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BACKGROUND: Hippeastrum species have a wide range of biological properties. In Argentina, this genus comprises ten widely distributed species. PURPOSE: To evaluate the antiparasitic and anticholinesterase activities and chemical profiles of seven Argentinean Hippeastrum species and determine the synergism between the major isolated alkaloid-montanine-and benznidazole in anti-Trypanosoma cruzi activity. METHODS: The antiparasitic activity was evaluated through antiproliferative and viability assays against T. cruzi epimastigotes. Synergism assays were performed using the Chou-Talalay method. AChE and BuChE inhibitory activities were also assessed. The alkaloid composition was obtained using GC-MS analysis. RESULTS: All extracts showed strong growth inhibition of T. cruzi epimastigote proliferation. The extracts from H. aglaiae, H. aulicum, and H. hybrid stand out for their potent and total growth inhibition, which was comparable to benznidazole. The H. reticulatum extract showed strong Acetylcholinesterase (AChE) inhibitory activities, while five species showed moderate Butyrylcholinesterase (BuChE) inhibition. Fifteen alkaloids were identified by means of GC-MS. Regarding the synergism assessment, the highest synergistic effect was obtained from the combination of montanine and benznidazole. CONCLUSION: Hippeastrum species bulb extracts from Argentina were shown to be a good source of antiparasitic alkaloids and cholinesterase inhibitors. The synergism between montanine and benznidazole emerges as a potential combination for future studies to treat Chagas disease.
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BACKGROUND: Cranioplasty is the used method in neurosurgery for repairing cranial bone defects. In our environment, the most widely used material is cryopreserved autologous bone (ABCp). OBJECTIVE: A retrospective observational study was proposed in order to analyze complications in patients who underwent decompressive craniectomy for brain trauma, with subsequent cranioplasty with ABCp. METHOD: Patients who underwent cranioplasties with consecutive ABCp performed at our institution over a four-year period (2016-2019) with subsequent follow-up were included, collecting multiple variables in relation to the appearance of complications. RESULTS: 113 cranioplasties were performed, of which 85.8% (n = 97) were performed with ABCp. Mainly with frontotemporoparietal bone defect (94.84%) performed late (> 3 months) in 91.76%. The complication rate was 16.49%, the most significant being the infection of the surgical site (n = 8, 8.24%), the presence of intracranial hematoma (n = 3, 3.09%) and the reabsorption of the autologous bone (n = 2, 2.06%), meriting surgical management in nine of them (9.27%). CONCLUSIONS: ABCp is a valid and safe option, which meets the basic characteristics to consider it the ideal material, with an acceptable rate of complications, biocompatible, with osteogenic potential, adequate protection of the brain and decrease in surgery costs.
ANTECEDENTES: La craneoplastia es el método utilizado en neurocirugía para reparar los defectos óseos craneanos. En nuestro medio, el material utilizado mayormente es el hueso autólogo criopreservado (HACp). OBJETIVO: Realizamos un estudio retrospectivo observacional para analizar las complicaciones en pacientes sometidos a craniectomía descompresiva por trauma craneoencefálico y realización de craneoplastia con HACp. MÉTODO: Se incluyeron pacientes que fueron sometidos a craneoplastias con HACp consecutivas realizadas en un periodo de 4 años (2016-2019) con seguimiento posterior, recabando múltiples variables en relación con la aparición de complicaciones. RESULTADOS: Se realizaron 113 craneoplastias, de las cuales el 85.8% (n = 97) fueron realizadas con HACp, principalmente con defecto óseo frontotemporoparietal (94.84%), realizadas de forma tardía (> 3 meses) en el 91.76%. El índice de complicaciones fue del 16.49%, siendo las más significativas la infección del sitio quirúrgico (n = 8, 8.24%), la presencia de hematoma endocraneano (n = 3, 3.09%) y la reabsorción del hueso autólogo (n = 2, 2.06%), ameritando manejo quirúrgico en nueve ocasiones (9.27%). CONCLUSIONES: El HACp es una opción válida y segura, la cual cumple con las características para considerarlo el material ideal, con un aceptable índice de complicaciones, biocompatible, con potencial osteogénico, adecuada protección encefálica y disminución de los costos de la cirugía.
