RESUMO
BACKGROUND: Type 2 diabetes mellitus (T2DM) is frequently associated with dyslipidemia, which corresponds to the increase in the triglycerides and fatty acid concentrations in tissues, such as the skeletal muscle. Also, T2DM molecular mechanism involves increasing in reactive oxygen species (ROS) production and oxidative stress. The use of herbal medicines such as Uncaria tomentosa (Ut) has been proposed as an auxiliary treatment for patients with T2DM. In this study, it was evaluated the effect of Ut aqueous extract on cell viability and ROS production, in skeletal myoblasts from C2C12 lineage exposed to the free fatty acid palmitate (PA). METHODS: Cells were incubated with PA in different concentrations ranging from 10 to 1000 µM, for 24 or 48 h, for cytotoxicity assay. Cell death, DNA fragmentation and ROS production assays were performed in cell cultures incubated with PA for 24 h, in the pre (preventive condition) or post treatment (therapeutic condition) with 250 µg/ml Ut aqueous extract, for 2 or 6 h. Cell death was evaluated by MTT method or flow cytometry. ROS generation was measured by fluorescence spectroscopy using the DCFDA probe. RESULTS: Cell viability was reduced to approximately 44% after the incubation with PA for 24 h from the concentration of 500 µM. In the incubation of cells with 500 µM PA and Ut extract for 6 h, in both conditions (preventive or therapeutic), it was observed an increase of 27 and 70% in cell viability respectively, in comparison to the cultures incubated with only PA. Also, the incubation of cultures with 500 µM PA, for 24 h, increased 20-fold the ROS formation, while the treatment with Ut extract, for 6 h, both in the preventive or therapeutic conditions, promoted decrease of 21 and 55%, respectively. CONCLUSION: The Ut extract was efficient in promoting cell protection against PA lipotoxicity and ROS generation, potentially preventing oxidative stress in C2C12 skeletal muscle cells. Since T2DM molecular mechanism involves oxidative stress condition and it is often associated with dyslipidemia and fatty acid accumulation in muscle tissue, these results open perspectives for the use of Ut as an auxiliary strategy for T2DM management.
Assuntos
Unha-de-Gato , Diabetes Mellitus Tipo 2 , Dislipidemias , Humanos , Espécies Reativas de Oxigênio/metabolismo , Palmitatos/toxicidade , Palmitatos/metabolismo , Unha-de-Gato/química , Unha-de-Gato/metabolismo , Músculo Esquelético , Água/químicaRESUMO
Medicines for chronic inflammation can cause gastric ulcers and hepatic and renal issues. An alternative treatment for chronic inflammation is that of natural bioactive compounds, which present low side effects. Extracts of Jatropha cordata (Ortega) Müll. Arg. have been evaluated for their cytotoxicity and anti-inflammatory activity; however, testing pure compounds would be of greater interest. Campesteryl palmitate, n-heptyl ferulate, palmitic acid, and a mixture of sterols, i.e., brassicasterol, campesterol, ß-sitosterol, and stigmasterol, were obtained from an ethyl acetate extract from J. cordata (Ortega) Müll. Arg. bark using column chromatography. The toxicity and in vitro anti-inflammatory activities were evaluated using RAW 264.7 murine macrophage cells. None of the products assessed exhibited toxicity. The sterol mixture exhibited greater anti-inflammatory activity than the positive control, and nitric oxide (NO) inhibition percentages were 37.97% and 41.68% at 22.5 µg/mL and 30 µg/mL, respectively. In addition, n-heptyl ferulate decreased NO by 30.61% at 30 µg/mL, while campesteryl palmitate did not show anti-inflammatory activity greater than the positive control. The mixture and n-heptyl ferulate showed NO inhibition; hence, we may conclude that these compounds have anti-inflammatory potential. Additionally, further research and clinical trials are needed to fully explore the therapeutic potential of these bioactive compounds and their efficacy in treating chronic inflammation.
