A modern, simple, and economical accessory for continuous L-format measurement of steady-state fluorescence anisotropy.

In this study, the pros and cons of the most relevant L-format devices reported in the literature for measuring steady-state fluorescence polarization/anisotropy are identified. Combining all this information, and with the use of modern elements for the acquisition, treatment, and recording of signals, a modern, simple, and economical L-format accessory is implemented to rapidly and continuously record steady-state fluorescence anisotropy. This device can be adapted to the majority of the commercial spectrofluorometers (or fluorometers). During the measurement, the emission polarizer is in permanent rotation by means of a Gimbal brushless DC motor, and as a result the recorded fluorescence signal is sinusoidal. The maximums and minimums of this signal, which are obtained with the help of LabVIEW tools, allow recording the fluorescence anisotropy. The LabVIEW applications developed for this investigation are freely available, so it is not necessary to have LabVIEW software.

Important methodological aspects that should be taken into account during the research of isolated mitochondria.

Isolated mitochondria have been widely used for the study of energetic functioning of these important cellular organelles in a physiological or pathophysiological state. This is due, on the one part, to the fact that isolated mitochondria are relatively easy to isolate from a great variety of animal tissues, and on the other part to the fact that their energetic functioning is relatively easy to study experimentally. Nevertheless, they have a great disadvantage because of the fact that these biological structures can easily undergo structural-functional changes during their in vitro handling, causing many conclusions reported in the research of isolated mitochondria to be merely the fruit of experimental artifacts. The present review describes a series of important methodological aspects that should be taken into account in order to obtain reliable results in the study of the energetic functioning (and of other aspects) of isolated mitochondria.

Early hyperglycemia following alloxan administration in vivo is not associated with altered hepatic mitochondrial function: acceptable model for type 1 diabetes?

Alloxan and oxidative stress, which have been detected in livers of laboratory animals shortly after in vivo alloxan administration, cause in vitro mitochondrial dysfunction, thus questioning alloxan diabetes as an acceptable model for type 1 diabetes, a model that cannot legitimately be used to investigate mitochondrial metabolism in a diabetic state. In the current study, the blood glucose concentration increased in the drug-treated group of Sprague-Dawley rats (compared with the placebo group) 45 or 60 min after alloxan treatment, whereas at 30 min the blood glucose concentration was unchanged. State 4, state 3, respiratory control, efficiency of oxidative phosphorylation, and mitochondrial ATP synthase activity, assayed using glutamate plus malate, pyruvate plus malate, or succinate as a substrate, were not negatively altered during the entire study. These results indicated that early increases of blood glucose concentration, after in vivo alloxan administration, did not lead to liver mitochondrial dysfunction, suggesting that alloxan diabetes can be used for the study of liver mitochondrial respiration in a diabetic state.

The Bioenergetics of Isolated Mitochondria from Different Animal Models for Diabetes.

Diabetes is a metabolic alteration characterized by a higher than normal blood glucose level. For the experimental study of the metabolic changes that occur during this illness, various animal models have been introduced: alloxan- and streptozotocin-injected animals, as well as depancreatized animals, as models for type 1 diabetes, and high-fat fed diabetic animals and laboratory animals with genetic diabetes as models for type 2 diabetes. All these models have been used to investigate specific events on the cellular and organ levels that occur as a consequence of diabetes. In particular, mitochondrial energy metabolism has been extensively studied using these experimental models for diabetes. The experimental results for the bioenergetics of isolated mitochondria harvested from different animal models for diabetes, with the exception of those obtained with high-fat fed diabetic animals, are conflicting; nevertheless, many researchers now consider mitochondrial energy dysfunction as one of the direct causes of the serious complications, in various organs and tissues, that are exhibited as a result of this illness. For this reason, it is important that future research clarify the true energy functional state of these organelles isolated from diabetic animals. In the present paper, the published data on this controversial but important issue of the energetic functioning of the mitochondria isolated from diabetic animals is reviewed. This paper also includes commentary on the status of current research and makes useful suggestions for the future direction of research on this topic.