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Lesões Encefálicas Traumáticas , Craniectomia Descompressiva , Procedimentos de Cirurgia Plástica , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/cirurgia , Craniectomia Descompressiva/efeitos adversos , Craniectomia Descompressiva/métodos , Humanos , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/cirurgia , Procedimentos de Cirurgia Plástica/métodos , Estudos Retrospectivos , Crânio/cirurgiaRESUMO
INTRODUCTION: Use of complementary and alternative medicine (CAM) therapies had been described in patients with disabling, chronic and painful conditions; these characteristics define the majority of vascular surgery (VS) entities. A lack of disclosure about CAM use from patients has been universally cited and may impact effective patient-doctor communication. Our primary objective was to describe CAM use, modalities, perceived benefits, safety, and associated factors among adult patients attending a VS outpatient clinic; we additionally explored patient's attitudes about CAM disclosure with their primary vascular surgeon. METHODS: This cross-sectional study invited 223 consecutive outpatients to an interview where the ICAM-Q (International Complementary and Alternative Medicine Questionnaire) and the PDRQ-9 (Patient-Doctor Relationship Questionnaire-9 items) were applied. In addition, sociodemographics, vascular disease and treatment-related information, comorbidity, and disease severity characteristics were obtained. Appropriated statistics was used; multiple logistic regression analysis identified factors associated to CAM use. All statistical tests were two-sided, and a p value ≤ 0.05 was considered significant. IRB approval was obtained. RESULTS: Patients recruited were primary females (69%) and had a median age of 65 years (54-75). Most frequent vascular diagnoses were chronic venous insufficiency (36.2%) and peripheral artery disease (26%). There were 104 (46.6%) patients who referred CAM use, primarily self-helped practices (96%), and use of herbal, vitamins, or homeopathic medicines (23.7%). Overall, the majority of the patients perceived CAM modalities helpful and 94.6% denied any adverse event. Female sex (OR: 1.768, 95% CI: 0.997-3.135, p = 0.051) and hospitalization during the previous year (OR: 3.173, 95% CI: 1.492-6.748, p = 0.003) were associated to CAM use. The majority of the patients (77%) agreed about CAM disclosure with their primary vascular surgeon; meanwhile, among CAM users, up to 54.9% did not disclose it, and their main reasons were "Doctor didn't ask" (32%) and "I consider it unnecessary" (16%). The patient-doctor relationship was rated by the patients with high scores. CONCLUSIONS: CAM use is frequent and perceived as safe and beneficial among VS outpatients; nonetheless, patients do not disclose CAM use with their primary vascular surgeons, and a wide range of reasons are given by the patients that prevent effective and open communication.
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Terapias Complementares , Adulto , Idoso , Terapias Complementares/efeitos adversos , Estudos Transversais , Feminino , Humanos , Pessoa de Meia-Idade , Pacientes Ambulatoriais , Encaminhamento e Consulta , Inquéritos e QuestionáriosRESUMO
ANTECEDENTES: Los abordajes quirúrgicos de la región selar han sido un reto para los neurocirujanos. Con la introducción de la técnica endoscópica se ha minimizado el trauma quirúrgico, pero esta técnica no se encuentra exenta de complicaciones. OBJETIVO: Describir nuestra experiencia institucional en pacientes sometidos a resección de macroadenomas hipofisarios por vía microquirúrgica endonasal transesfenoidal asistida por endoscopia con énfasis en las complicaciones resultantes. MÉTODO: Revisamos los expedientes electrónicos de 17 pacientes sometidos a dicho procedimiento de manera consecutiva entre 2017 y 2018, y recabamos variables como edad, sexo, clasificación imagenológica, tiempo de diagnóstico al momento de la cirugía, complicaciones posoperatorias y recurrencia. RESULTADOS: La edad promedio de los pacientes fue de 45.23 años y el 70.59% eran mujeres. Radiológicamente predominó el grado 2 Knosp (47.05%). La complicación principal fue diabetes insípida transitoria (23.52%), seguida por la fístula de líquido cefalorraquídeo (11.76%). CONCLUSIONES: El manejo microquirúrgico asistido por endoscopia ha demostrado ser un recurso valioso para tratar macroadenomas hipofisarios, pero presenta grados variables de complicaciones, por lo cual se requiere la adquisición de habilidades para el uso del endoscopio, la planeación preoperatoria adecuada y el manejo conjunto multidisciplinario con el fin de evitarlas. BACKGROUND: Surgical approaches to the sellar region have been a challenge for the neurosurgeon. With the introduction of the endoscopic technique, surgical trauma has been minimized, however it is not free of complications. OBJECTIVE: To describe our institutional experience in patients undergoing resection of pituitary macroadenomas by endoscopic-assisted microsurgical approach, with emphasis on the resulting complications. METHOD: We reviewed the electronic files of 17 patients who underwent this procedure consecutively between 2017 and 2018, collecting variables such as age, sex, imaging classification, time of diagnosis at the time of surgery, postoperative complications and recurrence. RESULTS: The average age of the patients was 45.23 years, with 70.59% women. Radiologically, grade 2 Knosp predominated (47.05%). The main complication was transient diabetes insipidus (23.52%) followed by cerebrospinal fluid fistula (11.76%). CONCLUSIONS: The endoscopic-assisted microsurgical approach has proven to be a valuable resource for treating pituitary macroadenomas, however it presents variable degrees of complications, which requires the acquisition of skills for the use of the endoscope, adequate preoperative planning, and multidisciplinary joint management in order to avoid them.