RESUMO
The cryopreservation reduces ram sperm quality, decreasing the pregnancy rate of ewes inseminated with thawed sperm. Hence, we aimed to improve the post-thaw quality of ram sperm replacing egg yolk on Tris-Glucose extender with different concentrations of LDL (2 or 8%), associated with the addition of 10 mM non-enzymatic antioxidants (ascorbic acid, hydroxytoluene butylate, ascorbyl palmitate, and trehalose). Semen samples were collected from six rams, split into different treatments, and frozen. After thawing, kinematic (CASA), structural (propidium iodide and carboxyfluorescein diacetate) and functional (hypoosmotic test) sperm membrane integrity was assessed. Total motility, VCL, and LIN were also assessed in thawed samples during 3 h of incubation (38 °C). The results showed that hydroxytoluene butylate at 10 mM in Tris-Glucose extender with 8% LDL improved velocity parameters immediately post-thaw compared with Tris-Glucose egg yolk extender, as well as prevented the reduction of total motility and VCL after incubation. There was no benefit of adding ascorbic acid and trehalose. Moreover, for the first time, it was shown the motility impairment promoted by ascorbyl palmitate to ram sperm.
RESUMO
Ascorbyl palmitate, an ascorbic acid ester, is an important amphipathic antioxidant that has several applications in foods, pharmaceuticals, and cosmetics. The enzymatic synthesis of ascorbyl palmitate is very attractive, but few efforts have been made to address its process scale-up and implementation. This study aimed at evaluating the enzymatic synthesis of ascorbyl palmitate in a rotating basket reactor operated in sequential batches. Different commercial immobilized lipases were tested, and the most suitable reaction conditions were established. Among those lipases studied were Amano Lipase PS, Lipozyme® TL IM, Lipozyme® Novo 40086, Lipozyme® RM IM and Lipozyme® 435. Initially, the enzymes were screened based on previously defined synthesis conditions, showing clear differences in behavior. Lipozyme® 435 proved to be the best catalyst, reaching the highest values of initial reaction rate and yield. Therefore, it was selected for the following studies. Among the solvents assayed, 2-methyl-2-butanol and acetone showed the highest yields, but the operational stability of the catalyst was better in 2-methyl-2-butanol. The tests in a basket reactor showed great potential for large-scale application. Yields remained over 80% after four sequential batches, and the basket allowed for easy catalyst recycling. The results obtained in basket reactor are certainly a contribution to the enzymatic synthesis of ascorbyl palmitate as a competitive alternative to chemical synthesis. This may inspire future cost-effectiveness studies of the process to assess its potential as a viable alternative to be implemented.
Assuntos
Ácido Ascórbico , Pentanóis , Solventes , Enzimas ImobilizadasRESUMO
The cryopreservation reduces ram sperm quality, decreasing the pregnancy rate of ewes inseminated with thawed sperm. Hence, we aimed to improve the post-thaw quality of ram sperm replacing egg yolk on Tris-Glucose extender with different concentrations of LDL (2 or 8%), associated with the addition of 10 mM non-enzymatic antioxidants (ascorbic acid, hydroxytoluene butylate, ascorbyl palmitate, and trehalose). Semen samples were collected from six rams, split into different treatments, and frozen. After thawing, kinematic (CASA), structural (propidium iodide and carboxyfluorescein diacetate) and functional (hypoosmotic test) sperm membrane integrity was assessed. Total motility, VCL, and LIN were also assessed in thawed samples during 3 h of incubation (38 °C). The results showed that hydroxytoluene butylate at 10 mM in Tris-Glucose extender with 8% LDL improved velocity parameters immediately post-thaw compared with Tris-Glucose egg yolk extender, as well as prevented the reduction of total motility and VCL after incubation. There was no benefit of adding ascorbic acid and trehalose. Moreover, for the first time, it was shown the motility impairment promoted by ascorbyl palmitate to ram sperm.(AU)
