Recibido en su versión modificada: 21 de mayo de 2008 Aceptado: 06 de junio de 2008 RESUMEN Intro... more Recibido en su versión modificada: 21 de mayo de 2008 Aceptado: 06 de junio de 2008 RESUMEN Introducción: Existen estudios controvertidos sobre la prevención de la enfermedad de Alzheimer y el uso de antiinflamatorios no esteroideos. El objetivo fue evaluar el efecto del ibuprofeno y ácido acetilsalicílico sobre el deterioro cognitivo, poder antioxidante total (PAT) e isoprostanos (8-iso-PGF2á) séricos. Material y métodos: Entre abril de 2004 y febrero de 2006, a 18 mujeres mayores de 55 años de edad se les realizó escrutinio con la Prueba Mínima del Estado Mental de Folstein (MMSE); Prueba Corta para la Evaluación de la Memoria y la Atención, Syndrome Kurtz Test (SKT) y Escala de Depresión Geriátrica de Yasevage. Fueron asignadas aleatoriamente para recibir 400 mg/día de ibuprofeno (n=9) o 500 mg/día de ácido acetilsalicílico (n=9) durante un año. En la visita basal, seis meses y al año se determinó PAT y 8-iso-PGF2á séricos. Resultados: A un año de intervención, en cinco mujeres (55.6%) el MMSE aumentó cuatro puntos con ácido acetilsalicílico comparado con tres (33.3%) de ibuprofeno (p=0.028). El PAT aumentó (p=0.01) y disminuyeron los 8-iso-PGF2á (p=0.01) en ambos grupos en comparación con los valores basales. Conclusiones: Ambos medicamentos mejoraron el estado cognitivo y el perfil oxidativo en la población estudiada. Palabras clave: Ácido acetilsalicílico, deterioro cognitivo, ibuprofeno, isoprostanos, poder antioxidante total SUMMARY Background: There is controversy about the prevention of Alzheimer's disease with nonsteroidal antiinflammatory drugs (NSAIDs). Our objective was to evaluate the effect of ibuprofen and acetylsalicylic acid on cognitive impairment, serum total antioxidant power (TAP) and isoprostane (8-iso-PGF2á). Methods: We applied from April 2004 to February 2006 a Folstein mini-mental state (MMSE), Syndrome Kurtz Test (SKT) and a geriatric depression scale (Yasevage) to eighteen eligible women. They were 55 years and older. All women (n=18) with normal cognitive state were randomized to ibuprofen 400 mg per day (n=9) and acetylsalicylic acid 500 mg per day (n=9) for one year. Serum TAP and 8-iso-PGF2á were performed at baseline, after six months and one year of treatment. Results: After one year of treatment with acetylsalicylic acid five women (55.6%) raised their score 4 points in MMSE compared with 3 points increased (33.3%) showed by the ibuprofen group. TAP increased (p=0.01) and 8-iso-PGF2á reduced (p=0.01) in both groups compared with baseline. Conclusions: Both drugs improved the cognitive state and oxidative status of our population.
Introduction. Recent studies indicate that decreased energy generation by mitochondria is a featu... more Introduction. Recent studies indicate that decreased energy generation by mitochondria is a feature that is common across neurodegenerative diseases. Patients and methods. In order to obtain direct evidence that mitochondrial functioning is altered, we measured the hydrolytic activity of F 0 F 1 -ATPase and its capacity to generate a stable proton gradient in submitochondrial particles in 29 patients diagnosed with probable Alzheimer's disease (AD). Submitochondrial particles were obtained from platelets of patients with a diagnosis of probable AD and from clinically healthy controls. Results. Data revealed that the hydrolytic activity of F 0 F 1 -ATPase increases significantly in patients with probable AD (41.7 ± 4.3 nmol PO 4 min -1 [mg protein] -1 , n = 29) as compared to the control subjects (29.1 ± 1.9 nmol PO 4 min -1 [mg protein] -1 , n = 29). It is important to note that, in the male population with probable AD, we found that hydrolytic activity of F 0 F 1 -ATPase increased as cerebral deterioration progressed. We also detected a lower pH gradient in the submitochondrial particles of patients with probable AD (0.28 ± 0.08 pH units, n = 25) as compared to the controls (0.5 ± 0.1 pH units, n = 20). Conclusions. Overall, these data point to an alteration in the functioning of the enzyme. [REV NEUROL 2005; 40: 81-5]
The circadian oscillations of many physiological processes provide an endogenous temporal program... more The circadian oscillations of many physiological processes provide an endogenous temporal program for the adaptive synchronization of mammals to the fluctuating external world. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system. The aim of the present work was investigate the effect of acute treatment of constant darkness on mitochondrial ATP synthase activities and membrane fluidity in liver from male rat. We found that ATP synthase activity was not changed by the treatment. However ATPase activity and membrane fluidity were significantly diminished and pH gradient driven by ATP hydrolysis was incremented, in comparison from samples from rats kept on normal light/dark cycles. Additionally, the treatment of constant darkness diminishes the passive proton permeability of the inner mitochondrial membrane. In conclusion constant darkness induces a more efficient coupling between proton transport and catalysis, and increment the efficiency of the enzyme because the ratio of ATP synthase/ATPase activity was higher. These results exhibited the physiological adaptation of liver mitochondria to acute treatment of constant darkness in order to satisfy the cellular energy demand.