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Complicações Pós-Operatórias , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
In the epididymis, lysosomal proteins of the epithelial cells are normally targeted from the Golgi apparatus to lysosomes for degradation, although their secretion into the epididymal lumen has been documented and associated with sperm maturation. In this study, cathepsin D (CatD) and prosaposin (PSAP) were examined in adult epididymis of control, and 2-day castrated rats without (Ct) and with testosterone replacement (Ct+T) to evaluate their expression and regulation within epididymal epithelial cells. By light microscope-immunocytochemistry, a quantitative increase in size of lysosomes in principal cells of Ct animals was noted from the distal initial segment to the proximal cauda. Androgen replacement did not restore the size of lysosomes to control levels. Western blot analysis revealed a significant increase in CatD expression in the epididymis of Ct animals, which suggested an upregulation of its expression in principal cells; androgens restored levels of CatD to that of controls. In contrast, PSAP expression in Ct animals was not altered from controls. Additionally, an increase in procathepsin D levels was noted from samples of the epididymal fluid of Ct compared to control animals, accompanied by an increased complex formation with PSAP. Moreover, an increased oligomerization of prosaposin was observed in the epididymal lumen of Ct rats, with changes reverted to controls in Ct+T animals. Taken together these data suggest castration causes an increased uptake of substrates that are acted upon by CatD in lysosomes of principal cells and in the lumen by procathepsin D. These substrates may be derived from apoptotic cells noted in the lumen of proximal regions and possibly by degenerating sperm in distal regions of the epididymis of Ct animals. Exploring the mechanisms by which lysosomal enzymes are synthesized and secreted by the epididymis may help resolve some of the issues originating from epididymal dysfunctions with relevance to sperm maturation.
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Androgênios/genética , Catepsina D/genética , Precursores Enzimáticos/genética , Saposinas/genética , Androgênios/metabolismo , Animais , Castração/efeitos adversos , Epididimo/crescimento & desenvolvimento , Epididimo/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/genética , Lisossomos/genética , Lisossomos/fisiologia , Masculino , Ratos , Espermatozoides/metabolismo , Testosterona/genética , Testosterona/metabolismoRESUMO
OBJECTIVES: Arteriovenous fistulas primary patency at one-year occurs in 43-85% of the patients with end-stage renal disease. The diagnosis attributable to end-stage renal disease has been suggested to impact arteriovenous fistulas outcomes. The objective was to compare primary patency at one week, 1, 3, 6, and 12 months of follow-ups, among systemic lupus erythematosus patients and two control groups; additionally, we evaluated the impact of systemic lupus erythematosus to predict early patency loss. METHODS: A retrospective review of charts from arteriovenous fistulas created between 2008 and 2017 was performed. One-hundred thirty-four patients were identified and classified according to end-stage renal disease attributable diagnosis as: systemic lupus erythematosus cases (N = 14), control-group-1 (91 patients with primarily diabetes and hypertension), and control-group-2 (29 patients with idiopathic end-stage renal disease). A case-control matched design (1:2:1) was proposed. Logistic regression analysis and Kaplan-Meier curves were used. Institutional Review Board approval was obtained. RESULTS: More systemic lupus erythematosus patients lost primary patency at 3 (28.6%) and 12 months (71.4%) than patients from control-groups-1 (vs. 3.6% and 35.7%, respectively) and -2 (vs. 0% and 14.3%, respectively), (p ≤ 0.011 for both). Days of primary patency survival were shorter in systemic lupus erythematosus patients (p = 0.003). Systemic lupus erythematosus diagnosis was the only factor associated with early patency loss, HR: 3.141, 95%CI: 1.161-8.493 (systemic lupus erythematosus diagnosis vs. control-group-1) and HR: 12.582, 95%CI: 1.582-100.035 (systemic lupus erythematosus diagnosis vs. control-group-2). CONCLUSIONS: Diagnosis attributable to end-stage renal disease has a major impact on arteriovenous fistula outcomes in patients. Systemic lupus erythematosus patients have an increased risk of arteriovenous fistulas patency loss within the first six months of follow-up. Patients with idiopathic end-stage renal disease had an excellent one year arteriovenous fistula patency survival.
