RESUMO
Ultraviolet A (UVA) radiation, present in sunlight, can induce cell redox imbalance leading to cellular damage and even cell death, compromising skin health. Here, we evaluated the in vitro antioxidant and photochemoprotective effect of dithiothreitol (DTT). DTT neutralized the free radicals 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+), 2,2-diphenyl-1-picrylhydrazyl (DPPH·), and superoxide anion (O2·-) in in vitro assays, as well as the ferric ion (Fe3+) in the ferric reducing antioxidant power (FRAP) assay. We also evaluated the effect of DTT pre-treatment in L929 dermal fibroblasts and DTT (50 and 100 µM) led to greater cell viability following UVA-irradiation compared to cells that were untreated. Furthermore, the pre-treatment of cells with DTT prevented the increase of intracellular reactive oxygen species (ROS) production, including hydrogen peroxide (H2O2), lipid peroxidation, and DNA condensation, as well as the decrease in mitochondrial membrane potential (Δψm), that occurred following irradiation in untreated cells. The endogenous antioxidant system of cells was also improved in irradiated cells that were DTT pre-treated compared to the untreated cells, as the activity of the superoxide dismutase (SOD) and catalase (CAT) enzymes remained as high as non-irradiated cells, while the activity levels were depleted in the untreated irradiated cells. Furthermore, DTT reduced necrosis in UVA-irradiated fibroblasts. Together, these results showed that DTT may have promising use in the prevention of skin photoaging and photodamage induced by UVA, as it provided photochemoprotection against the harmful effects of this radiation, reducing oxidative stress and cell death, due mainly to its antioxidant capacity.
Assuntos
Antioxidantes , Peróxido de Hidrogênio , Humanos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Ditiotreitol/farmacologia , Ditiotreitol/metabolismo , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Pele/efeitos da radiação , Raios Ultravioleta , Necrose , FibroblastosRESUMO
Experiments were conducted to investigate whether supplementation of cryopreservation medium with ascorbate, dithiothreitol (DTT) or an inhibitor of caspase-3 (z-DEVD-fmk) could improve post-thaw survival of bovine embryos produced in vitro (IVP). For all experiments, embryos were harvested on day 7 after insemination and subjected to controlled-rate freezing in medium containing 1.5 M ethylene glycol and treatments as described below. In experiments 1-3, embryos were cryopreserved in freezing medium with ascorbate (0, 0.1, 0.3 or 0.5 mM), DTT (0, 50, 100 or 200 µM) and z-DEVD-fmk (0, 50, 100 or 200 µM), respectively. Post-thaw survival was assessed at 24, 48 and 72 h. For experiments 4-5, embryos were cryopreserved in freezing medium with or without 0.1 mM ascorbate. At 24 h post-thaw, embryo total cell number, DNA fragmentation and levels of reactive oxygen species (ROS) were evaluated. Embryos subjected to freezing and thawing in medium supplemented with 0.1 mM ascorbate had greater (p < .05) re-expansion rates at 24, 48 and 72 h and hatching rate at 72 h as compared to embryos not treated with ascorbate. Post-thaw cryosurvival was not affected by the addition of either DTT or z-DEVD-fmk to medium used for cryopreservation. Embryos cryopreserved in medium supplemented with 0.1 mM ascorbate had reduced (p < .001) levels of intracellular ROS and fewer (p < .001) cells with DNA fragmentation. In conclusion, post-thaw survival of bovine IVP embryos is enhanced by supplementation of freezing medium with ascorbate.