Tumor necrosis factor-alpha and interleukin-6 levels among patients suffering a bile duct injury ... more Tumor necrosis factor-alpha and interleukin-6 levels among patients suffering a bile duct injury during cholecystectomy Background: During cholecystectomy, the bile duct may be injured. When this complication occurs, Kupffer cells are activated and produce tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL6) to phagocyte toxic products Aim: To measure serum levels of TNF-a and IL-6 among patients that suffered a bile duct injury after a cholecystectomy. Patients and Methods: Serum levels of TNF-a and IL-6 were measured prior to the bile-enteric derivation and after one year of follow-up, in 31 patients that had a complete bile duct obstruction after open or laparoscopic cholecystectomy and in 5 healthy controls. Results: At baseline TNF-a levels in healthy subjects and patients with bile duct injury were 0 and 43.9 ± 2.9 ng/mL, respectively (p < 0.01). At one year of follow up, TNF-a became undetectable among patients. At baseline, the values for IL-6 among healthy controls and patients were 3.0 ± 2. 0 and 72.0 ± 94.7 pg/mL respectively, (p < 0,004). After one year of follow up, IL-6 levels decreased to 6.4 ± 0.3 pg/mL among patients. Conclusions: TNF-a and IL-6 levels were elevated before bile-enteric derivation among patients with bile duct injury and became normal one year later. (Rev Med Chile 2010; 138: 1259-1263.
En estudios epidemiológicos se ha encontrado un mayor riesgo de padecer la enfermedad de Parkinso... more En estudios epidemiológicos se ha encontrado un mayor riesgo de padecer la enfermedad de Parkinson (EP) con la exposición ambiental a factores tales como sustancias derivadas de los procesos industriales, utilización de productos agroquímicos, o vivir en un medio rural. La hipótesis de que ciertos tóxicos ambientales pudieran ser el origen de la EP está apoyada por el descubrimiento de que compuestos químicos como los herbicidas paraquat, diquat y el fungicida maneb son selectivamente tóxicos en las neuronas dopaminérgicas nigroestriadas. Asimismo, uno de los insecticidas producidos por plantas, como la rotenona y el producto secundario de la síntesis de heroína sintética (1-metil-4-fenil-1,2,3,6tetrahidropiridina) puede reproducir en modelos animales los hallazgos neuroquímicos, histopatológicos y clínicos propios de la EP. De manera interesante, existen similitudes en la estructura química del paraquat y del MPTP. Evidencia recien muestra que la inflamación y el estrés oxidativo tienen un papel esencial en el desarrollo de la EP. Por lo que en el laboratorio, encontramos que en un modelo animal, la melatonina disminuye los incrementos en los productos de oxidación lipídica, de los metabolitos del óxido nítrico y la actividad de la ciclo-oxigenasa 2, que son inducidos por una inyección intraperitoneal de MPTP. Lo que sugiere que el efecto neuroprotector de la melatonina son parcialmente atribuidos a su acción antioxidante y anti-inflamatoria.
Background: post-cholecystectomy bile duct injuries are identified by the onset of jaundice as we... more Background: post-cholecystectomy bile duct injuries are identified by the onset of jaundice as well as elevated bilirubin and alkaline phosphatase levels during the peri-operative period. It is unknown how serum oxidative stress markers are modified in patients with post-cholecystectomy bile duct injuries.
Objective: To determine the oxidative stress markers in serum from patients with relapsing-remitt... more Objective: To determine the oxidative stress markers in serum from patients with relapsing-remitting multiple sclerosis. Methods: Blood samples from healthy controls and 22 patients 15 women (7 aged from 20 to 30 and 8 were > 40 years old) and 7 men (5 aged from 20 to 30 and 2 were > 40 years old) fulfilling the McDonald Criteria and classified as having Relapsing-Remitting Multiple Sclerosis accordingly with Lublin were collected for oxidative stress markers quantification. Results: Nitric oxide metabolites (nitrates/nitrites), lipid peroxidation products (malondialdehyde plus 4-hidroxialkenals), and glutathione peroxidase activity were significantly increased in serum of subjects with relapsing-remitting multiple sclerosis in comparison with that of healthy controls. These data support the hypothesis that multiple sclerosis is a component closely linked to oxidative stress.
Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis t... more Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn't been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.
can lead to changes in the synthesis of melatonin. In vitro studies have shown that this hormone ... more can lead to changes in the synthesis of melatonin. In vitro studies have shown that this hormone modulates the efficiency of mitochondrial respiration. Therefore, this work examined whether the efficiency of mitochondrial respiration changes in rats that have been subjected to constant illumination or darkness for a short period. Methods: Rats were randomly distributed in three groups: Control, Constant Illumination (72 hours) and Constant Darkness (72 hours). Upon completion of treatment, rats were sacrificed and mitochondria from the pineal gland, Harderian gland, thymus and spleen were isolated. Subsequently, mitochondrial respiratory control was quantified from the removed tissues in the three experimental groups. Results: Our findings show that brief treatments of continued illumination or continued darkness had no significant effect on mitochondrial respiratory control in spleen, thymus or Harderian glands. In contrast, we observed a slight increase in mitochondrial respiratory control in the pineal gland of animals exposed to constant illumination. Conclusions: Our results suggest that brief treatment with continuous light or darkness does not have a significant effect on the efficiency of mitochondrial activity in spleen, thymus or Harderian gland. This is probably due to the endogenous circadian rhythms that tightly regulate mitochondrial enzymatic activity in these tissues.
Multiple sclerosis (MS) exhibits many of the hallmarks of an inflammatory autoimmune disorder inc... more Multiple sclerosis (MS) exhibits many of the hallmarks of an inflammatory autoimmune disorder including breakdown of the blood-brain barrier (BBB), the recruitment of lymphocytes, microglia, and macrophages to lesion sites, the presence of multiple lesions, generally being more pronounced in the brain stem and spinal cord, the predominantly perivascular location of lesions, the temporal maturation of lesions from inflammation through demyelination, to gliosis and partial remyelination, and the presence of immunoglobulin in the central nervous system and cerebrospinal fluid. Lymphocytes activated in the periphery infiltrate the central nervous system to trigger a local immune response that ultimately damages myelin and axons. Pro-inflammatory cytokines amplify the inflammatory cascade by compromising the BBB, recruiting immune cells from the periphery, and activating resident microglia. inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in the demyelination and free radical-mediated tissue injury in the pathogenesis of MS. The inflammatory environment in demyelinating lesions leads to the generation of oxygen-and nitrogen-free radicals as well as proinflammatory cytokines which contribute to the development and progression of the disease. Inflammation can lead to oxidative stress and vice versa. Thus, oxidative stress and inflammation are involved in a self-perpetuating cycle.