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Derivação Arteriovenosa Cirúrgica , Falência Renal Crônica/terapia , Lúpus Eritematoso Sistêmico/complicações , Nefrite Lúpica/terapia , Diálise Renal , Adolescente , Adulto , Derivação Arteriovenosa Cirúrgica/efeitos adversos , Feminino , Oclusão de Enxerto Vascular/etiologia , Oclusão de Enxerto Vascular/fisiopatologia , Humanos , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/etiologia , Lúpus Eritematoso Sistêmico/diagnóstico , Nefrite Lúpica/diagnóstico , Nefrite Lúpica/etiologia , Masculino , Pessoa de Meia-Idade , Diálise Renal/efeitos adversos , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Grau de Desobstrução Vascular , Adulto JovemRESUMO
BACKGROUND: Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model. PURPOSE: Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets. METHODS: The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine. RESULTS: Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy. CONCLUSION: Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.
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Lactonas/farmacologia , Sesquiterpenos de Germacrano/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Glutationa/metabolismo , Hemina/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mikania/química , NADH NADPH Oxirredutases/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Esteróis/biossíntese , Superóxido Dismutase/metabolismo , Trypanosoma cruzi/patogenicidade , Trypanosoma cruzi/ultraestruturaRESUMO
BACKGROUND: Leishmaniasis and Chagas disease are life-threatening illnesses caused by the protozoan parasites Leishmania spp. and Trypanosoma cruzi, respectively. They are known as "neglected diseases" due to the lack of effective drug treatments and the scarcity of research work devoted to them. Therefore, the development of novel and effective drugs is an important and urgent need. Natural products are an important source of bioactive molecules for the development of new drugs. In this study, we evaluated the activity of enhydrin, uvedalin and polymatin B, three sesquiterpene lactones (STLs) isolated from Smallanthus sonchifolius, on Leishmania mexicana (MNYC/BZ/62/M) and Trypanosoma cruzi (Dm28c). In addition, the in vivo trypanocidal activity of enhydrin and uvedalin and the effects of these STLs on parasites' ultrastructure were evaluated. METHODS: The inhibitory effect of the three STLs on the growth of L. mexicana amastigotes and promastigotes as well as T. cruzi epimastigotes was evaluated in vitro. The changes produced by the STLs on the ultrastructure of parasites were examined by transmission electron microscopy (TEM). Enhydrin and uvedalin were also studied in a murine model of acute T. cruzi infection (RA strain). Serum activities of the hepatic enzymes alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase were used as biochemical markers of hepatotoxicity. RESULTS: The three compounds exhibited leishmanicidal activity on both parasite forms with IC50 values of 0.42-0.54 µg/ml for promastigotes and 0.85-1.64 µg/ml for intracellular amastigotes. Similar results were observed on T. cruzi epimastigotes (IC50 0.35-0.60 µg/ml). The TEM evaluation showed marked ultrastructural alterations, such as an intense vacuolization and mitochondrial swelling in both L. mexicana promastigotes and T. cruzi epimastigotes exposed to the STLs. In the in vivo study, enhydrin and uvedalin displayed a significant decrease in circulating parasites (50-71%) and no signs of hepatotoxicity were detected. CONCLUSIONS: Enhydrin, uvedalin and polymatin B possess significant leishmanicidal and trypanocidal activity on different parasite stages. These results show that these compounds may provide valuable leads for the development of new drugs against these neglected parasitic diseases.