Assuntos
Animais , Masculino , Preservação do Sêmen/veterinária , Ovinos/fisiologia , Análise do Sêmen/veterinária , Hidroxitolueno Butilado/administração & dosagem , Lipoproteínas/análise , AntioxidantesRESUMO
Modern lifestyles, including lack of physical activity and poor nutritional habits, are driving the rapidly increasing prevalence of obesity and type 2 diabetes. Increased levels of free fatty acids (FFAs), particularly saturated FFAs, in obese individuals have been linked to pancreatic ß-cell failure. This process, termed lipotoxicity, involves activation of several stress responses, including ER stress and oxidative stress. However, the molecular underpinnings and causal relationships between the disparate stress responses remain unclear. Here we employed transgenic mice, expressing a genetically-encoded cytosolic H2O2 sensor, roGFP2-Orp1, to monitor dynamic changes in H2O2 levels in pancreatic islets in response to chronic palmitate exposure. We identified a transient increase in H2O2 levels from 4 to 8 h after palmitate addition, which was mirrored by a concomitant decrease in cellular NAD(P)H levels. Intriguingly, islets isolated from NOX2 knock-out mice displayed no H2O2 transient upon chronic palmitate treatment. Furthermore, NOX2 knockout rescued palmitate-dependent impairment of insulin secretion, calcium homeostasis and viability. Chemical inhibition of NOX activity protected islets from palmitate-induced impairment in insulin secretion, however had no detectable impact upon the induction of ER stress. In summary, our results reveal that transient NOX2-dependent H2O2 production is a likely cause of early palmitate-dependent lipotoxic effects.
Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ilhotas Pancreáticas , Animais , Peróxido de Hidrogênio , Insulina , Camundongos , NADPH Oxidase 2/genética , Palmitatos/toxicidadeRESUMO
Fasciola hepatica is helminth parasite found around the world that causes fasciolosis, a chronic disease affecting mainly cattle, sheep, and occasionally humans. Triclabendazole is the drug of choice to treat this parasite. However, the continuous use of this drug has led to the development of parasite resistance and, consequently, the limitation of its effectiveness. Hence, vaccination appears as an attractive option to develop. In this work, we evaluated the potential of F. hepatica Kunitz-type molecule (FhKTM) as an antigen formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate ester (Coa-ASC16) and the synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) during an experimental model of fasciolosis in mice, and we further dissected the immune response associated with host protection. Our results showed that immunization of mice with FhKTM/CpG-ODN/Coa-ASC16 induces protection against F. hepatica challenge by preventing liver damage and improving survival after F. hepatica infection. FhKTM/CpG-ODN/Coa-ASC16-immunized mice elicited potent IFN-γ and IL-17A with high levels of antigen-specific IgG1, IgG2a, and IgA serum antibodies. Strikingly, IL-17A blockade during infection decreased IgG2a and IgA antibody levels as well as IFN-γ production, leading to an increase in mortality of vaccinated mice. The present study highlights the potential of a new vaccine formulation to improve control and help the eradication of F. hepatica infection, with potential applications for natural hosts such as cattle and sheep.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Fasciola hepatica/imunologia , Fasciolíase/prevenção & controle , Proteínas de Helminto/farmacologia , Interferon gama/imunologia , Interleucina-17/imunologia , Vacinas/farmacologia , Animais , Fasciolíase/imunologia , Feminino , Proteínas de Helminto/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Vacinas/imunologiaRESUMO