In severe acute pancreatitis (SAP) inflammatory processes foster necrosis, cellular lysis and lib... more In severe acute pancreatitis (SAP) inflammatory processes foster necrosis, cellular lysis and liberation of vasoactive substances associated with multiple organ failure. The effects of vacuum-assisted closure and Drotrecogin alpha on inflammatory cytokines were evaluated in SAP patients with infected necrosis. Methods: Forty-six patients were included in three groups: Group 1, necrosectomy and abdominal cavity washing; Group 2, necrosectomy plus vacuum-assisted closure (VAC), and Group 3, necrossectomy plus VAC plus Drotrecogin alpha. Immunoreactive IL-32, TNF-α, IL-6, TGF-β and IL-2 cytokines were quantified with ELISA method. Results: IL-32 was significantly increased in all patients, predominantly the non-survivor of Group 3 (p < 0.0001). Group 2 maintained increased IL-32 levels throughout. Peak TNF-α was observed in non-survivors of Groups 1 and 2, with a frank tendency to decrease in Group 3. The IL-6 was increased, sustained throughout the study, peaking at the onset in non-survivors. At the end IL-6 tended to diminish, predominantly in survivors. TNF-α and IL-6 were significantly increased on hospitalization, with a maximum peak in non-survivors of all groups. Initial values of TGF-β were significantly increased in survivors of the three groups, and were significantly diminished in non-survivors; affecting pancreas regeneration and favoring sys-temic inflammation, with possible multiple-organ repercussions. IL-2 levels were elevated, predominantly in non-survivors of Group 1. There was positive correlation between the increase IL-32 and TNF-α, and negative correlation between the increase in TNF-α and decrease in TGF-β; and, a tendency for negative correlation between the IL-2 increased and TGF-β levels. Conclusion: We found a generalized, sustained inflammatory state that fosters a torpid outcome in SAP patients.
Introduction: In bile duct injuries (BDI), cholestasis and cholangitis can alter the fibrinolytic... more Introduction: In bile duct injuries (BDI), cholestasis and cholangitis can alter the fibrinolytic system by promoting an increase of extracellular matrix depositions which favor an imbalance between metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Materials and Methods: Levels of PAI-1, MMP-3, MMP-8, TIMP-1 and TIMP-2 in 35 patients with post-cholecystectomy BDI by complete biliary obstruction were measured and compared to a healthy control group. Sirius red staining and immune staining for MMP-3 and MMP-8 were also undertaken in liver biopsies. Results: Levels of PAI-1, TIMP-1, TIMP-2 and MMP-8 were higher in BDI than healthy controls: 15 ± 2 ng/mL vs 7.1 ± 2 ng/mL (p < 0.024); 539 ± 64 ng/mL vs 256 ± 13 ng/mL (p < 0.003); 178.4 ± 79.8 ng/mL vs 98.6 ± 2 ng/mL; and 90 ± 10 ng/mL vs 6.8 ± 2 ng/mL (p < 0.002), respectively. However, serum levels of MMP-3 expression in BDI were decreased 9.0 ± 1 vs. healthy controls 20.3 ± 6 ng/mL (p < 0.001). Morphometric analysis showed that Sirius red staining in BDI was increased 121548.6 ± 20545.4 µm 2 vs. 22865.7 ± 3865 µm 2 in healthy controls (p < 0.0001), MMP-8 deposition in liver was higher in BDI compared to controls: 233015.2 ± 32953.3 µm 2 vs. 30744.2 ± 5810.2 µm 2 (p < 0.0001). MMP-3 liver staining in BDI compared to healthy controls was not significant 130837.9 ± 17,484 µm 2 vs. 116337.9 ± 24803.3 µm 2 (p < 0.55). These results suggest an imbalance between fibrogenic/fibrinolytic protein levels. Interestingly, expression of the fibrinolytic protein MMP-8 was increased in serum and liver biopsies in BDI. Conclusion: We found an imbalance of profibrogenic molecules which promote extracellular matrix deposition. The over-expression of fibrinolytic proteins such as MMP-8 could limit liver fibrosis, preventing hepatic dysfunction in post-cholecystectomy BDI.
Background: Diabetic retinopathy (DR) is a preventable cause of visual disability. The aims of th... more Background: Diabetic retinopathy (DR) is a preventable cause of visual disability. The aims of the present study were to investigate levels and behavior oxidative stress markers and mitochondrial function in non-proliferative DR (NPDR) and to establish the correlation between the severity of NPDR and markers of oxidative stress and mitochondrial function. Methods: In a transverse analysis, type 2 diabetes mellitus (T2DM) patients with mild, moderate and severe non-proliferative DR (NPDR) were evaluated for markers of oxidative stress (i.e. products of lipid peroxidation (LPO) and nitric oxide (NO) catabolites) and antioxidant activity (i.e. total antioxidant capacity (TAC), catalase, and glutathione peroxidase (GPx) activity of erythrocytes). Mitochondrial function was also determined as the fluidity of the submitochondrial particles of platelets and the hydrolytic activity of F0/F1-ATPase. Results: Levels of LPO and NO were significantly increased in T2DM patients with severe NPDR (3.19 ± 0.05 μmol/mL and 45.62 ± 1.27 pmol/mL, respectively; P < 0.007 and P < 0.0001 vs levels in health volunteers, respectively), suggesting the presence of oxidative stress. TAC had significant decrease levels with minimum peak in severe retinopathy with 7.98 ± 0.48 mEq/mL (P < 0.0001). In contrast with TAC, erythrocyte catalase and GPx activity was increased in patients with severe NPDR (139.4 ± 4.4 and 117.13 ± 14.84 U/mg, respectively; P < 0.0001 vs healthy volunteers for both), suggesting an imbalance between oxidants and antioxidants. The fluidity of membrane submitochondrial particles decreased significantly in T2DM patients with mild, moderate, or severe NPDR compared with that in healthy volunteers (P < 0.0001 for all). Furthermore, there was a significant increase in the hydrolytic activity of the F0/F1-ATPase in T2DM patients with mild NPDR (265.07 ± 29.55 nmol/PO4; P < 0.0001 vs healthy volunteers), suggesting increased catabolism. Conclusions: Patients with NPDR exhibit oxidative deregulation with decreased membrane fluidity of submitochondrial particles and increased systemic catabolism (mitochondrial dysfunction) with the potential for generalized systemic damage in T2DM.