Assuntos
Criopreservação , Embrião de Mamíferos , Animais , Caspase 3 , Inibidores de Caspase , Bovinos , Criopreservação/veterinária , Ditiotreitol/farmacologia , Fertilização in vitro/veterinária , Espécies Reativas de OxigênioRESUMO
Nowadays, the nutraceutical agent sulforaphane (SFN) shows great versatility in turning on different cellular responses. Mainly, this isothiocyanate acts as a master regulator of cellular homeostasis due to its antioxidant response and cytoplasmic, mitochondrial, and endoplasmic reticulum (ER) protein modulation. Even more, SFN acts as an effective strategy to counteract oxidative stress, apoptosis, and ER stress, among others as seen in different injury models. Particularly, ER stress is buffered by the unfolded protein response (UPR) activation, which is the first instance in orchestrating the recovery of ER function. Interestingly, different studies highlight a close interrelationship between ER stress and oxidative stress, two events driven by the accumulation of reactive oxygen species (ROS). This response inevitably perpetuates itself and acts as a vicious cycle that triggers the development of different pathologies, such as cardiovascular diseases, neurodegenerative diseases, and others. Accordingly, it is vital to target ER stress and oxidative stress to increase the effectiveness of clinical therapies used to treat these diseases. Therefore, our study is focused on the role of SFN in preserving cellular homeostasis balance by regulating the ER stress response through the Nrf2-modulated antioxidant pathway.
Assuntos
Antioxidantes , Isotiocianatos , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Apoptose , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Homeostase , Isotiocianatos/metabolismo , Isotiocianatos/farmacologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Sulfóxidos , Resposta a Proteínas não DobradasRESUMO
Environmental stresses disturb the endoplasmic reticulum (ER) protein folding. However, primary metabolic responses induced by ER stress remain unclear. Thus, we investigated the morphophysiological and metabolomic changes under ER stress, induced by dithiothreitol (DTT) and tunicamycin (TM) treatments in sorghum seedlings from 24 to 96 h. The ER stress caused lipid peroxidation and increased the expression of SbBiP1, SbPDI, and SbIRE1. The development impairment was more pronounced in roots than in shoots as distinct metabolomic profiles were observed. DTT decreased root length, lateral roots, and root hair, while TM decreased mainly the root length. At 24 h, under ER stresses, the glutamic acid and o-acetyl-serine were biomarkers in the shoots. While homoserine, pyroglutamic acid, and phosphoric acid were candidates for roots. At the latest time (96 h), kestose and galactinol were key metabolites for shoots under DTT and TM, respectively. In roots, palatinose, trehalose, and alanine were common markers for DTT and TM late exposure. The accumulation of sugars such as arabinose and kestose occurred mainly in roots in the presence of DTT at a later time, which also inhibited glycolysis and the tricarboxylic acid cycle (TCA). Amino acid metabolism was induced, which also contributed TCA components decreasing, such as succinate in shoots and citrate in roots. Thus, our study may provide new insights into primary metabolism modulated by ER stress and seedling development.
Assuntos
Estresse do Retículo Endoplasmático , Sorghum , Ditiotreitol , Plântula , TunicamicinaRESUMO
BACKGROUND: Red oak pollen is an important cause of allergic respiratory disease and it is widely distributed in North America and central Europe. To date, however, red oak pollen allergens have not been identified. Here, we describe the allergenic protein profile from red oak pollen. METHODS: Total proteins were extracted from red oak pollen using a modified phenolic extraction method, and, subsequently, proteins were separated by two-dimensional gel electrophoresis (2DE) for both total protein stain (Coomassie Blue) and immunoblotting. A pool of 8 sera from red oak sensitive patients was used to analyze blotted proteins. Protein spots were analyzed by Mass Spectrometry. RESULTS: Electrophoretic pattern of total soluble proteins showed higher intensity bands in the regions of 26-40 and 47-52 kDa. Two dimensional immunoblots using pool sera from patients revealed four allergenic proteins spots with molecular masses in the range from 50 to 55 kDa. Mass spectrometry analysis identified 8 proteins including Enolase 1 and Enolase 1 chloroplastic, Xylose isomerase (X1 isoform), mitochondrial Aldehyde dehydrogenase, UTP-Glusose-1-phosphate uridylyltransferase, Betaxylosidase/alpha-l-arabinofuranosidase and alpha- and beta subunits of ATP synthase. CONCLUSIONS: This study has identified for first time 8 IgE binding proteins from red oak pollen. These findings will pave the way towards the development of new diagnostic and therapeutic modalities for red oak allergy.