High-fat diet (HFD) feeding is deleterious to hypothalamic tissue, leading to inflammation and lipotoxicity, as well as contributing to central insulin resistance. Autophagy is a process that restores cellular homeostasis by degrading malfunctioning organelles and proteins. Chronic HFD-feeding down-regulates hypothalamic autophagy. However, the effects of short-term HFD-feeding and the saturated fatty acid palmitate (PA) on hypothalamic autophagy and in neurones that express neuropeptide Y (NPY) and agouti-related peptide remains unknown. Therefore, we assessed hypothalamic autophagy after 1 and 3 days of HFD-feeding. We also injected PA i.c.v and analysed the modulation of autophagy in hypothalamic tissue. Both interventions resulted in changes in autophagy-related gene profiles without significant differences in protein content of p62 and LC3B-II, markers of the autophagy pathway. When we assessed native NPY neurones in brain slices from PA-treated animals, we observed increased levels of Atg7 and LC3B protein in response to PA treatment, indicating the induction of autophagy. We then tested the direct effects of fatty acids using the immortalised hypothalamic NPY-expressing neuronal cell model mHypoE-46. We found that PA, but not palmitoleate (PO) (a monounsaturated fatty acid), was able to induce autophagy. Co-treatment with PA and PO was able to block the PA-mediated induction of autophagy, as assessed by flow cytometry. When the de novo ceramide synthesis pathway was blocked with myriocin pre-treatment, we observed a decrease in PA-mediated induction of autophagy, although there was no change with the toll-like receptor 4 inhibitor, TAK-242. Taken together, these findings provide evidence that saturated and unsaturated fatty acids can differentially regulate hypothalamic autophagy and that ceramide synthesis may be an important mediator of those effects. Understanding the mechanisms by which dietary fats affect autophagy in neurones involved in the control of energy homeostasis will provide potential new pathways for targeting and containing the obesity epidemic.
Assuntos
Autofagia/efeitos dos fármacos , Ácidos Graxos/farmacologia , Neurônios/efeitos dos fármacos , Animais , Autofagia/genética , Células Cultivadas , Dieta Hiperlipídica , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Camundongos , Neurônios/metabolismo , Neuropeptídeo Y/metabolismo , Ácido Palmítico/farmacologia , Fatores de TempoRESUMO
BACKGROUND: Paliperidone palmitate is a long-acting, second-generation antipsychotic (SGA) indicated for the treatment of acute exacerbations and maintenance treatment of adults with schizophrenia. This study addressed the response to paliperidone palmitate in Latin American patients with acute symptoms and recently diagnosed schizophrenia. OBJECTIVE: Explore the efficacy and tolerability of paliperidone palmitate administered once a month for 4 months in patients with acute phase and recent diagnosis (within 1-6 years) of schizophrenia in 3 Latin American countries. METHODS: This was a non-randomized, open-label, multicenter study with paliperidone palmitate injected intramuscularly in the deltoid muscle at an initial loading dose of 150 mg eq. (234 mg) on day 1 and 100 mg eq. (156 mg) on day 8 (± 4 days). The recommended maintenance dose was 75 mg eq. (117 mg) from day 36 to day 92. Efficacy was evaluated with PANSS and CGI-S. The last observation carried forward (LOCF) was used for efficacy analysis for imputation of missing data; no adjustments were made for multiplicity. Adverse events were evaluated during treatment. RESULTS: The patient retention rate was 84.0% (144 patients received study drug; 121 finished the study). The percentage of patients with a reduction of at least 30% in PANSS total score compared to baseline gradually increased during the study, and at the end, 78.4% of patients showed response. The PANSS total score and CGI-S scores decreased significantly from baseline to LOCF endpoint (P <0.0001 for both); significant reduction in PANSS total score was observed at day 8 and persisted to the end of the study. Most common adverse events were muscle rigidity (11.8%), akathisia (11.1%), injection-site pain (7.6%), weight gain (7.6%), and insomnia (7.6%). CONCLUSION: Paliperidone palmitate was efficacious in Latin American patients studied with an acute exacerbation and recent diagnosis of schizophrenia, and no new safety signals were identified.
