The heat shock protein 60 promotes progesterone synthesis in mitochondria of JEG-3 cells.

Progesterone synthesis in human placenta is essential to maintain pregnancy. The limiting step in placental progesterone synthesis is cholesterol transport from the cytoplasm to the inner mitochondrial membrane. Multiple proteins located in mitochondrial contact sites seem to play a key role in this process. Previously, our group identified the heat shock protein 60 (HSP60) as part of mitochondrial contact sites in human placenta, suggesting its participation in progesterone synthesis. Here, we examined the role of HSP60 in progesterone synthesis. Our results show that over-expression of HSP60 in human placental choriocarcinoma cells (JEG-3) and human embryonic kidney 293 cells (HEK293) promotes progesterone synthesis. Furthermore, incubation of the HSP60 recombinant protein with intact isolated mitochondria from JEG-3 cells also promotes progesterone synthesis in a dose-related fashion. We also show that HSP60 interacts with STARD3 and P450scc proteins from mitochondrial membrane contact sites. Finally, we show that the HSP60 recombinant protein binds cholesterol. Ours results demonstrate that HSP60 participates in mitochondrial progesterone synthesis. These findings provide novel insights into progesterone synthesis in the human placenta and its role in maintaining pregnancy.

Recollection and familiarity for words and faces: a study comparing Remember-Know judgements and the Process Dissociation Procedure.

Measures of recollection and familiarity often differ depending on the paradigm utilised. Remember-Know (R-K) and Process Dissociation Procedure (PDP) methods have been commonly used but rarely compared within a single study. In the current experiments, R-K and PDP were compared by examining the effect of attention at study and time to respond at test on recollection and familiarity using the same experimental procedures for each paradigm. We also included faces in addition to words to test the generality of the findings often obtained using words. The results from the R-K paradigm revealed that recollection and familiarity were similarly affected by attention at study and time to respond at test. However, in the case of PDP, the measures of recollection and familiarity showed a different pattern of results. The effects observed for recollection were similar to those obtained with the R-K method, whereas familiarity was affected by time to respond but not by attention at study. These results are discussed in relation to the controlled-automatic processing distinction and the contribution of each paradigm to research on recognition memory.

Even a Chronic Mild Hyperglycemia Affects Membrane Fluidity and Lipoperoxidation in Placental Mitochondria in Wistar Rats.

It is known the deleterious effects of diabetes on embryos, but the effects of diabetes on placenta and its mitochondria are still not well known. In this work we generated a mild hyperglycemia model in female wistar rats by intraperitoneal injection of streptozotocin in 48 hours-old rats. The sexual maturity onset of the female rats was delayed around 6-7 weeks and at 16 weeks-old they were mated, and sacrificed at day 19th of pregnancy. In placental total tissue and isolated mitochondria, the fatty acids composition was analyzed by gas chromatography, and lipoperoxidation was measured by thiobarbituric acid reactive substances. Membrane fluidity in mitochondria was measured with the excimer forming probe dipyrenylpropane and mitochondrial function was measured with a Clark-type electrode. The results show that even a chronic mild hyperglycemia increases lipoperoxidation and decreases mitochondrial function in placenta. Simultaneously, placental fatty acids metabolism in total tissue is modified but in a different way than in placental mitochondria. Whereas the chronic mild hyperglycemia induced a decrease in unsaturated to saturated fatty acids ratio (U/S) in placental total tissue, the ratio increased in placental mitochondria. The measurements of membrane fluidity showed that fluidity of placenta mitochondrial membranes increased with hyperglycemia, showing consistency with the fatty acids composition through the U/S index. The thermotropic characteristics of mitochondrial membranes were changed, showing lower transition temperature and activation energies. All of these data together demonstrate that even a chronic mild hyperglycemia during pregnancy of early reproductive Wistar rats, generates an increment of lipoperoxidation, an increase of placental mitochondrial membrane fluidity apparently derived from changes in fatty acids composition and consequently, mitochondrial malfunction.