Background. Dementia affects memory, thinking, language, judgment, and behavior. Depression, is c... more Background. Dementia affects memory, thinking, language, judgment, and behavior. Depression, is common in older adults with dementia. The concomitance of dementia and depression increases disability with impaired activities of daily living (ADL), increasing the chances of institutionalization and mortality. Methods. Cross-sectional study of a population 60 years and older who live in the State of Jalisco, Mexico. A total of 1142 persons were assessed regarding their cognitive function, emotional state, and physical performance. Door-to-door interview technique was assigned in condition with multistage probability random sampling. Cognitive function, depression and functional disability were assessed by applying standardized Minimental State Examination (Folstein), Geriatric Depression Scale, and the Katz index, respectively. Diagnosis of dementia was performed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, the Fourth Edition. Data were analyzed using SPSS software. Results. Prevalence of demency was 9.5% (63.35% women, and 36.7% men). Demency was associated with being woman, being older than 70 years, low level of education, not having the economic benefit of retirement, being single or living without a partner, low level of education, suffering from depression and have functional disability in ADL. Conclusion. Dementia is more common in women and is related to depression and disability.
Objective: To evaluate and compare the intake of lipids and (A, E, and C) vitamins in patients wi... more Objective: To evaluate and compare the intake of lipids and (A, E, and C) vitamins in patients with and without possible neurodegenerative diseases. Methods: Twenty adults with possible Alzheimer's disease or Parkinson's disease and 41 control subjects (50-89 years old) from a rural region were studied. Dietary intake was evaluated with the analysis of macronutrients and micronutrients conducted by a food frequency questionnaire and 24 hours dietary record. Analyses were adjusted for age, sex, body mass index, and energy intake. Through interrogation and use of medical record form of health secretary we obtained information about the sociodemographic characteristics. Multivariate analysis of variance to allow for covariated adjustment was used. Results: Patients had a lower energy intake, vitamin C (P = 0.016), fruits (P < 0.001), vegetables (P = 0.037), and oils and fat (P = 0.002), than the controls. Interestingly, the C vitamin intake in patients was still higher than the recommended. Patients had a higher consumption of cereals (P = 0.017), high-animal fat diet (P = 0.024), and whole milk (P < 0.001); 2.4% of the controls smoke and 5% are alcohol consumers. Eighty-five percent of patients and 78% of the controls do not have physical activity. Family history of subjects in this study indicated chronic diseases. Conclusion: The subjects included in this study had a high intake of C vitamin, this is due to the consumption of fruits and vegetables. However, patients with possible Alzheimer's or Parkinson's disease had a lower intake of fruits and vegetables, which could be due to type of food to which they have access.
Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracocc... more Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied. Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin. Among the oxyanions, sulfite induced the higher increase in ATPase activity. Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the V max . There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme. Dithiothreitol did not affect the ATPase activity. ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin. In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin. It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit : is differently exposed to trypsin.
Maize (Zea mays 1.) embryos, isolated from the developing seed and incubated in dilute buffer, sh... more Maize (Zea mays 1.) embryos, isolated from the developing seed and incubated in dilute buffer, show reduced triacylglycerol (TAC) synthesis, and accumulation stops after 24 h. Synthesis and accumulation can be maintained at high levels if the incubation medium contains abscisic acid (ABA) and/or a high osmotic concentration. Radiolabeled free fatty acids accumulate at higher levels in embryos that contain less TAC, and acetyl coenzyme A carboxylase activity remains essentially unchanged under all of the conditions tested. In contrast, the activities of the acyltransferases required for TAC synthesis remain high only in embryos incubated with ABA and/or a high osmotic concentration. Dose-response curves showed that 4 p~ of ABA or mannitol at -1.0 MPa elicits a full response; both values are within the range considered to be physiological. The TAC synthesis capacity and diacylglycerol acyltransferase activity lost by pretreatment of the embryos can be restored by re-exposure to ABA or high osmoticum. Cermination is not involved because isolated scutellum halves showed the same changes in enzyme activity found in the whole embryo but did not germinate. Our results provide direct evidence for the regulation of TAC-synthesizing activities in maize embryos by ABA and the osmotic potential of the environment.
Isolation of subcellular fractions from dry structures such as seeds or their tissues is difficul... more Isolation of subcellular fractions from dry structures such as seeds or their tissues is difficult. In the present work, plasma membranes were isolated from dry maize (Zea mays L.) embryos with an enrichment of 11-fold as estimated by glucan synthase II (GSII, EC 2.4.1.34) activity and a purity of 78 to 90% as judged by the sensitivity of ATP hydrolysis to vanadate, a specific inhibitor of the plasma membrane H+-ATPase (EC 3.6.1.35). The procedure involved a double homogenization of the dry embryos and the addition of a 1500-g supernatant to an aqueous polyethyleneglycol-dextran two-phase partitioning system; the optimal ratio of polyethyleneglycol-dextran for purification of plasma membranes from dry seeds was 6.8/6.8% (w/w). In the isolated membranes a trace of a tonoplast enzyme marker (tonoplast H+-ATPase, EC 3.6.1.3) could be detected, but there were negligible amounts of mitochondrial and rough endoplasmic reticulum markers, H+-ATPase (EC 3.6.1.34) and diacylglycerol acyltrans-ferase (EC 2.3.1.20), respectively. The technique could also be used in hydrated embryos. The entire procedure can be carried out in 5 to 6 h. The resulting preparation is stable for at least 2 months at −70°C. The membranes of dry and hydrated embryos exhibited a high level of vanadate-sensitive ATPase activity that was increased by lysophosphatidylcholine.