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Lactonas/farmacologia , Leishmania mexicana/efeitos dos fármacos , Sesquiterpenos de Germacrano/química , Sesquiterpenos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Asteraceae/química , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Modelos Animais de Doenças , Lactonas/administração & dosagem , Lactonas/efeitos adversos , Lactonas/uso terapêutico , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/ultraestrutura , Leishmaniose/tratamento farmacológico , Leishmaniose/parasitologia , Fígado/efeitos dos fármacos , Camundongos , Microscopia Eletrônica de Transmissão , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Sesquiterpenos/administração & dosagem , Sesquiterpenos/efeitos adversos , Sesquiterpenos/química , Sesquiterpenos/uso terapêutico , Sesquiterpenos de Germacrano/farmacologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/ultraestruturaRESUMO
Glycosidases may play a role in sperm maturation during epididymal transit. In this work, we describe the interaction of these enzymes with bull spermatozoa. We found that ß-galactosidase associated to spermatozoa can be released under low ionic strength conditions, whereas the interaction of N-acetyl-ß-D-glucosaminidase and ß-glucuronidase with spermatozoa appeared to be stronger. On the other hand, α-mannosidase and α-fucosidase cannot be removed from the gametes. In addition, part of N-acetyl-ß-D-glucosaminidase, ß-galactosidase, and ß-glucuronidase can also be released by mannose-6-phosphate. Taking into account these data, we explored the presence of cation-independent- and cation-dependent-mannose-6-phosphate receptors in the spermatozoa and found that cation-independent mannose-6-phosphate receptor is highly expressed in bull spermatozoa and cation-dependent-mannose-6-phosphate receptor is expressed at a lesser extent. In addition, by immunofluorescence, we observed that cation-independent-mannose-6-phosphate receptor is mostly located at the acrosomal zone, whereas cation-dependent-mannose-6-phosphate receptor presents a different distribution pattern on spermatozoa during the epididymal transit. N-acetyl-ß-D-glucosaminidase and ß-glucuronidase isolated from epididymal fluid interacted mostly with cation-independent-mannose-6-phosphate receptor, while ß-galactosidase was recognized by both receptors. We concluded that glycosidases might play different roles in bull spermatozoa and that mannos-6-phosphate receptors may act as recruiters of some enzymes. J. Cell. Biochem. 117: 2464-2472, 2016. © 2016 Wiley Periodicals, Inc.
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Epididimo/metabolismo , Glicosídeo Hidrolases/metabolismo , Manosefosfatos/metabolismo , Receptor IGF Tipo 2/metabolismo , Espermatozoides/metabolismo , Animais , Western Blotting , Bovinos , Imunofluorescência , MasculinoRESUMO
Secochiliolide acid (1) isolated from the Patagonian shrub Nardophyllum bryoides, was used as a scaffold for the preparation of a series of nine derivatives. Compound 1 and its derivatives were tested against Trypanosoma cruzi epimastigotes grown in liquid media. It was first observed that secochiliolide acid (1) inhibited the proliferation of the parasites, with an IC50 of 2 µg/mL. Six of the synthesized derivatives were also active with IC50's between 2 and 7 µg/mL which are comparable to that of the commercial drug benznidazole (2.5 µg/mL). These results indicate that the carboxyl group is not essential for the bioactivity of 1, while the presence of the tetrasubstituted exocyclic double bond seems to be important. Moreover, the presence of the furan and spirolactone rings is not essential for the bioactivity per se, but is important in combination with other structural fragments present in the molecule.
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Diterpenos/química , Diterpenos/farmacologia , Propionatos/química , Propionatos/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Asteraceae/química , Doença de Chagas/tratamento farmacológico , Humanos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
Leishmaniasis is a worldwide parasitic disease, caused by monoflagellate parasites of the genus Leishmania. In the search for more effective agents against these parasites, the identification of molecular targets has been attempted to ensure the efficiency of drugs and to avoid collateral damages on the host's cells. In this work, we have investigated some of the mechanisms of action of a group of natural sesquiterpene lactones that are effective against Leishmania mexicana mexicana promastigotes. We first observed that the antiproliferative effect of mexicanin I (Mxc), dehydroleucodine (DhL), psilostachyin (Psi), and, at lesser extent, psilostachyin C (Psi C) is blocked by 1.5 mM reduced glutathione. The reducing agent was also able to reverse the early effect of the compounds, suggesting that lactones may react with intracellular sulfhydryl groups. Moreover, we have shown that all the sesquiterpene lactones, except Psi C, significantly decreased the endogenous concentration of glutathione within the parasite. Consistent with these findings, the active sesquiterpene lactones increased between 2.7 and 5.4 times the generation of ROS by parasites. These results indicate that the induction of oxidative stress is at least one of the mechanisms of action of DhL, Mxc, and Psi on parasites while Psi C would act by another mechanism.