Dextran causes aggregation of mitochondria and influences their oxidoreductase activities and light scattering.

It has been reported that dextrans diminish the intermembrane space of mitochondria, increase the number of contact sites between the inner and the outer mitochondrial membranes, decrease the outer membrane permeability to adenosine 5(')-diphosphate, and change the kinetic properties of mitochondrial kinases. In the present work the influence of dextran M40 (5% w/v) on the oxidoreductase activities of the inner and outer membranes of mitochondria, the interaction of cytochrome c with mitochondrial membranes, and the light scattering by rat liver mitochondria were studied. No influence of dextran on the release of cytochrome c from mitochondria or its interaction with mitochondrial membranes was observed. Decreases in the NADH-oxidase (to 80+/-2% of the control), NADH-cytochrome c reductase (to 26+/-2%), succinate-cytochrome c reductase (to 70+/-5%), and NADH-ferricyanide reductase (to 75+/-3%) activities induced by dextran, which may be due to the mitochondrial aggregation, were observed. The formation of aggregates was registered by light scattering, confirmed by light microscopy, and explained within the framework of the Gouy-Chapman theory of the electrical double layer. The observed mitochondrial aggregation seems to be useful also for understanding the mechanisms of mitochondrial condensation and perinuclear clustering during apoptosis.

Mitochondrial oligomycin-sensitive ATPase during isoproterenol-induced cell injury of myocardium

En el presente trabajo se investigó la actividad enzimática de la ATPasa mitocondrial sensible a la oligomicina durante una lesión celular del miocardio inducida por el isoproterenol, usando homogenados de corazón de rata y una técnica potenciométrica. La actividad enzimática de la ATPasa mitocondrial sensible a la oligomicina, al igual que la acción inhibidora de la oligomicina sobre esta enzima, no muestra alteraciones significativas durante el tratamiento con isoproterenol. Estos resultados no concuerdan con la hipótesis sobre posibles modificaciones en la configuración activa de la ATPasa mitocondrial durante una lesión celular del miocardio inducida por el isoproterenol.

Activation of mitochondrial oxidative phosphorylation during (+/-)-isoproterenol-induced cell injury of myocardium

En el presente trabajo se investigaron las actividades hidrolítica y sintetizadora de la ATPasa mitocondrial durante una lesión celular del miocardio inducida por el isoproterenol (67 mg/kg de peso corporal). La investigación fue un estudio secuencial (72 h), en la cual fueron usados homogenados de corazón de rata y una técnica potenciométrica. Las actividades hidrolíticas en homogenados de ratas tratadas con (+/-)-isoproterenol fueron esta-dísticamente iguales, durante todo el estudio secuencial, a la actividad hidrolítica en homo-genados normales. La actividad sintetizadora de la ATPasa mitochondrial (fosforilación oxidativa mitocondrial) aumentó a las 3, 6 y 18 h (35, 48 y 23 por ciento, respectivamente) con respecto al control después de la administración de (+/-)-isoproterenol. A las 12 h y 21-72 h, después de la administración de la droga, los datos no muestran diferencia entre la actividad sintetizadora de la ATPasa mitocondrial en homogenados de ratas tratadas con (+/-)-isoproterenol y la actividad sintetizadora en homogenados normales. Los hechos de que las actividades sintetizadoras e hidrolíticas de la ATPasa mitocondrial, en homogenados de ratas tratadas con (+/-)-isoproterenol, nunca fueron más bajas que las actividades sintetizadora e hidrolítica en homogenados normales, y de que una activación de la fosforilación oxidativa mitocondrial ocurre a algunos tiempos después del tratamiento con (+/-)-isoproterenol indican que es posible que no tengan lugar modificaciones considerables y "negativas" en la configuración activa de la ATPasa mitocondrial durante una lesión celular del miocardio inducida por el (+/-)-isoproterenol (67 mg/kg de peso corporal).