RESUMO
The lack of tissue selectivity of anticancer drugs generates intense collateral and adverse effects of cancer patients, making the incorporation of vitamins or micronutrients into the diet of individuals to reduce side or adverse effects of antineoplastics. The study aimed to evaluate the effects of retinol palmitate (RP) on the toxicogenic damages induced by cyclophosphamide (CPA), doxorubicin (DOX) and its association with the AC protocol (CPA + DOX), in Sarcoma 180 (S-180) tumor cell line, using the micronuclei test with a block of cytokinesis (CBMN); and in non-tumor cells derived from Mus musculus using the comet assay. The results suggest that CPA, DOX and AC protocol induced significant toxicogenic damages (P < 0.05) on the S-180 cells by induction of micronuclei, cytoplasmic bridges, nuclear buds, apoptosis, and cell necrosis, proving their antitumor effects, and significant damage (P < 0.001) to the genetic material of peripheral blood cells of healthy mice, proving the genotoxic potential of these drugs. However, RP modulated the toxicogenic effects of antineoplastic tested both in the CBMN test (P < 0.05), at the concentrations of 1, 10 and 100 IU/mL; as in the comet assay (P < 0.001) at the concentration of 100 IU/kg for the index and frequency of genotoxic damage. The accumulated results suggest that RP reduced the action of antineoplastics in non-tumor cells as well as the cytotoxic, mutagenic, and cell death in neoplastic cells.
Assuntos
Antineoplásicos/toxicidade , Diterpenos/farmacologia , Vitamina A/análogos & derivados , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Ensaio Cometa , Ciclofosfamida/efeitos adversos , Ciclofosfamida/toxicidade , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/efeitos adversos , Doxorrubicina/toxicidade , Interações Medicamentosas , Humanos , Camundongos , Testes para Micronúcleos , Mutagênese/efeitos dos fármacos , Ésteres de Retinil , Vitamina A/farmacologiaRESUMO
BACKGROUND: Free fatty acids (FFAs) are known for their dual effects on insulin secretion and pancreatic ß-cell survival. Short-term exposure to FFAs, such as palmitate, increases insulin secretion. On the contrary, long-term exposure to saturated FFAs results in decreased insulin secretion, as well as triggering oxidative stress and endoplasmic reticulum (ER) stress, culminating in cell death. The effects of FFAs can be mediated either via their intracellular oxidation and consequent effects on cellular metabolism or via activation of the membrane receptor GPR40. Both pathways are likely to be activated upon both short- and long-term exposure to FFAs. However, the precise role of GPR40 in ß-cell physiology, especially upon chronic exposure to FFAs, remains unclear. METHODS: We used the GPR40 agonist (GW9508) and antagonist (GW1100) to investigate the impact of chronically modulating GPR40 activity on BRIN-BD11 pancreatic ß-cells physiology and function. RESULTS: We observed that chronic activation of GPR40 did not lead to increased apoptosis, and both proliferation and glucose-induced calcium entry were unchanged compared to control conditions. We also observed no increase in H2O2 or superoxide levels and no increase in the ER stress markers p-eIF2α, CHOP and BIP. As expected, palmitate led to increased H2O2 levels, decreased cell viability and proliferation, as well as decreased metabolism and calcium entry. These changes were not counteracted by the co-treatment of palmitate-exposed cells with the GPR40 antagonist GW1100. CONCLUSIONS: Chronic activation of GPR40 using GW9508 does not negatively impact upon BRIN-BD11 pancreatic ß-cells physiology and function. The GPR40 antagonist GW1100 does not protect against the deleterious effects of chronic palmitate exposure. We conclude that GPR40 is probably not involved in mediating the toxicity associated with chronic palmitate exposure.