Recibido en su versión modificada: 21 de mayo de 2008 Aceptado: 06 de junio de 2008 RESUMEN Intro... more Recibido en su versión modificada: 21 de mayo de 2008 Aceptado: 06 de junio de 2008 RESUMEN Introducción: Existen estudios controvertidos sobre la prevención de la enfermedad de Alzheimer y el uso de antiinflamatorios no esteroideos. El objetivo fue evaluar el efecto del ibuprofeno y ácido acetilsalicílico sobre el deterioro cognitivo, poder antioxidante total (PAT) e isoprostanos (8-iso-PGF2á) séricos. Material y métodos: Entre abril de 2004 y febrero de 2006, a 18 mujeres mayores de 55 años de edad se les realizó escrutinio con la Prueba Mínima del Estado Mental de Folstein (MMSE); Prueba Corta para la Evaluación de la Memoria y la Atención, Syndrome Kurtz Test (SKT) y Escala de Depresión Geriátrica de Yasevage. Fueron asignadas aleatoriamente para recibir 400 mg/día de ibuprofeno (n=9) o 500 mg/día de ácido acetilsalicílico (n=9) durante un año. En la visita basal, seis meses y al año se determinó PAT y 8-iso-PGF2á séricos. Resultados: A un año de intervención, en cinco mujeres (55.6%) el MMSE aumentó cuatro puntos con ácido acetilsalicílico comparado con tres (33.3%) de ibuprofeno (p=0.028). El PAT aumentó (p=0.01) y disminuyeron los 8-iso-PGF2á (p=0.01) en ambos grupos en comparación con los valores basales. Conclusiones: Ambos medicamentos mejoraron el estado cognitivo y el perfil oxidativo en la población estudiada. Palabras clave: Ácido acetilsalicílico, deterioro cognitivo, ibuprofeno, isoprostanos, poder antioxidante total SUMMARY Background: There is controversy about the prevention of Alzheimer's disease with nonsteroidal antiinflammatory drugs (NSAIDs). Our objective was to evaluate the effect of ibuprofen and acetylsalicylic acid on cognitive impairment, serum total antioxidant power (TAP) and isoprostane (8-iso-PGF2á). Methods: We applied from April 2004 to February 2006 a Folstein mini-mental state (MMSE), Syndrome Kurtz Test (SKT) and a geriatric depression scale (Yasevage) to eighteen eligible women. They were 55 years and older. All women (n=18) with normal cognitive state were randomized to ibuprofen 400 mg per day (n=9) and acetylsalicylic acid 500 mg per day (n=9) for one year. Serum TAP and 8-iso-PGF2á were performed at baseline, after six months and one year of treatment. Results: After one year of treatment with acetylsalicylic acid five women (55.6%) raised their score 4 points in MMSE compared with 3 points increased (33.3%) showed by the ibuprofen group. TAP increased (p=0.01) and 8-iso-PGF2á reduced (p=0.01) in both groups compared with baseline. Conclusions: Both drugs improved the cognitive state and oxidative status of our population.
Introduction. Recent studies indicate that decreased energy generation by mitochondria is a featu... more Introduction. Recent studies indicate that decreased energy generation by mitochondria is a feature that is common across neurodegenerative diseases. Patients and methods. In order to obtain direct evidence that mitochondrial functioning is altered, we measured the hydrolytic activity of F 0 F 1 -ATPase and its capacity to generate a stable proton gradient in submitochondrial particles in 29 patients diagnosed with probable Alzheimer's disease (AD). Submitochondrial particles were obtained from platelets of patients with a diagnosis of probable AD and from clinically healthy controls. Results. Data revealed that the hydrolytic activity of F 0 F 1 -ATPase increases significantly in patients with probable AD (41.7 ± 4.3 nmol PO 4 min -1 [mg protein] -1 , n = 29) as compared to the control subjects (29.1 ± 1.9 nmol PO 4 min -1 [mg protein] -1 , n = 29). It is important to note that, in the male population with probable AD, we found that hydrolytic activity of F 0 F 1 -ATPase increased as cerebral deterioration progressed. We also detected a lower pH gradient in the submitochondrial particles of patients with probable AD (0.28 ± 0.08 pH units, n = 25) as compared to the controls (0.5 ± 0.1 pH units, n = 20). Conclusions. Overall, these data point to an alteration in the functioning of the enzyme. [REV NEUROL 2005; 40: 81-5]
The circadian oscillations of many physiological processes provide an endogenous temporal program... more The circadian oscillations of many physiological processes provide an endogenous temporal program for the adaptive synchronization of mammals to the fluctuating external world. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system. The aim of the present work was investigate the effect of acute treatment of constant darkness on mitochondrial ATP synthase activities and membrane fluidity in liver from male rat. We found that ATP synthase activity was not changed by the treatment. However ATPase activity and membrane fluidity were significantly diminished and pH gradient driven by ATP hydrolysis was incremented, in comparison from samples from rats kept on normal light/dark cycles. Additionally, the treatment of constant darkness diminishes the passive proton permeability of the inner mitochondrial membrane. In conclusion constant darkness induces a more efficient coupling between proton transport and catalysis, and increment the efficiency of the enzyme because the ratio of ATP synthase/ATPase activity was higher. These results exhibited the physiological adaptation of liver mitochondria to acute treatment of constant darkness in order to satisfy the cellular energy demand.
Tumor necrosis factor-alpha and interleukin-6 levels among patients suffering a bile duct injury ... more Tumor necrosis factor-alpha and interleukin-6 levels among patients suffering a bile duct injury during cholecystectomy Background: During cholecystectomy, the bile duct may be injured. When this complication occurs, Kupffer cells are activated and produce tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL6) to phagocyte toxic products Aim: To measure serum levels of TNF-a and IL-6 among patients that suffered a bile duct injury after a cholecystectomy. Patients and Methods: Serum levels of TNF-a and IL-6 were measured prior to the bile-enteric derivation and after one year of follow-up, in 31 patients that had a complete bile duct obstruction after open or laparoscopic cholecystectomy and in 5 healthy controls. Results: At baseline TNF-a levels in healthy subjects and patients with bile duct injury were 0 and 43.9 ± 2.9 ng/mL, respectively (p < 0.01). At one year of follow up, TNF-a became undetectable among patients. At baseline, the values for IL-6 among healthy controls and patients were 3.0 ± 2. 0 and 72.0 ± 94.7 pg/mL respectively, (p < 0,004). After one year of follow up, IL-6 levels decreased to 6.4 ± 0.3 pg/mL among patients. Conclusions: TNF-a and IL-6 levels were elevated before bile-enteric derivation among patients with bile duct injury and became normal one year later. (Rev Med Chile 2010; 138: 1259-1263.