Evaluación de la equivalencia de tres versiones paralelas del Fototest / Evaluation of the equivalence of three parallel versions of the Phototest

Introducción: el Fototest es un test cognitivo muy breve y aplicable a analfabetos que se ha mostrado válido y coste- efectivo en la detección del deterioro cognitivo y la demencia y en el seguimiento de estos pacientes. Las aplicaciones repetidas de un test cognitivo pueden inducir mejorías en el rendimiento debido al fenómeno «efecto de la práctica »; este efecto puede minimizarse con el uso de formas paralelas. Nuestro objetivo es evaluar la equivalencia de tres versiones paralelas del Fototest. Material y métodos: estudio transversal en una muestra de conveniencia; los sujetos se distribuyeron aleatoriamente en tres grupos a los que se aplicó respectivamente la versión original (Fototest- 1) y dos versiones paralelas (Fototest-2 y Fototest-3) del Fototest, que diferían en los objetos por denominar y recordar. Análisis estadístico: estadística descriptiva univariada y comparación entre grupos con ANOVA o χ2 según tipo de variables. Resultados: 223 sujetos (65,3 % mujeres) con una edad de 58,0 ± 16,8 (media ± DE) años, distribuidos aleatoriamente en tres grupos de 75 (Fototest-1), 76 (Fototest-2) y 72 (Fototest-3) sujetos; estos grupos no diferían entre sí en edad, sexo o nivel educativo. No hay diferencias significativas entre los grupos en las puntuaciones parciales (denominación, fluencia hombres, fluencia mujeres, recuerdo libre y recuerdo facilitado), subtotales (fluencia total, recuerdo total) y total del Fototest (Fototest-1: 37,8 ± 5,6, Fototest-2: 36,8 ± 7,5, Fototest-3: 37,4 ± 5,8; p = 0,66). Discusión: las tres versiones del Fototest son equivalentes e intercambiables, lo que puede facilitar la labor del explorador y contrarrestar el «efecto de la práctica » asociado al uso repetido (AU)

Introduction: The Phototest is a very short cognitive test that is applicable to illiterates and has proven to be valid and cost-effective for the detection of cognitive impairment and dementia and for the follow-up of these patients. Repeated applications of a cognitive test may induce improvements in performance due to «practice effects», which can be minimized by the use of parallel forms. Our objective was to evaluate the equivalence of three parallel versions of the Phototest. Material and Methods: Cross-sectional study of a convenience sample; participants were randomly distributed into three groups for the respective application of the original version of the Phototest (Phototest-1) and two parallel versions (Phototest-2 and Phototest-3), which differ in the objects to be named and recalled. Statistical analysis: univariate descriptive statistics and comparison among groups using ANOVA or the chi-square test according to the type of variable. Results: 223 participants (65.3% females) with a mean±SD of 58.0±16.8 yrs were randomly distributed among three groups of 75 (Phototest-1), 76 (Phototest-2) and 72 (Phototest-3) participants, with no significant inter-group differences in age, sex, or educational level. The groups did not significantly differ in partial Phototest scores (naming, fluency of males, fluency of females, free recall or cued recall), subtotal scores (total fluency, total recall) or total scores (Phototest-1: 37.8±5.6, Fototest-2: 36.8±7.5, Fototest-3: 37.4±5.8; p=0.66). Discussion: The three versions of Phototest are equivalent and interchangeable, which can assist the work of the examiner and counteract the 'practice effects' associated with repeated applications (AU)

Mitochondrial proteases act on STARD3 to activate progesterone synthesis in human syncytiotrophoblast.

BACKGROUND: STARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied. METHODS: Progesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondrial proteases cleave STARD3 into a 28-kDa fragment that stimulates progesterone synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis. RESULTS: STARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in progesterone synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22(R)-hydroxycholesterol increased progesterone synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport. CONCLUSION: Metalloproteases from human placental mitochondria are involved in steroidogenesis through the proteolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding. GENERAL SIGNIFICANCE: Mitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in progesterone synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells.

Coenzyme Q10 supplementation improves metabolic parameters, liver function and mitochondrial respiration in rats with high doses of atorvastatin and a cholesterol-rich diet.

