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
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
Seizures increase prostaglandin and cytokine levels in the brain. However, it remains to be determined whether cyclooxygenase-2 (COX-2) derived metabolites play a role in seizure-induced cytokine increase in the brain and whether anticonvulsant activity is shared by all COX-2 inhibitors. In this study we investigated whether three different COX-2 inhibitors alter pentylenetetrazol (PTZ)-induced seizures and increase of interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) levels in the hippocampus and cerebral cortex of mice. Adult male albino Swiss mice received nimesulide, celecoxib or etoricoxib (0.2, 2 or 20mg/kg in 0.1% carboxymethylcellulose (CMC) in 5% Tween 80, p.o.). Sixty minutes thereafter the animals were injected with PTZ (50mg/kg, i.p.) and the latency to myoclonic jerks and to generalized tonic-clonic seizures were recorded. Twenty minutes after PTZ injection animals were killed and cytokine levels were measured. PTZ increased cytokine levels in the cerebral cortex and hippocampus. While celecoxib and nimesulide attenuated PTZ -induced increase of proinflammatory cytokines in the cerebral cortex, etoricoxib did not. Nimesulide was the only COX-2 inhibitors that attenuated PTZ-induced seizures. This effect coincided with an increase of IL-10 levels in the cerebral cortex and hippocampus, constituting circumstantial evidence that IL-10 increase may be involved in the anticonvulsant effect of nimesulide.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase 2/farmacologia , Citocinas/metabolismo , Hipocampo/efeitos dos fármacos , Pentilenotetrazol/efeitos adversos , Convulsões/tratamento farmacológico , Convulsões/metabolismo , Animais , Córtex Cerebral/metabolismo , Inibidores de Ciclo-Oxigenase 2/uso terapêutico , Hipocampo/metabolismo , Inflamação/metabolismo , Masculino , Camundongos , Convulsões/induzido quimicamenteRESUMO
Persistent activation of GABAA receptors triggers compensatory changes in receptor function that are relevant to physiological, pathological and pharmacological conditions. Chronic treatment of cultured neurons with GABA for 48h has been shown to produce a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine site interactions with a half-time of 24-25h. Down-regulation is the result of a transcriptional repression of GABAA receptor subunit genes and depends on activation of L-type voltage-gated calcium channels. The mechanism of this uncoupling is currently unknown. We have previously demonstrated that a single brief exposure of rat primary neocortical cultures to GABA for 5-10min (t½=3min) initiates a process that results in uncoupling hours later (t½=12h) without a change in receptor number. Uncoupling is contingent upon GABAA receptor activation and independent of voltage-gated calcium influx. This process is accompanied by a selective decrease in subunit mRNA levels. Here, we report that the brief GABA exposure induces a decrease in the percentage of α3-containing receptors, a receptor subtype that exhibits a high degree of coupling between GABA and benzodiazepine binding sites. Initiation of GABA-induced uncoupling is prevented by co-incubation of GABA with high concentrations of sucrose suggesting that it is dependent on a receptor internalization step. Moreover, results from immunocytochemical and biochemical experiments indicate that GABA exposure causes an increase in GABAA receptor endocytosis. Together, these data suggest that the uncoupling mechanism involves an initial increase in receptor internalization followed by activation of a signaling cascade that leads to selective changes in receptor subunit levels. These changes might result in the assembly of receptors with altered subunit compositions that display a lower degree of coupling between GABA and benzodiazepine sites. Uncoupling might represent a homeostatic mechanism that negatively regulates GABAergic transmission under physiological conditions in which synaptic GABAA receptors are transiently activated for several minutes.
Assuntos
Benzodiazepinas/farmacologia , Neurônios/efeitos dos fármacos , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/farmacologia , Animais , Benzodiazepinas/metabolismo , Sítios de Ligação/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Flunitrazepam/farmacocinética , Antagonistas GABAérgicos/farmacologia , Moduladores GABAérgicos/farmacocinética , Regulação da Expressão Gênica/efeitos dos fármacos , Picrotoxina/farmacologia , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genética , Fatores de Tempo , Trítio/farmacocinéticaRESUMO
Phosphatidylinositol-4 kinase (PI-4K) is responsible for the generation of phosphatidylinositol-4 phosphate (PtdIns(4)P), a bioactive signaling molecule involved in several biological functions. In this study, we show that sphingosine modulates the activity of the PI-4K isoform associated with the basolateral membranes (BLM) from kidney proximal tubules. Immunoblotting with an anti-α subunit PI-4K polyclonal antibody revealed the presence of two bands of 57 and 62kDa in the BLM. BLM-PI-4K activity retains noteworthy biochemical properties; it is adenosine-sensitive, not altered by wortmanin, and significantly inhibited by Ca(2+) at the µM range. Together, these observations indicate the presence of a type II PI-4K. Endogenous phosphatidylinositol (PI) alone reaches PI-4K half-maximal activity, revealing that even slight modifications in PI levels at the membrane environment promote significant variations in BLM-associated-PI-4K activity. ATP-dependence assays suggested that the Mg.ATP(2-) complex is the true substrate of the enzyme and that free Mg(2+) is an essential cofactor. Another observation indicated that higher concentrations of free ATP are inhibitory. BLM-associated-PI-4K activity was ~3-fold stimulated in the presence of increasing concentration of sphingosine, while in concentrations higher than 0.4mM, in which S1P is pronouncedly formed, there was an inhibitory effect on PtdIns(4)P formation. We propose that a tightly coupled regulatory network involving phosphoinositides and sphingolipids participate in the regulation of key physiological processes in renal BLM carried out by PI-4K.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Membrana Celular/metabolismo , Glicerofosfolipídeos/metabolismo , Túbulos Renais Proximais/enzimologia , Esfingolipídeos/metabolismo , Esfingosina/farmacologia , Animais , Immunoblotting , Túbulos Renais Proximais/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , SuínosRESUMO
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.