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Clathrin-mediated endocytosis plays an important role in the maintenance of neuronal integrity in the synaptic terminals. Here we studied the effect of anomalous polyglutamine expansion in huntingtin on the interaction of coat proteins with membranes, in areas of mouse brain or in cultured striatal cells. We observed that this anomaly induces a redistribution of AP-2, but not other coat proteins, from the membrane to the cytosol in the striatum, and in the cultured striatal cells. It was also noted that huntingtin associates with AP-2, and that this association decreases due to the mutation in huntingtin. This decreased receptor-mediated endocytosis, measured by the internalization of transferrin in the mutated cells. It was also confirmed that huntingtin mutation made the cells more vulnerable to the action of quinolinic acid, with an increasing degradation of the AP-2 alpha subunits. On the basis of these results, we conclude that abnormal polyglutamine expansion in huntingtin affects clathrin-mediated endocytosis, and may be one of the pathogenic mechanisms of neurodegeneration.
Assuntos
Corpo Estriado/citologia , Endocitose/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Neurônios/fisiologia , Proteínas Nucleares/genética , Fator de Transcrição AP-2/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Clatrina/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Proteína Huntingtina , Imunoprecipitação , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Ácido Quinolínico/farmacologia , Estatísticas não Paramétricas , Fatores de Tempo , Transferrina/farmacologiaRESUMO
Chagas disease is a major health problem in Latin America and is caused by the parasitic protozoan Trypanosoma cruzi. Although many drugs have been used to alleviate the disease, these have been ineffective in the chronic phase and have also presented numerous side effects on patients. In this study we tested the effect of three sesquiterpene lactones (dehydroleucodine, helenalin and mexicanin) and a diterpene (5-epi-icetexone) on parasites (Y-strain) grown in host cells. At 48 h of treatment, the number of amastigotes inside the cells was lower than in the controls. This effect was observable at concentrations of 1.5-3.8 µM, which are of low cytotoxicity to host cells. In addition, the compounds caused a decrease in the percentage of infected cells. The treatments also reduced the presence of trypomastigotes in the extracellular medium. In all cases, helenalin was the most potent. The number of parasites per cell at 24h indicates the occurrence of multiple infection, which would also be affected by the compounds. However, we should not discard an effect on the proliferation and survival of parasites within the host cells. On the other hand, an additional effect on the differentiation of parasites and/or the survival of extracellular trypomastigotes might be possible. We conclude that these compounds are very effective against T. cruzi possibly by multiple mechanisms.
Assuntos
Antiprotozoários/farmacologia , Diterpenos/farmacologia , Lactonas/farmacologia , Sesquiterpenos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiprotozoários/química , Chlorocebus aethiops , Diterpenos/química , Lactonas/química , Sesquiterpenos/química , Células VeroRESUMO
It is well known that clathrin-mediated endocytosis is crucial for the normal functioning and integrity of neurons in the central nervous system. In this study we attempted to correlate the expression of coat proteins with development in different areas of rat brain. By Western blot, we studied the expression of AP-2, GGA1 and GGA2 in striatum, cerebellum, brain stem, cerebral cortex and hippocampus of newborn rats and during post-natal development; 5, 15, 30, 60, 90 or 150 days after birth. We observed that the expression of the α2 subunit of AP-2 increased substantially between the 15th and 30th day after birth in all areas studied, excepting the cerebellum and cortex. On the other hand, the expression of the α1 subunit does not change significantly during the development in any of the areas under study. We also noted that the expression of the µ2 subunit did not follow the pattern of α2 during development. In general terms, the expression of GGA1 and GGA2 followed a similar pattern to that of AP-2, although these proteins increased significantly in the cerebral cortex from the 15th day after birth. Moreover, presenilin-1, a protein associated with aging and neurodegeneration, shows an expression pattern similar to coat proteins in the striatum and cortex. These results suggest that proteins that conform the intracellular transport machinery in the brain cells seems to accompany development, according to the maturation of the different brain areas.