RESUMO
This work reports the characterisation of caseinolytic and milk-clotting activities of proteases extracted from ripe fruits of Morinda citrifolia L., as a potential of their use in cheese production. Noni puree extract (NPE) was obtained by homogenising the fresh puree in 150â¯mM NaCl/50â¯mM sodium phosphate buffer (pH 7.0). The resulting protein concentration was of 0.367⯱â¯0.006â¯mg/mL, and an electrophoretic profile of the extract revealed protein bands ranging from 14 to 55â¯kDa. The proteolytic activity of NPE was higher when the extract had been previously incubated at pH 6.0 (8.859⯱â¯0.216â¯U/mg), whereas the optimum caseinolytic activity was observed at 50⯰C. Noni puree proteases were strongly (98%) inhibited by iodoacetamide and E-64, suggesting the presence of only cysteine proteases in the crude extract. NPE proteases showed a milk-clotting activity (MCA) of 238.80⯱â¯5.29â¯U/mL, a specific milk-clotting activity (SMCA) of 9950.17⯱â¯220.74â¯U/mg, and an SMCA/PA ratio of 1124.31⯱â¯24.94, this last being comparable to those of commercial calf rennet. The cheese manufactured using NPE presented brittle and soft texture, high humidity, and showed sanitary conditions compatible with current Brazilian regulations. The product showed a slightly bitter taste, but still good acceptability, rating between 6 and 7 in the hedonic scale for flavour, texture, and overall acceptance. Lastly, there was 60% of positive purchase intent, demonstrating that noni fruit is a promising source of milk-clotting enzymes for the dairy industry.
Assuntos
Queijo , Cisteína Proteases/metabolismo , Frutas/metabolismo , Leite/metabolismo , Morinda/metabolismo , Extratos Vegetais/metabolismo , Animais , Brasil , Manipulação de Alimentos/métodosRESUMO
Many tropical trees have high canopies and their leaves are not accessible. Thus, the use of tissue from a more accessible organ (cambium) for DNA extraction may be an alternative for molecular studies. We adapted a feasible methodology for extracting genomic DNA from cambium tissue harvested in the field for the assessment with PCR. We tested three storage conditions (two buffers and a silica gel) and four periods of time after harvest. We used previously described protocols and tested them on three species that occur in Amazonian forests and other biomes: Anadenanthera peregrina var. peregrina, Cedrela fissilis, and Ceiba speciosa. Our protocol obtained suitable PCR-grade genomic DNA for DNA sequencing and microsatellite genotyping. We recommend the use of silica for long-term storage and the buffer with ascorbic acid for short-term storage.(AU)
Muitas árvores tropicais possuem dossel alto e folhas não facilmente acessíveis. O uso de tecido de um órgão mais acessível (câmbio) para extração de DNA pode ser uma alternativa para estudos moleculares. Nós adaptamos uma metodologia viável para extrair DNA genômico de tecido cambial coletado no campo para avaliação com PCR. Testamos três condições de armazenamento (dois tampões e sílica gel) e quatro períodos após a coleta. Utilizamos protocolos descritos anteriormente e os testamos em três espécies encontradas em florestas amazônicas e outros biomas: Anadenanthera peregrina var. peregrina, Cedrela fissilis e Ceiba speciosa. Nosso protocolo foi eficaz na obtenção de DNA adequado para sequenciamento e genotipagem de microssatélites. Recomendamos o uso de sílica para armazenamento de longo prazo e o tampão com ácido ascórbico para curto prazo.(AU)
Assuntos
Floresta Úmida , Ácido Ascórbico , DNA/isolamento & purificaçãoRESUMO
Pathological α-synuclein (α-syn) overexpression and iron (Fe)-induced oxidative stress (OS) are involved in the death of dopaminergic neurons in Parkinson's disease (PD). We have previously characterized the role of triacylglycerol (TAG) formation in the neuronal response to Fe-induced OS. In this work we characterize the role of the α-syn variant A53T during Fe-induced injury and investigate whether lipid metabolism has implications for neuronal fate. To this end, we used the N27 dopaminergic neuronal cell line either untransfected (UT) or stably transfected with pcDNA3 vector (as a transfection control) or pcDNA-A53T-α-syn (A53T α-syn). The overexpression of A53T α-syn triggered an increase in TAG content mainly due to the activation of Acyl-CoA synthetase. Since fatty acid (FA) ß-oxidation and phospholipid content did not change in A53T α-syn cells, the unique consequence of the increase in FA-CoA derivatives was their acylation in TAG moieties. Control cells exposed to Fe-induced injury displayed increased OS markers and TAG content. Intriguingly, Fe exposure in A53T α-syn cells promoted a decrease in OS markers accompanied by α-syn aggregation and elevated TAG content. We report here new evidence of a differential role played by A53T α-syn in neuronal lipid metabolism as related to the neuronal response to OS.