BACKGROUND: The aim of this study was to evaluate the actions of coenzyme Q10 (CoQ10) on rats with a cholesterol-rich diet (HD) and high doses of atorvastatin (ATV, 0.2, 0.56 or 1.42 mg/day). METHODS: Two experiments were done, the first one without coenzyme Q10 supplementation. On the second experiment all groups received coenzyme Q10 0.57 mg/day as supplement. After a 6-week treatment animals were sacrificed, blood and liver were analyzed and liver mitochondria were isolated and its oxygen consumption was evaluated in state 3 (phosphorylating state) and state 4 (resting state) in order to calculate the respiratory control (RC). RESULTS: HD increased serum and hepatic cholesterol levels in rats with or without CoQ10. ATV reduced these values but CoQ10 improved even more serum and liver cholesterol. Triacylglycerols (TAG) were also lower in blood and liver of rats with ATV + CoQ10. HDL-C decreased in HD rats. Treatment with ATV maintained HDL-C levels. However, these values were lower in HD + CoQ10 compared to control diet (CD) + CoQ10. RC was lessened in liver mitochondria of HD. The administration of ATV increased RC. All groups supplemented with CoQ10 showed an increment in RC. In conclusion, the combined administration of ATV and CoQ10 improved biochemical parameters, liver function and mitochondrial respiration in hypercholesterolemic rats. CONCLUSIONS: Our results suggest a potential beneficial effect of CoQ10 supplementation in hypercholesterolemic rats that also receive atorvastatin. This beneficial effect of CoQ10 must be combined with statin treatment in patient with high levels of cholesterol.

Diagnostic accuracy, effectiveness and cost for cognitive impairment and dementia screening of three short cognitive tests applicable to illiterates.

BACKGROUND: Illiteracy, a universal problem, limits the utilization of the most widely used short cognitive tests. Our objective was to assess and compare the effectiveness and cost for cognitive impairment (CI) and dementia (DEM) screening of three short cognitive tests applicable to illiterates. METHODS: Phase III diagnostic test evaluation study was performed during one year in four Primary Care centers, prospectively including individuals with suspicion of CI or DEM. All underwent the Eurotest, Memory Alteration Test (M@T), and Phototest, applied in a balanced manner. Clinical, functional, and cognitive studies were independently performed in a blinded fashion in a Cognitive Behavioral Neurology Unit, and the gold standard diagnosis was established by consensus of expert neurologists on the basis of these results. Effectiveness of tests was assessed as the proportion of correct diagnoses (diagnostic accuracy [DA]) and the kappa index of concordance (k) with respect to gold standard diagnoses. Costs were based on public prices at the time and hospital accounts. RESULTS: The study included 139 individuals: 47 with DEM, 36 with CI, and 56 without CI. No significant differences in effectiveness were found among the tests. For DEM screening: Eurotest (k = 0.71 [0.59-0.83], DA = 0.87 [0.80-0.92]), M@T (k = 0.72 [0.60-0.84], DA = 0.87 [0.80-0.92]), Phototest (k = 0.70 [0.57-0.82], DA = 0.86 [0.79-0.91]). For CI screening: Eurotest (k = 0.67 [0.55-0.79]; DA = 0.83 [0.76-0.89]), M@T (k = 0.52 [0.37-0.67]; DA = 0.80 [0.72-0.86]), Phototest (k = 0.59 [0.46-0.72]; DA = 0.79 [0.71-0.86]). There were no differences in the cost of DEM screening, but the cost of CI screening was significantly higher with M@T (330.7 ± 177.1 €, mean ± sd) than with Eurotest (294.1 ± 195.0 €) or Phototest (296.0 ± 196. 5 €). Application time was shorter with Phototest (2.8 ± 0.8 min) than with Eurotest (7.1 ± 1.8 min) or M@T (6.8 ± 2.2 min). CONCLUSIONS: Eurotest, M@T, and Phototest are equally effective. Eurotest and Phototest are both less expensive options but Phototest is the most efficient, requiring the shortest application time.

Effectiveness and costs of phototest in dementia and cognitive impairment screening.