En estudios epidemiológicos se ha encontrado un mayor riesgo de padecer la enfermedad de Parkinso... more En estudios epidemiológicos se ha encontrado un mayor riesgo de padecer la enfermedad de Parkinson (EP) con la exposición ambiental a factores tales como sustancias derivadas de los procesos industriales, utilización de productos agroquímicos, o vivir en un medio rural. La hipótesis de que ciertos tóxicos ambientales pudieran ser el origen de la EP está apoyada por el descubrimiento de que compuestos químicos como los herbicidas paraquat, diquat y el fungicida maneb son selectivamente tóxicos en las neuronas dopaminérgicas nigroestriadas. Asimismo, uno de los insecticidas producidos por plantas, como la rotenona y el producto secundario de la síntesis de heroína sintética (1-metil-4-fenil-1,2,3,6tetrahidropiridina) puede reproducir en modelos animales los hallazgos neuroquímicos, histopatológicos y clínicos propios de la EP. De manera interesante, existen similitudes en la estructura química del paraquat y del MPTP. Evidencia recien muestra que la inflamación y el estrés oxidativo tienen un papel esencial en el desarrollo de la EP. Por lo que en el laboratorio, encontramos que en un modelo animal, la melatonina disminuye los incrementos en los productos de oxidación lipídica, de los metabolitos del óxido nítrico y la actividad de la ciclo-oxigenasa 2, que son inducidos por una inyección intraperitoneal de MPTP. Lo que sugiere que el efecto neuroprotector de la melatonina son parcialmente atribuidos a su acción antioxidante y anti-inflamatoria.
Background: post-cholecystectomy bile duct injuries are identified by the onset of jaundice as we... more Background: post-cholecystectomy bile duct injuries are identified by the onset of jaundice as well as elevated bilirubin and alkaline phosphatase levels during the peri-operative period. It is unknown how serum oxidative stress markers are modified in patients with post-cholecystectomy bile duct injuries.
Objective: To determine the oxidative stress markers in serum from patients with relapsing-remitt... more Objective: To determine the oxidative stress markers in serum from patients with relapsing-remitting multiple sclerosis. Methods: Blood samples from healthy controls and 22 patients 15 women (7 aged from 20 to 30 and 8 were > 40 years old) and 7 men (5 aged from 20 to 30 and 2 were > 40 years old) fulfilling the McDonald Criteria and classified as having Relapsing-Remitting Multiple Sclerosis accordingly with Lublin were collected for oxidative stress markers quantification. Results: Nitric oxide metabolites (nitrates/nitrites), lipid peroxidation products (malondialdehyde plus 4-hidroxialkenals), and glutathione peroxidase activity were significantly increased in serum of subjects with relapsing-remitting multiple sclerosis in comparison with that of healthy controls. These data support the hypothesis that multiple sclerosis is a component closely linked to oxidative stress.
Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis t... more Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn't been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD.
can lead to changes in the synthesis of melatonin. In vitro studies have shown that this hormone ... more can lead to changes in the synthesis of melatonin. In vitro studies have shown that this hormone modulates the efficiency of mitochondrial respiration. Therefore, this work examined whether the efficiency of mitochondrial respiration changes in rats that have been subjected to constant illumination or darkness for a short period. Methods: Rats were randomly distributed in three groups: Control, Constant Illumination (72 hours) and Constant Darkness (72 hours). Upon completion of treatment, rats were sacrificed and mitochondria from the pineal gland, Harderian gland, thymus and spleen were isolated. Subsequently, mitochondrial respiratory control was quantified from the removed tissues in the three experimental groups. Results: Our findings show that brief treatments of continued illumination or continued darkness had no significant effect on mitochondrial respiratory control in spleen, thymus or Harderian glands. In contrast, we observed a slight increase in mitochondrial respiratory control in the pineal gland of animals exposed to constant illumination. Conclusions: Our results suggest that brief treatment with continuous light or darkness does not have a significant effect on the efficiency of mitochondrial activity in spleen, thymus or Harderian gland. This is probably due to the endogenous circadian rhythms that tightly regulate mitochondrial enzymatic activity in these tissues.
Multiple sclerosis (MS) exhibits many of the hallmarks of an inflammatory autoimmune disorder inc... more Multiple sclerosis (MS) exhibits many of the hallmarks of an inflammatory autoimmune disorder including breakdown of the blood-brain barrier (BBB), the recruitment of lymphocytes, microglia, and macrophages to lesion sites, the presence of multiple lesions, generally being more pronounced in the brain stem and spinal cord, the predominantly perivascular location of lesions, the temporal maturation of lesions from inflammation through demyelination, to gliosis and partial remyelination, and the presence of immunoglobulin in the central nervous system and cerebrospinal fluid. Lymphocytes activated in the periphery infiltrate the central nervous system to trigger a local immune response that ultimately damages myelin and axons. Pro-inflammatory cytokines amplify the inflammatory cascade by compromising the BBB, recruiting immune cells from the periphery, and activating resident microglia. inflammation-associated oxidative burst in activated microglia and macrophages plays an important role in the demyelination and free radical-mediated tissue injury in the pathogenesis of MS. The inflammatory environment in demyelinating lesions leads to the generation of oxygen-and nitrogen-free radicals as well as proinflammatory cytokines which contribute to the development and progression of the disease. Inflammation can lead to oxidative stress and vice versa. Thus, oxidative stress and inflammation are involved in a self-perpetuating cycle.