Assuntos
Benzoatos/farmacologia , Células Secretoras de Insulina/metabolismo , Metilaminas/farmacologia , Propionatos/farmacologia , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzoatos/administração & dosagem , Cálcio/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Metilaminas/administração & dosagem , Palmitatos/toxicidade , Propionatos/administração & dosagem , Pirimidinas/administração & dosagem , Ratos , Receptores Acoplados a Proteínas G/efeitos dos fármacosRESUMO
Fluorescence quenching is widely used to obtain association constants between proteins and ligands. This methodology is based on assumption that ground-state complex between protein and ligand is responsible for quenching. Here, we call the attention about the risk of using the temperature criterion for decision of applying or not fluorescence quenching data to measure association constants. We demonstrated that hydrophobic effect can be the major force involved in the interaction and, as such, superposes the well-established rationalization that host/guest complexation is weakened at higher temperatures due to loss of translational and rotational degrees of freedom. To do so, the complexation of bovine serum albumin with octyl gallate was studied by steady-state, time-resolved fluorescence spectroscopy and isothermal titration calorimetry. The results clearly demonstrated the complexation, even though the Stern-Volmer constant increased at higher temperatures (1.6 × 104 and 4.1 × 105 mol-1 L at 20°C and 40°C), which could suggest a simple dynamic process and not complexation. The entropy-driven feature of the interaction was demonstrated by the unfavorable enthalpy (∆H° = 104.4 kJmol-1 ) but favorable entropy (∆S° = 447.5 Jmol-1 K-1 ). The relevance of the ligand hydrophobicity was also evaluated by comparing ascorbic acid and its ester ascorbyl palmitate. Docking simulations showed a higher number of hydrophobic contacts and lower energy poses for the esters, confirming the experimental results. In conclusion, the well-established rationalization that host/guest complexation is weakened at higher temperatures is not straightforward for protein-ligand interactions. Hence, the temperature effect for a decision between static and dynamic quenching and its use to decide if a complexation at ground state is taking place between ligand and protein should not be used.
Assuntos
Albuminas/química , Ácido Gálico/análogos & derivados , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/química , Entropia , Ácido Gálico/química , Interações Hidrofóbicas e Hidrofílicas , Temperatura , TermodinâmicaRESUMO
Obesity has been associated with cognitive decline, atrophy of brain regions related to learning and memory, and higher risk of developing dementia. However, the molecular mechanisms underlying these neurological alterations are still largely unknown. Here, we investigate the effects of palmitate, a saturated fatty acid present at high amounts in fat-rich diets, in the brain. Palmitate is increased in the cerebrospinal fluid (CSF) of overweight and obese patients with amnestic mild cognitive impairment. In mice, intracerebroventricular infusion of palmitate impairs synaptic plasticity and memory. Palmitate induces astroglial and microglial activation in the mouse hippocampus, and its deleterious impact is mediated by microglia-derived tumor necrosis factor alpha (TNF-α) signaling. Our results establish that obesity is associated with increases in CSF palmitate. By defining a pro-inflammatory mechanism by which abnormal levels of palmitate in the brain impair memory, the results further suggest that anti-inflammatory strategies may attenuate memory impairment in obesity.
Assuntos
Transtornos da Memória/etiologia , Obesidade/líquido cefalorraquidiano , Palmitatos/líquido cefalorraquidiano , Fator de Necrose Tumoral alfa/metabolismo , Animais , Humanos , Transtornos da Memória/patologia , Camundongos , Obesidade/patologiaRESUMO
Objective: To analyze the efficacy and safety of paliperidone palmitate 3-monthly (PP3M) in Latin American patients with schizophrenia vs. rest-of-world (ROW). Methods: We analyzed data from two multinational, double-blind (DB), randomized, controlled phase 3 studies including patients with schizophrenia (DSM-IV-TR) previously stabilized on PP1M/PP3M (open-label [OL] phase). Patients were randomized to PP3M or PP1M (noninferiority study A) and PP3M or placebo (study B) in DB phase. The subgroup analysis included Latin American (Argentina, Brazil, Colombia, Mexico) patients. Primary efficacy endpoints were relapse-free rates (study A) and time-to-relapse (study B). Results: In study A, 63/71 (88.7%) and in study B 38/43 (88.4%) Latin American patients completed the DB phase. In study A, relapse-free percentage was similar in Latin America (PP3M: 