofóbicas e Hidrofílicas , Radical Hidroxila/química , Oxirredução , Reologia , Proteínas de Frutos do Mar/metabolismo , Solubilidade , Compostos de Sulfidrila/análise , Água/químicaRESUMO
Plastic pollution is prevalent worldwide and affects marine wildlife from urbanized beaches to pristine oceanic islands. However, the ecological basis and mechanisms that result in marine animal ingestion of plastic debris are still relatively unknown, despite recent advances. We investigated the relationship between scavenging behavior and plastic ingestion using green turtles, Chelonia mydas, as a model. Diet analysis of C. mydas showed that sea turtles engaging in scavenging behavior ingested significantly more plastic debris than individuals that did not engage in this foraging strategy. We argue that opportunistic scavenging behavior, an adaptive behavior in most marine ecosystems, may now pose a threat to a variety of marine animals due to the current widespread plastic pollution found in oceans.
Assuntos
Decapodiformes/metabolismo , Ingestão de Alimentos , Comportamento Alimentar , Plásticos/metabolismo , Tartarugas/metabolismo , Poluentes da Água/metabolismo , Animais , Oceano Atlântico , Ecossistema , Cadeia Alimentar , Conteúdo Gastrointestinal/química , ResíduosRESUMO
BACKGROUND: The giant squid (Dosidicus gigas) has been proposed as raw material to obtain myofibrillar protein concentrates. However, it has been observed that colloidal systems formed from squid proteins have limited stability. Therefore, the isolation and characterization of the actomyosin-paramyosin isolated (API) complex were performed, because they are the main proteins to which functionality has been attributed. RESULTS: Densitogram analysis revealed 45% of actin, 38% of myosin and 17% of paramyosin. The amino acid profile indicates a higher proportion of acidic amino acids, which gives a higher negative charge; this was supported by the zeta potential. Total sulfhydryl (TSH) content was lower compared with proteins of other aquatic species. CONCLUSION: The higher percentage of actin in relation to myosin, the presence of paramyosin, as well as the low content of sulfhydryl groups, could comprise the main causes of the low technological functional property of proteins from D. gigas mantle. © 2017 Society of Chemical Industry.
Assuntos
Actomiosina/química , Decapodiformes/química , Tropomiosina/química , Actinas/química , Actinas/metabolismo , Actomiosina/metabolismo , Animais , Decapodiformes/metabolismo , Estabilidade Proteica , Alimentos Marinhos/análise , Tropomiosina/metabolismoRESUMO
In squid nerves, MgATP modulation of the Na(+)/Ca(2+) exchanger requires the presence of a cytosolic protein which becomes phosphorylated during the process. This factor has been recently identified. Mass spectroscopy and Western blot analysis established that it is a member of the lipocalin superfamily of lipid-binding proteins (LBP or FABP) of 132 amino acids. We called it regulatory protein of squid nerve sodium/calcium exchanger (ReP1-NCXSQ, access to GenBank EU981897).ReP1-NCXSQ was cloned, expressed, and purified. Circular dichroism, far-UV, and infrared spectroscopy suggest a secondary structure, predominantly of beta-sheets. The tertiary structure prediction provides ten beta-sheets and two alpha-helices, characteristic of most of LPB. Functional experiments showed that, to be active, ReP1-NCXSQ must be phosphorylated by MgATP, through the action of a kinase present in the plasma membrane. Moreover, PO4-ReP1-NCXSQ can stimulate the exchanger in the absence of ATP. An additional crucial observation was that, in proteoliposomes containing only the purified Na(+)/Ca(2+) exchanger, PO4-ReP1-NCXSQ promotes activation; therefore, this upregulation has no other requirement than a lipid membrane and the incorporated exchanger protein.Recently, we solved the crystal structure of ReP1-NCXSQ which was as predicted: a "barrel" consisting of ten beta-sheets and two alpha-helices. Inside the barrel is the fatty acid coordinated by hydrogen bonds with Arg126 and Tyr128. Point mutations showed that neither Tyr20Ala, Arg58Val, Ser99Ala, nor Arg126Val is necessary for protein phosphorylation or activity. On the other hand, Tyr128 is essential for activity but not for phosphorylation. We can conclude that (1) for the first time, a role of an LBP is demonstrated in the metabolic regulation of an ion exchanger; (2) phosphorylation of this LBP can be separated from the activation capacity; and (3) Tyr128, a candidate to coordinate lipid binding inside the barrel, is essential for activity.