In severe acute pancreatitis (SAP) inflammatory processes foster necrosis, cellular lysis and lib... more In severe acute pancreatitis (SAP) inflammatory processes foster necrosis, cellular lysis and liberation of vasoactive substances associated with multiple organ failure. The effects of vacuum-assisted closure and Drotrecogin alpha on inflammatory cytokines were evaluated in SAP patients with infected necrosis. Methods: Forty-six patients were included in three groups: Group 1, necrosectomy and abdominal cavity washing; Group 2, necrosectomy plus vacuum-assisted closure (VAC), and Group 3, necrossectomy plus VAC plus Drotrecogin alpha. Immunoreactive IL-32, TNF-α, IL-6, TGF-β and IL-2 cytokines were quantified with ELISA method. Results: IL-32 was significantly increased in all patients, predominantly the non-survivor of Group 3 (p < 0.0001). Group 2 maintained increased IL-32 levels throughout. Peak TNF-α was observed in non-survivors of Groups 1 and 2, with a frank tendency to decrease in Group 3. The IL-6 was increased, sustained throughout the study, peaking at the onset in non-survivors. At the end IL-6 tended to diminish, predominantly in survivors. TNF-α and IL-6 were significantly increased on hospitalization, with a maximum peak in non-survivors of all groups. Initial values of TGF-β were significantly increased in survivors of the three groups, and were significantly diminished in non-survivors; affecting pancreas regeneration and favoring sys-temic inflammation, with possible multiple-organ repercussions. IL-2 levels were elevated, predominantly in non-survivors of Group 1. There was positive correlation between the increase IL-32 and TNF-α, and negative correlation between the increase in TNF-α and decrease in TGF-β; and, a tendency for negative correlation between the IL-2 increased and TGF-β levels. Conclusion: We found a generalized, sustained inflammatory state that fosters a torpid outcome in SAP patients.
Introduction: In bile duct injuries (BDI), cholestasis and cholangitis can alter the fibrinolytic... more Introduction: In bile duct injuries (BDI), cholestasis and cholangitis can alter the fibrinolytic system by promoting an increase of extracellular matrix depositions which favor an imbalance between metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Materials and Methods: Levels of PAI-1, MMP-3, MMP-8, TIMP-1 and TIMP-2 in 35 patients with post-cholecystectomy BDI by complete biliary obstruction were measured and compared to a healthy control group. Sirius red staining and immune staining for MMP-3 and MMP-8 were also undertaken in liver biopsies. Results: Levels of PAI-1, TIMP-1, TIMP-2 and MMP-8 were higher in BDI than healthy controls: 15 ± 2 ng/mL vs 7.1 ± 2 ng/mL (p < 0.024); 539 ± 64 ng/mL vs 256 ± 13 ng/mL (p < 0.003); 178.4 ± 79.8 ng/mL vs 98.6 ± 2 ng/mL; and 90 ± 10 ng/mL vs 6.8 ± 2 ng/mL (p < 0.002), respectively. However, serum levels of MMP-3 expression in BDI were decreased 9.0 ± 1 vs. healthy controls 20.3 ± 6 ng/mL (p < 0.001). Morphometric analysis showed that Sirius red staining in BDI was increased 121548.6 ± 20545.4 µm 2 vs. 22865.7 ± 3865 µm 2 in healthy controls (p < 0.0001), MMP-8 deposition in liver was higher in BDI compared to controls: 233015.2 ± 32953.3 µm 2 vs. 30744.2 ± 5810.2 µm 2 (p < 0.0001). MMP-3 liver staining in BDI compared to healthy controls was not significant 130837.9 ± 17,484 µm 2 vs. 116337.9 ± 24803.3 µm 2 (p < 0.55). These results suggest an imbalance between fibrogenic/fibrinolytic protein levels. Interestingly, expression of the fibrinolytic protein MMP-8 was increased in serum and liver biopsies in BDI. Conclusion: We found an imbalance of profibrogenic molecules which promote extracellular matrix deposition. The over-expression of fibrinolytic proteins such as MMP-8 could limit liver fibrosis, preventing hepatic dysfunction in post-cholecystectomy BDI.
Background: Diabetic retinopathy (DR) is a preventable cause of visual disability. The aims of th... more Background: Diabetic retinopathy (DR) is a preventable cause of visual disability. The aims of the present study were to investigate levels and behavior oxidative stress markers and mitochondrial function in non-proliferative DR (NPDR) and to establish the correlation between the severity of NPDR and markers of oxidative stress and mitochondrial function. Methods: In a transverse analysis, type 2 diabetes mellitus (T2DM) patients with mild, moderate and severe non-proliferative DR (NPDR) were evaluated for markers of oxidative stress (i.e. products of lipid peroxidation (LPO) and nitric oxide (NO) catabolites) and antioxidant activity (i.e. total antioxidant capacity (TAC), catalase, and glutathione peroxidase (GPx) activity of erythrocytes). Mitochondrial function was also determined as the fluidity of the submitochondrial particles of platelets and the hydrolytic activity of F0/F1-ATPase. Results: Levels of LPO and NO were significantly increased in T2DM patients with severe NPDR (3.19 ± 0.05 μmol/mL and 45.62 ± 1.27 pmol/mL, respectively; P < 0.007 and P < 0.0001 vs levels in health volunteers, respectively), suggesting the presence of oxidative stress. TAC had significant decrease levels with minimum peak in severe retinopathy with 7.98 ± 0.48 mEq/mL (P < 0.0001). In contrast with TAC, erythrocyte catalase and GPx activity was increased in patients with severe NPDR (139.4 ± 4.4 and 117.13 ± 14.84 U/mg, respectively; P < 0.0001 vs healthy volunteers for both), suggesting an imbalance between oxidants and antioxidants. The fluidity of membrane submitochondrial particles decreased significantly in T2DM patients with mild, moderate, or severe NPDR compared with that in healthy volunteers (P < 0.0001 for all). Furthermore, there was a significant increase in the hydrolytic activity of the F0/F1-ATPase in T2DM patients with mild NPDR (265.07 ± 29.55 nmol/PO4; P < 0.0001 vs healthy volunteers), suggesting increased catabolism. Conclusions: Patients with NPDR exhibit oxidative deregulation with decreased membrane fluidity of submitochondrial particles and increased systemic catabolism (mitochondrial dysfunction) with the potential for generalized systemic damage in T2DM.