97%, PP1M: 100%) and ROW (PP3M: 91%, PP1M: 89%). In study B, median time-to-relapse was not estimable in the Latin American subgroup for either placebo or PP3M groups, nor for the ROW PP3M group; the median time-to-relapse in the ROW placebo group was 395 days. Caregiver burden improved in patients switching from oral antipsychotics (OL baseline) to PP3M/PP1M in DB phase (Involvement Evaluation Questionnaire score mean ± SD change, -9.4±15.16; p < 0.001). Treatment emergent adverse events with PP3M during DB phase were similar in Latin America (study A: 24/34 [70.6%]; study B: 15/21 [71.4%]) and ROW (study A: 318/470 [67.7%]; study B: 84/139 [60.4%]) subgroups. Conclusion: PP3M was efficacious and showed no new safety concerns in patients with schizophrenia from Latin America, corroborating ROW findings. Clinical trial registration: NCT01515423, NCT01529515
Assuntos
Humanos , Masculino , Feminino , Adolescente , Adulto , Idoso , Adulto Jovem , Esquizofrenia/tratamento farmacológico , Antipsicóticos/administração & dosagem , Palmitato de Paliperidona/administração & dosagem , Recidiva , Fatores de Tempo , Efeito Placebo , Método Duplo-Cego , Inquéritos e Questionários , Reprodutibilidade dos Testes , Resultado do Tratamento , Estimativa de Kaplan-Meier , Prevenção Secundária , América Latina , Pessoa de Meia-IdadeRESUMO
Ascorbyl palmitate is a fatty acid ester endowed with antioxidant properties, used as a food additive and cosmetic ingredient, which is presently produced by chemical synthesis. Ascorbyl palmitate was synthesized from ascorbic acid and palmitic acid with a Pseudomonas stutzeri lipase immobilized on octyl silica, and also with the commercial immobilized lipase Novozym 435. The latter was selected for optimizing the reaction conditions because of its high reactivity and stability in the solvent 2-methyl-2-butanol used as reaction medium. The reaction of the synthesis was studied considering temperature and molar ratio of substrates as variables and synthesis yield as response parameter. The highest yield in the synthesis of ascorbyl palmitate was 81%, obtained at 55 °C and an ascorbic acid to palmitic acid molar ratio of 1:8, both variables having a strong effect on yield. The synthesized ascorbyl palmitate was purified to 94.4%, with a purification yield of 84.2%. The use of generally recognized as safe (GRAS) certified solvents with a polarity suitable for the solubilization of the compounds made the process a viable alternative for the synthesis and downstream processing of ascorbyl palmitate.
Assuntos
Antineoplásicos/síntese química , Ácido Ascórbico/análogos & derivados , Enzimas Imobilizadas , Lipase/química , Antineoplásicos/química , Ácido Ascórbico/síntese química , Ácido Ascórbico/química , Técnicas de Química Sintética , Estabilidade de Medicamentos , Enzimas Imobilizadas/química , SolventesRESUMO
Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p-nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p<0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well.
Assuntos
Candida parapsilosis/citologia , Candida parapsilosis/fisiologia , Lipase , Nitrogênio , Palmitatos/análiseRESUMO
Recent studies have been trying to find out how diet and metabolic changes such as dyslipidaemia, hyperglycaemia, and hyperinsulinaemia can stimulate cancer progression. This investigation aimed to evaluate the effect of high concentrations of fatty acids and/or glucose in tumour prostate cells, focusing on the proliferation/migration profile and oxidative stress. PC3 cells were treated with high concentration of saturated fatty acid (palmitate, 100 µM), glucose (220 mg/dL), or both for 24 or 48 h. Results demonstrated that PC3 cells showed a significant increase in proliferation after 48 h of treatment with glucose and palmitate+glucose. Cell proliferation was associated with reduced levels of AMPK phosphorylation in glucose group at 24 and 48 h of treatment, while palmitate group presented this result only after 48 h of treatment. Also, there was a significant increase in cell migration between time 0 and 48 h after all treatments, except in the control. Catalase activity was increased by palmitate in the beginning of treatment, while glucose presented a later effect. Also, nitrite production was increased by glucose only after 48 h, and the total antioxidant activity was enhanced by palmitate in the initial hours. Thus, we conclude that the high concentration of the saturated fatty acid palmitate and glucose in vitro influences PC3 cells and stimulates cellular activities related to carcinogenesis such as cell proliferation, migration, and oxidative stress in different ways. Palmitate presents a rapid and initial effect, while a glucose environment stimulates cells later on, maintaining high levels of cell proliferation.