Assuntos
Ferro/toxicidade , Neurônios/metabolismo , alfa-Sinucleína/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/genética , Gotículas Lipídicas/metabolismo , Mutação , Neurônios/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transfecção/métodos , Triglicerídeos/metabolismo , alfa-Sinucleína/genéticaRESUMO
The clinical use of paclitaxel as a chemotherapeutic agent is limited by the severe acute and chronic hypersensitivity caused when it is administered via intraperitoneal or intravenous routes. Thus far, evidence has suggested that transient receptor potential vanilloid-1 (TRPV1) has a key role in the chronic neuropathy induced by paclitaxel. Despite this, the role of TRPV1 in paclitaxel -related acute nociception, especially the development of visceral nociception, has not been evaluated. Thus, the goal of this study was to evaluate the participation of TRPV1 in a model of acute nociception induced by paclitaxel in rats and mice. A single intraperitoneal (i.p.) paclitaxel administration (1â¯mg/kg, i.p.) produced an immediate visceral nociception response 1â¯h after administration, caused mechanical and heat hypersensitivity, and diminished burrowing behaviour 24â¯h after administration. These nociceptive responses were reduced by SB-366791 treatment (0.5â¯mg/kg, i.p., a TRPV1 antagonist). In addition, TRPV1-positive sensory fibre ablation (using resiniferatoxin, 200⯵g/kg, s.c.) reduced visceral nociception and mechanical or heat hypersensitivity caused by paclitaxel injection. Similarly, TRPV1 deficient mice showed a pronounced reduction in mechanical allodynia to paclitaxel acute injection and did not develop heat hypersensitivity. Moreover, 24â¯h after its injection, paclitaxel induced chemical hypersensitivity to capsaicin (a TRPV1 agonist, 0.01 nmol/site) and increased TRPV1 immunoreactivity in the dorsal root ganglion and sciatic nerve. In conclusion, TRPV1 is involved in mechanical and heat hypersensitivity and spontaneous-pain behaviour induced 24â¯h after a single paclitaxel injection. This receptor is also involved in visceral nociception induced immediately after paclitaxel administration.
Assuntos
Nociceptividade/efeitos dos fármacos , Paclitaxel/efeitos adversos , Canais de Cátion TRPV/metabolismo , Dor Aguda/induzido quimicamente , Dor Aguda/metabolismo , Dor Aguda/fisiopatologia , Animais , Masculino , Camundongos , Ratos , Xantofilas/farmacologiaRESUMO
StAP3 is a plant aspartic protease with cytotoxic activity toward a broad spectrum of pathogens, including potato and human pathogen microorganisms, and cancer cells, but not against human T cells, human red blood cells or plant cells. For this reason, StAP3 could be a promising and potential drug candidate for future therapies. In this work, the improvement of the performance of StAP3 was achieved by means of a modification with PEG. The separation of a mono-PEGylated StAP3 fraction was easily performed by gel filtration chromatography. The mono-PEGylated StAP3 fraction was studied in terms of in vitro antimicrobial activity, exhibiting higher antimicrobial activity against Fusarium solani spores and Bacillus cereus, but slightly lower activity against Escherichia coli than native protein. Such increase in antifungal activity has not been reported previously for a PEGylated plant protein. In addition, PEGylation did not affect the selective cytotoxicity of StAP3, since no hemolytic activity was observed.