Background. Dementia affects memory, thinking, language, judgment, and behavior. Depression, is c... more Background. Dementia affects memory, thinking, language, judgment, and behavior. Depression, is common in older adults with dementia. The concomitance of dementia and depression increases disability with impaired activities of daily living (ADL), increasing the chances of institutionalization and mortality. Methods. Cross-sectional study of a population 60 years and older who live in the State of Jalisco, Mexico. A total of 1142 persons were assessed regarding their cognitive function, emotional state, and physical performance. Door-to-door interview technique was assigned in condition with multistage probability random sampling. Cognitive function, depression and functional disability were assessed by applying standardized Minimental State Examination (Folstein), Geriatric Depression Scale, and the Katz index, respectively. Diagnosis of dementia was performed according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders, the Fourth Edition. Data were analyzed using SPSS software. Results. Prevalence of demency was 9.5% (63.35% women, and 36.7% men). Demency was associated with being woman, being older than 70 years, low level of education, not having the economic benefit of retirement, being single or living without a partner, low level of education, suffering from depression and have functional disability in ADL. Conclusion. Dementia is more common in women and is related to depression and disability.
Objective: To evaluate and compare the intake of lipids and (A, E, and C) vitamins in patients wi... more Objective: To evaluate and compare the intake of lipids and (A, E, and C) vitamins in patients with and without possible neurodegenerative diseases. Methods: Twenty adults with possible Alzheimer's disease or Parkinson's disease and 41 control subjects (50-89 years old) from a rural region were studied. Dietary intake was evaluated with the analysis of macronutrients and micronutrients conducted by a food frequency questionnaire and 24 hours dietary record. Analyses were adjusted for age, sex, body mass index, and energy intake. Through interrogation and use of medical record form of health secretary we obtained information about the sociodemographic characteristics. Multivariate analysis of variance to allow for covariated adjustment was used. Results: Patients had a lower energy intake, vitamin C (P = 0.016), fruits (P < 0.001), vegetables (P = 0.037), and oils and fat (P = 0.002), than the controls. Interestingly, the C vitamin intake in patients was still higher than the recommended. Patients had a higher consumption of cereals (P = 0.017), high-animal fat diet (P = 0.024), and whole milk (P < 0.001); 2.4% of the controls smoke and 5% are alcohol consumers. Eighty-five percent of patients and 78% of the controls do not have physical activity. Family history of subjects in this study indicated chronic diseases. Conclusion: The subjects included in this study had a high intake of C vitamin, this is due to the consumption of fruits and vegetables. However, patients with possible Alzheimer's or Parkinson's disease had a lower intake of fruits and vegetables, which could be due to type of food to which they have access.
Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracocc... more Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied. Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin. Among the oxyanions, sulfite induced the higher increase in ATPase activity. Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the V max . There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme. Dithiothreitol did not affect the ATPase activity. ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin. In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin. It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit : is differently exposed to trypsin.
Maize (Zea mays 1.) embryos, isolated from the developing seed and incubated in dilute buffer, sh... more Maize (Zea mays 1.) embryos, isolated from the developing seed and incubated in dilute buffer, show reduced triacylglycerol (TAC) synthesis, and accumulation stops after 24 h. Synthesis and accumulation can be maintained at high levels if the incubation medium contains abscisic acid (ABA) and/or a high osmotic concentration. Radiolabeled free fatty acids accumulate at higher levels in embryos that contain less TAC, and acetyl coenzyme A carboxylase activity remains essentially unchanged under all of the conditions tested. In contrast, the activities of the acyltransferases required for TAC synthesis remain high only in embryos incubated with ABA and/or a high osmotic concentration. Dose-response curves showed that 4 p~ of ABA or mannitol at -1.0 MPa elicits a full response; both values are within the range considered to be physiological. The TAC synthesis capacity and diacylglycerol acyltransferase activity lost by pretreatment of the embryos can be restored by re-exposure to ABA or high osmoticum. Cermination is not involved because isolated scutellum halves showed the same changes in enzyme activity found in the whole embryo but did not germinate. Our results provide direct evidence for the regulation of TAC-synthesizing activities in maize embryos by ABA and the osmotic potential of the environment.
Isolation of subcellular fractions from dry structures such as seeds or their tissues is difficul... more Isolation of subcellular fractions from dry structures such as seeds or their tissues is difficult. In the present work, plasma membranes were isolated from dry maize (Zea mays L.) embryos with an enrichment of 11-fold as estimated by glucan synthase II (GSII, EC 2.4.1.34) activity and a purity of 78 to 90% as judged by the sensitivity of ATP hydrolysis to vanadate, a specific inhibitor of the plasma membrane H+-ATPase (EC 3.6.1.35). The procedure involved a double homogenization of the dry embryos and the addition of a 1500-g supernatant to an aqueous polyethyleneglycol-dextran two-phase partitioning system; the optimal ratio of polyethyleneglycol-dextran for purification of plasma membranes from dry seeds was 6.8/6.8% (w/w). In the isolated membranes a trace of a tonoplast enzyme marker (tonoplast H+-ATPase, EC 3.6.1.3) could be detected, but there were negligible amounts of mitochondrial and rough endoplasmic reticulum markers, H+-ATPase (EC 3.6.1.34) and diacylglycerol acyltrans-ferase (EC 2.3.1.20), respectively. The technique could also be used in hydrated embryos. The entire procedure can be carried out in 5 to 6 h. The resulting preparation is stable for at least 2 months at −70°C. The membranes of dry and hydrated embryos exhibited a high level of vanadate-sensitive ATPase activity that was increased by lysophosphatidylcholine.
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