Assuntos
Envelhecimento/fisiologia , Encéfalo , Proteínas do Capsídeo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Encéfalo/anatomia & histologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Presenilina-1/metabolismo , Ratos , Ratos WistarRESUMO
Numerous natural compounds have been used against Trypanosoma cruzi, the causative agent of Chagas' disease. Here, we studied the effect of the diterpene 5-epi-icetexone on growth and morphology of parasites synchronized with hydroxyurea, at different periods of time after removal of the nucleotide. We observed that the diterpene does not affect the growth of the parasites when added within 10 h after removal of hydroxyurea, but the compound was effective on growth when added to the cultures after 12 h. Thymidine incorporation was somewhat inhibited when the diterpene was added at 12 h after removal of hydroxyurea, possibly on the transition S/G2. Using transmission electron microscopy we observed that the diterpene induced a delay in the progression of cell division. We conclude that the compound, at cytostatic dose, affects the cell cycle of T. cruzi, possibly in the transition S/G2 phase and cell division. Further studies will focus to identify the molecular targets for the action of 5-epi-icetexone.
Assuntos
Ciclo Celular/efeitos dos fármacos , Doença de Chagas/parasitologia , Diterpenos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Humanos , Hidroxiureia/farmacologia , Timidina/metabolismo , Fatores de Tempo , Trypanosoma cruzi/citologia , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
The co-existence of two types of mannose-6-phosphate receptors (CD-MPR and CI-MPR) in most cell types is still not well explained. Some evidence suggests that the CI-MPR could be actively involved in the regulation of growth factors in the early stages of mammalian organ development. In this study, it was demonstrated that both receptors are distributed in a non-overlapping fashion in rat liver, and that the distribution of CI-MPR changes over a percoll gradient between newborn and adult animals. By using marker proteins it was observed that in newborns the CI-MPR is located both in intracellular fractions and in fractions that coincide with a plasma membrane marker, whereas in adults it is only detected in intracellular fractions. It was also noted that N-acetyl-ß-D-glucosaminidase distribution is closer to CI-MPR than to CD-MPR and that acid phosphatase did not match with any receptor. This evidence may also suggest that both receptors have different functions, mainly at early stages in the development of organs.
Assuntos
Fígado/metabolismo , Receptor IGF Tipo 2/metabolismo , Animais , Animais Recém-Nascidos , Fígado/crescimento & desenvolvimento , Organogênese , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 2/genéticaRESUMO
In this study, the antiprotozoal activity of the sesquiterpene lactone psilostachyin C was investigated. This natural compound was isolated from Ambrosia scabra by bioassay-guided fractionation and was identified by spectroscopic techniques. Psilostachyin C exerted in vitro trypanocidal activity against Trypanosoma cruzi epimastigotes, trypomastigotes and amastigotes, with 50% inhibitory concentration (IC(50)) values of 0.6, 3.5 and 0.9 µg/mL, respectively, and displayed less cytotoxicity against mammalian cells, with a 50% cytotoxic concentration (CC(50)) of 87.5 µg/mL. Interestingly, this compound induced ultrastructural alterations, as seen by transmission electron microscopy, in which vacuolisation and a structural appearance resembling multivesicular bodies were observed even at a concentration as low as 0.2 µg/mL. In an in vivo assay, a significant reduction in the number of circulating parasites was found in T. cruzi-infected mice treated with psilostachyin C for 5 days compared with untreated mice (7.4 ± 1.2 × 10(5)parasites/mL vs. 12.8 ± 2.0 × 10(5)parasites/mL) at the peak of parasitaemia. According to these results, psilostachyin C may be considered a promising template for the design of novel trypanocidal agents. In addition, psilostachyin C inhibited the growth of Leishmania mexicana and Leishmania amazonensis promastigotes (IC(50)=1.2 µg/mL and 1.5 µg/mL, respectively).