RESUMO
In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-ß superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Transdução de Sinais , Animais , Sequência de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Embrião não Mamífero , Elementos Facilitadores Genéticos , Ligação Proteica , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Glutathione (GSH) displays a broad range of functions, among them a role as a neuromodulator with some neuroprotective properties. Taking into account that oxidative stress has been associated with depressive disorders, this study investigated the possibility that GSH, a major cell antioxidant, elicits an antidepressant-like effect in mice. Thus, GSH was administered by i.c.v. route to mice that were tested in the forced swimming test and in the tail suspension test, two predictive tests for antidepressant drug activity. In addition, GSH metabolism and the redox environment were modulated in order to study the possible mechanisms underlying the effects of GSH in the forced swimming test. The administration of GSH decreased the immobility time in the forced swimming test (300-3000nmol/site) and tail suspension test (100-1000nmol/site), consistent with an antidepressant-like effect. GSH depletion elicited by l-buthionine sulfoximine (3.2µmol/site, i.c.v.) did not alter the antidepressant-like effect of GSH, whereas the inhibition of extracellular GSH catabolism by acivicin (100nmol/site, i.c.v.) prevented the antidepressant-like effect of GSH. Moreover, a sub-effective dose (0.01nmol/site, i.c.v.) of the oxidizing agent DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) potentiated the effect of GSH (100nmol/site, i.c.v.), while the pretreatment (25-100mg/kg, i.p.) with the reducing agent DTT (dl-dithiothreitol) prevented the antidepressant-like effect of GSH (300nmol/site, i.c.v.). DTNB (0.1nmol/site, i.c.v.), produced an antidepressant-like effect, per se, which was abolished by DTT (25mg/kg, i.p.). The results show, for the first time, that centrally administered GSH produces an antidepressant-like effect in mice, which can be modulated by the GSH metabolism and the thiol/disulfide reagents. The redox environment may constitute a new venue for future antidepressant-drug development.
Assuntos
Antidepressivos , Depressão/psicologia , Glutationa/farmacologia , Natação/psicologia , Animais , Antimetabólitos/farmacologia , Antioxidantes/metabolismo , Butionina Sulfoximina/farmacologia , Ácido Ditionitrobenzoico/farmacologia , Ditiotreitol/farmacologia , Feminino , Glutationa/administração & dosagem , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Glutationa Transferase/metabolismo , Elevação dos Membros Posteriores/psicologia , Injeções Intraventriculares , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Oxirredução , gama-Glutamiltransferase/metabolismoRESUMO
Iron accumulation and oxidative stress are hallmarks of retinas from patients with age-related macular degeneration (AMD). We have previously demonstrated that iron-overloaded retinas are a good in vitro model for the study of retinal degeneration during iron-induced oxidative stress. In this model we have previously characterized the role of cytosolic phospholipase A2 (cPLA2) and calcium-independent isoform (iPLA2). The aim of the present study was to analyze the implications of Group V secretory PLA2 (sPLA2), another member of PLA2 family, in cyclooxygenase (COX)-2 and nuclear factor kappa B (NF-κB) regulation. We found that sPLA2 is localized in cytosolic fraction in an iron concentration-dependent manner. By immunoprecipitation (IP) assays we also demonstrated an increased association between Group V sPLA2 and COX-2 in retinas exposed to iron overload. However, COX-2 activity in IP assays was observed to decrease in spite of the increased protein levels observed. p65 (RelA) NF-κB levels were increased in nuclear fractions from retinas exposed to iron. In the presence of ATK (cPLA2 inhibitor) and YM 26734 (sPLA2 inhibitor), the nuclear localization of both p65 and p50 NF-κB subunits was restored to control levels in retinas exposed to iron-induced oxidative stress. Membrane repair mechanisms were also analyzed by studying the participation of acyltransferases in phospholipid remodeling during retinal oxidation stress. Acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylserine (PS), were observed to show an inhibited acylation profile in retinas exposed to iron while phosphatidylethanolamine (PE) showed the opposite. The use of PLA2 inhibitors demonstrated that PS is actively deacylated during iron-induced oxidative stress. Results from the present study suggest that Group V sPLA2 has multiple intracellular targets during iron-induced retinal degeneration and that the specific role of sPLA2 could be related to inflammatory responses by its participation in NF-κB and COX-2 regulation.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Fosfolipases A2 do Grupo V/fisiologia , Degeneração Macular/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Retina/efeitos dos fármacos , Acetilação , Acetiltransferases/metabolismo , Animais , Western Blotting , Bovinos , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Compostos Ferrosos/toxicidade , Fosfolipases A2 do Grupo V/antagonistas & inibidores , Sobrecarga de Ferro/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipases A/metabolismo , Fosfolipases A/fisiologia , Retina/metabolismoRESUMO
The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control.