Assuntos
Glucose/metabolismo , Palmitatos/metabolismo , Neoplasias da Próstata/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácidos Graxos/metabolismo , Glucose/efeitos adversos , Glucose/fisiologia , Humanos , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Masculino , Células PC-3/efeitos dos fármacos , Palmitatos/farmacologia , Fosforilação , Próstata/metabolismoRESUMO
In this work, a stable nanocarrier for the anti-cancer drug docetaxel was rational designed. The nanocarrier was developed based on the solid lipid nanoparticle preparation process aiming to minimize the total amount of excipients used in the final formulations. A particular interest was put on the effects of the polymers in the final composition. In this direction, two poloxoamers -Pluronic F127 and F68- were selected. Some poloxamers are well known to be inhibitors of the P-glycoprotein efflux pump. Additionally, their poly-ethylene-oxide blocks can help them to escape the immune system, making the poloxamers appealing to be present in a nanoparticle designed for the treatment of cancer. Within this context, a factorial experiment design was used to achieve the most suitable formulations, and also to identify the effects of each component on the final (optimized) systems. Two final formulations were chosen with sizes < 250 nm and PDI < 0.2. Then, using dynamic light scattering and nanotracking techniques, the stability of the formulations was assessed during six months. Structural studies were carried on trough different techniques: DSC, x-ray diffraction, FTIR-AR and Molecular Dynamics. The encapsulation efficiency of the anticancer drug docetaxel (> 90%) and its release dynamics from formulations were measured, showing that the polymer-lipid nanoparticle is suitable as a drug delivery system for the treatment of cancer.
Assuntos
Docetaxel/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Lipídeos/química , Nanopartículas/química , Polímeros/química , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Varredura Diferencial de Calorimetria , Docetaxel/química , Docetaxel/farmacocinética , Portadores de Fármacos/química , Desenho de Fármacos , Liberação Controlada de Fármacos , Microscopia Eletrônica de Transmissão , Nanopartículas/ultraestrutura , Poloxâmero/química , Polietilenoglicóis/química , Difração de Raios XRESUMO
There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.
Assuntos
Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interferon Tipo I/metabolismo , Oligodesoxirribonucleotídeos/imunologia , Adjuvantes Imunológicos , Animais , Antígenos/química , Citocinas/metabolismo , Humanos , Imunidade Humoral , Camundongos , Camundongos Knockout , Nanoestruturas , Oligodesoxirribonucleotídeos/química , Ovalbumina/imunologia , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Excessive fat consumption increases the level of fatty acids (FAs) in the blood, which reach the hypothalamus and damage the circuit related to energy balance. In the present study, we used palmitate in a primary culture of purified astrocytes to mimic the fat-rich environment found in obesity. Our results showed increased glial fibrillary acidic protein (GFAP) reactivity in hypothalamic astrocytes compared to cortical astrocytes. In addition, palmitate-treated astrocytes showed no significant changes in cytokine expression and an upregulation of glutathione in the culture medium that may serve as an intrinsic neuroprotective property against excess FA. Additionally, purified hypothalamic neurons were incubated with palmitate-treated astrocyte-conditioned medium (MPAL). MPAL treated-neurons exhibited a reduction in excitatory synapses and enhanced neuritogenesis. Our results suggest that hypothalamic astrocytes react to palmitate differently than cortical astrocytes and influence the behavior of the neural network related to energy balance. Our work brings a better understanding of the interactions among hypothalamic neurons in a high FA environment, similarly to obesity induced by a high-fat diet.