Assuntos
Antiprotozoários/farmacologia , Extratos Vegetais/farmacologia , Sesquiterpenos/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Ambrosia/química , Animais , Antiprotozoários/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Doença de Chagas/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Humanos , Concentração Inibidora 50 , Leishmania mexicana/efeitos dos fármacos , Masculino , Camundongos , Microscopia Eletrônica de Transmissão , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/toxicidade , Doenças dos Roedores/tratamento farmacológico , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/toxicidade , Resultado do Tratamento , Trypanosoma cruzi/ultraestruturaRESUMO
It is believed that the mammalian epididymis participates in the maturation of the sperm due to its secretory activity. High concentrations of several secreted acid hydrolases are found in the epididymal lumen. Moreover, some of these enzymes are secreted by the epididymal epithelium in an androgen-dependent fashion. In this study, we attempted to discern whether mannose-6-phosphate receptors (MPRs) regulate transport and secretion of lysosomal enzymes in the rat epididymis, and if these events are altered when the animals are subjected to hormonal manipulation. We observed that expression of cation-dependent MPR (CD-MPR) and cation-independent MPR (CI-MPR) increased significantly in caudal epididymis of castrated rats by immunoblot. This increase was corroborated by quantitation of MPRs, by binding assays. This change could be due to androgen deprivation, as a similar effect was observed after treatment with the anti-androgenic drug flutamide. Furthermore, we observed that the CD-MPR was redistributed to the apical area of the epithelium on castrated rats by immunohistochemistry, which is compatible with the redistribution of the receptors toward lighter fractions in a Percoll gradient. Consistent with a possible involvement of the CD-MPR in the secretion, we observed an increase in pro-cathepsin D levels in epididymal fluid after castration. We conclude that the CD-MPR might be regulated by hormones and that this receptor might be involved in the secretion of specific enzymes into the rat epididymis.
Assuntos
Epididimo/metabolismo , Orquiectomia , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Western Blotting , Catepsina D/metabolismo , Di-Hidrotestosterona/metabolismo , Ensaio de Imunoadsorção Enzimática , Epididimo/efeitos dos fármacos , Estradiol/metabolismo , Flutamida/farmacologia , Imuno-Histoquímica , Lisossomos/enzimologia , Lisossomos/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptor IGF Tipo 2 , Testosterona/metabolismo , alfa-Manosidase/metabolismoRESUMO
The significance of the coexistence of 2 mannose-6-phosphate receptors (MPRs) in most cell types still remains poorly understood. In this study, expression of the cation-dependent MPR (CD-MPR) and the cation-independent MPR (CI-MPR) was measured by Western blot in rat organs at 3 ages, i.e. in newborn and 10- and 90-day-old rats. It was observed that expression of the CI-MPR tends to diminish from newborns to adults in 5 of the 6 organs studied, whereas the CD-MPR did not show a clear tendency over time. In pancreas, conversely, both MPRs increased progressively from newborns to adults. The activity of 2 acid hydrolases was also measured at the different ages, and a low correlation was found with the expression of the 2 MPRs. With the exception of the pancreas, it is possible that the CI-MPR is mostly occupied with the clearance of insulin-like growth factor-II at early stages of development, and that later both MPRs may participate in the maturation of the lysosomal apparatus. We propose that in the pancreas, both receptors may be involved in increasing the proteolytic activity of this exocrine gland during postnatal development.
Assuntos
Crescimento e Desenvolvimento , Especificidade de Órgãos , Receptor IGF Tipo 2/metabolismo , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Hidrolases/metabolismo , Membranas/enzimologia , Ratos , Ratos Sprague-DawleyRESUMO
In this paper, the effects of 3 natural sesquiterpene lactones, i.e., helenalin (Hln), mexicanin (Mxc), and dehydroleucodine (DhL), were evaluated using cultured Leishmania mexicana promastigotes. It was observed that the compounds inhibited the in vitro growth of the parasites at relatively low concentrations. The effect was rapid and irreversible with an estimated IC50 of 2-4 microM, while all the lactones were more effective than ketoconazole. Moreover, these compounds exhibited low cytotoxicity for mammalian cells. Hln induced strong vacuolization of the parasite cytoplasm, although pericellular microtubules were preserved. The 3 lactones induced DNA fragmentation as judged by the high labeling with the fluorescent TUNEL method, which was confirmed by electrophoresis on agarose gels. The ability of the parasites to invade Vero cells was also decreased by exposure to low concentrations of the compounds. We conclude that these compounds can affect the parasite's life cycle, possibly through multiple mechanisms. Identification of the molecular targets of these natural products and their effects on amastigotes should be determined to evaluate the possible therapeutic use of the compounds against leishmaniasis.