Assuntos
Fabaceae/química , Insetos/efeitos dos fármacos , Inseticidas/farmacologia , Proteínas de Plantas/farmacologia , Sementes/química , Inibidores da Tripsina/farmacologia , Tripsina/metabolismo , Sequência de Aminoácidos , Animais , Quimotripsina/metabolismo , Insetos/metabolismo , Inseticidas/química , Inseticidas/isolamento & purificação , Larva/efeitos dos fármacos , Dose Letal Mediana , Peso Molecular , Papaína/antagonistas & inibidores , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificaçãoRESUMO
Tryparedoxins (TXNs) are multipurpose oxidoreductases from trypanosomatids that transfer reducing equivalents from trypanothione to various thiol proteins. In Trypanosoma cruzi, two genes coding for TXN-like proteins have been identified: TXNI, previously characterized as a cytoplasmic protein, and TXNII, a putative tail-anchored membrane protein. In this work, we performed a comparative functional characterization of T. cruzi TXNs. Particularly, we cloned the gene region coding for the soluble version of TXNII for its heterologous expression. The truncated recombinant protein (without its 22 C-terminal transmembrane amino acids) showed TXN activity. It was also able to transfer reducing equivalents from trypanothione, glutathione, or dihydrolipoamide to various acceptors, including methionine sulfoxide reductases and peroxiredoxins. The results support the occurrence and functionality of a second tryparedoxin, which appears as a new component in the redox scenario for T. cruzi.
Assuntos
Glutationa/metabolismo , Tiorredoxinas/genética , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Expressão Gênica , Glutationa/análogos & derivados , Oxirredução , Proteína Dissulfeto Redutase (Glutationa) , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Espermidina/análogos & derivados , Espermidina/metabolismo , Tiorredoxinas/metabolismoRESUMO
Maspin is a tumor suppressor with many biological activities, multiple ligands and different subcellular localizations. Its underlying molecular mechanism remains elusive. We hypothesized that phosphorylation might regulate maspin localization and function. Using two-dimensional gel electrophoresis with different focusing power followed by Western blot we identified four different maspin forms with the same molecular weight (42 kDa), but different isoelectric points. Three of these forms were sensitive to acidic phosphatase treatment, suggesting that they are phosphorylated. Sodium peroxidovanadate treatment, a protein-tyrosine phosphatase inhibitor, resulted in a rapid increase in maspin protein levels and cytoplasmic accumulation. These data show that there are three different maspin tyrosine phosphoforms. Inhibition of tyrosine phosphatases increased maspin protein levels and leads to its cytoplasmic accumulation.
RESUMO
It has recently been proposed that hypothalamic glial cells sense glucose levels and release lactate as a signal to activate adjacent neurons. GK (glucokinase), the hexokinase involved in glucose sensing in pancreatic beta-cells, is also expressed in the hypothalamus. However, it has not been clearly determined if glial and/or neuronal cells express this protein. Interestingly, tanycytes, the glia that cover the ventricular walls of the hypothalamus, are in contact with CSF (cerebrospinal fluid), the capillaries of the arcuate nucleus and adjacent neurons; this would be expected for a system that can detect and communicate changes in glucose concentration. Here, we demonstrated by Western-blot analysis, QRT-PCR [quantitative RT-PCR (reverse transcription-PCR)] and in situ hybridization that GK is expressed in tanycytes. Confocal microscopy and immuno-ultrastructural analysis revealed that GK is localized in the nucleus and cytoplasm of beta1-tanycytes. Furthermore, GK expression increased in these cells during the second week of post-natal development. Based on this evidence, we propose that tanycytes mediate, at least in part, the mechanism by which the hypothalamus detects changes in glucose concentrations.