Assuntos
Decapodiformes , Proteínas do Tecido Nervoso , Trocador de Sódio e Cálcio , Animais , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Clonagem Molecular , Decapodiformes/química , Decapodiformes/genética , Decapodiformes/metabolismo , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Fosforilação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Trocador de Sódio e Cálcio/química , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/isolamento & purificação , Trocador de Sódio e Cálcio/metabolismoRESUMO
This work shows, for the first time, a properly metabolically regulated squid nerve Na(+)/Ca(2+) exchanger (NCXSQ1) heterologous expressed in Saccharomyces cerevisiae. The exchanger was fused to the enhanced green fluorescence protein (eGFP) on its C-terminus and had two tags, a Strep-tag II and 6 histidines, added to the N-terminal region (ST-6HB-NCXSQ1-eGFP). The eGFP fluorescence signal co-localized with that of the plasma membrane marker FM1-43 in whole cells that displayed an uptake of Ca(2+) with the expected characteristics of the reverse Na(+)/Ca(2+) exchange mode. Similar to squid nerve membrane vesicles, inside-out yeast plasma membrane vesicles (ISOV) showed a Ca(2+)(i) regulation of the forward mode that was modulated by previously phosphorylated regulatory cytosolic protein (ReP1-NCXSQ). On the other hand, a close association between NCXSQ1 and ReP1-NCXSQ, estimated by co-immunoprecipitation, was independent of ReP1-NCXSQ phosphorylation. An additional crucial observation was that in proteoliposomes containing only the ST-6HB-NCXSQ1-eGFP protein, Na(+)/Ca(2+) exchange was stimulated by phosphorylated ReP1-NCXSQ; i.e., this up-regulation needs no other requirement besides the lipid membrane and the exchanger protein. Finally, this work provides a potential approach to obtain enough purified NCXSQ1 for structural and biochemical studies which have been delayed due to the lack of sufficient material.
Assuntos
Decapodiformes/metabolismo , Saccharomyces cerevisiae , Trocador de Sódio e Cálcio/metabolismo , Animais , Membrana Celular/metabolismo , Lipossomos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trocador de Sódio e Cálcio/genética , Regulação para CimaRESUMO
In a previous work we demonstrated that, in dialyzed squid axons, an impairment of the Ca2+(i)-regulatory site affected the apparent affinities for external Na+ and Ca2+ in a way opposite to that predicted by the exiting (ping-pong) models for the exchangers. In the present work, we used model simulations and actual experiments where the Ca2+(i)-regulatory remained always saturated while [Ca2+](i) was either limiting or near saturating for the internal Ca2+ transport sites. Under these conditions, both the theoretical and experimental transport activation curves for external Na+ and Ca2+ were those expected from the current kinetic schemes. These observations have two important implications: on the one hand, they confirm the ping-pong translocation schemes for Na+/Ca2+ exchange. On the other, they call for caution in interpreting kinetic data in membrane transport systems possessing intracellular ionic and/or metabolic regulation.
Assuntos
Axônios/metabolismo , Decapodiformes/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Animais , Cálcio/metabolismo , Cinética , Modelos Biológicos , Sódio/metabolismoRESUMO
The extrasynaptic region of the squid giant nerve fiber exhibits neuron-Schwann cell interactions that appear to involve glutamate as a mediator. In an earlier work, it was demonstrated that the periaxonal sheaths of such nerves (where the Schwann cells are located) possess the capacity to transport glutamate. However there was no information available about the possible fate of the glutamate incorporated into the sheaths. In this study, it is demonstrated that the periaxonal sheaths of the extrasynaptic region of squid giant nerves are capable of metabolizing glutamate. Sheaths incubated with 10 microM [1-14C] glutamate produced [14C] O2 in a manner proportional to time and estimated cell water volume. At least 45% of this CO2 production was determined to be independent of transaminase catalyzed isotopic exchange, thus reflecting real degradation (decarboxylation) of glutamate. It was also demonstrated that the sheaths were capable of glutamine synthesis. Taken together, the findings of our laboratory indicate not only that the Schwann cells of the sheaths fulfil the requirements for a site for the uptake and metabolism of transmitter glutamate in the squid giant nerve but also that certain metabolic characteristics associated with the neuro-glial unit around synapses are also found in non-synaptic areas of nerve fibers.
Assuntos
Glutamatos/metabolismo , Fibras Nervosas/metabolismo , Animais , Axônios/metabolismo , Decapodiformes/metabolismo , Bainha de Mielina/metabolismoRESUMO
The storage (-28 degrees C) of squids prior to thermal treatment (steaming at 100 degrees C and autoclaving at 115 degrees C) was found to exert no significant influence on the available lysine and tryptophan contents of the flesh stored for no longer than 9-10 months. During a longer period of storage the contents of the studied amino acids decreased by 22.2% on an average. The -SH groups content and weight losses after thermal treatment were increasing with the time of frozen storage. The colour of squid flesh, as measured with a and b indicators (LAB system), changed most in the autoclaved samples, the colour changing towards yellow-red.