BACKGROUND: To assess and compare the effectiveness and costs of Phototest, Mini Mental State Examination (MMSE), and Memory Impairment Screen (MIS) to screen for dementia (DEM) and cognitive impairment (CI). METHODS: A phase III study was conducted over one year in consecutive patients with suspicion of CI or DEM at four Primary Care (PC) centers. After undergoing all screening tests at the PC center, participants were extensively evaluated by researchers blinded to screening test results in a Cognitive-Behavioral Neurology Unit (CBNU). The gold standard diagnosis was established by consensus of expert neurologists. Effectiveness was assessed by the proportion of correct diagnoses (diagnostic accuracy [DA]) and by the kappa index of concordance between test results and gold standard diagnoses. Costs were based on public prices and hospital accounts. RESULTS: The study included 140 subjects (48 with DEM, 37 with CI without DEM, and 55 without CI). The MIS could not be applied to 23 illiterate subjects (16.4%). For DEM, the maximum effectiveness of the MMSE was obtained with different cutoff points as a function of educational level [k = 0.31 (95% Confidence interval [95%CI], 0.19-0.43), DA = 0.60 (95%CI, 0.52-0.68)], and that of the MIS with a cutoff of 3/4 [k = 0.63 (95%CI, 0.48-0.78), DA = 0.83 (95%CI, 0.80-0.92)]. Effectiveness of the Phototest [k = 0.71 (95%CI, 0.59-0.83), DA = 0.87 (95%CI, 0.80-0.92)] was similar to that of the MIS and higher than that of the MMSE. Costs were higher with MMSE (275.9 ± 193.3€ [mean ± sd euros]) than with Phototest (208.2 ± 196.8€) or MIS (201.3 ± 193.4€), whose costs did not significantly differ. For CI, the effectiveness did not significantly differ between MIS [k = 0.59 (95%CI, 0.45-0.74), DA = 0.79 (95%CI, 0.64-0.97)] and Phototest [k = 0.58 (95%CI, 0.45-0.74), DA = 0.78 (95%CI, 0.64-0.95)] and was lowest for the MMSE [k = 0.27 (95%CI, 0.09-0.45), DA = 0.69 (95%CI, 0.56-0.84)]. Costs were higher for MMSE (393.4 ± 121.8€) than for Phototest (287.0 ± 197.4€) or MIS (300.1 ± 165.6€), whose costs did not significantly differ. CONCLUSION: MMSE is not an effective instrument in our setting. For both DEM and CI, the Phototest and MIS are more effective and less costly, with no difference between them. However, MIS could not be applied to the appreciable percentage of our population who were illiterate.

Steroidogenesis in BeWo cells: role of protein kinase A and benzodiazepines.

The peripheral benzodiazepine receptor and protein kinase A have been proposed to modulate placental steroidogenesis. Binding of the radioactive benzodiazepine PK 11195 has been observed in membranes isolated from whole human placenta, but the presence of the peripheral benzodiazepine receptors, now called translocator protein, does not seem to be indispensable. We hypothesized that cAMP analogs could induce the translocator protein expression in BeWo cells increasing steroidogenesis in the presence of benzodiazepines. The effect of two benzodiazepines and of 8-Br-cAMP on steroidogenesis in BeWo cells or in isolated human placental mitochondria was studied. Benzodiazepines did not modify progesterone synthesis in either system. Progesterone increased three times in BeWo cells incubated in the presence of 8-Br-cAMP. The translocator protein was not identified by western blot in mitochondria isolated from either the human placenta or BeWo cells but it was present in isolated rat testicular mitochondria. Neither was it observed in isolated mitochondria from BeWo cells incubated with 8-Br-cAMP. An inhibitor of protein kinase A activity, H89, at 25 microM inhibited 90% the steroidogenesis in BeWo cells, even in the presence of 8-Br-cAMP, but protein phosphorylation in mitochondria increased in the presence of H89, suggesting that protein kinase A modulates the phosphorylation cycle of mitochondrial proteins. The results suggest that placental steroidogenesis is regulated via activation of protein kinase A modulated by cAMP.

Metabolismo energético y esteroidogénico de la plancenta humana / Energetic and steroidogenic metabolism at the human placenta

Las células del sinciciotrofoblasto obtienen el ATP a través de la glucólisis anaerobia. Sin embargo, aunque las mitocondrias de la placenta sintetizan ATP, éste no participa en los procesos citoplasmáticos. Nuestros datos muestran la presencia de una ATP-difosfohidrolsa (apirasa) asociada a las mitocondrias de la placenta, que se inhibe por vanadato y FSBA. En este trabajo proponemos la hipótesis de que la apirasa y el ATP que sintetizan las mitocondrias de las células del sinciciotrofoblasto están asociados al transporte de colesterol necesario para la síntesis de progesterona, y que el uso del ATP y la actividad de la apirasa están asociados a los puntos de unión mitocondriales.

El colesterol es esencial en el desarrollo embrionario y en el crecimiento celular / Cholesterol as an essential factor for embryonic and cellular growth

El colesterol es indispensable para el desarrollo y crecimiento celular. En varios organismos, como en los insectos, el colesterol se considera una vitamina, ya que debe ser ingerido en los alimentos. En Drosophila, el colesterol es esencial para la embriogénesis y en su ausencia estas moscas no desarrollan las alas. Esto se debe a la interacción entre el colesterol y las proteínas hedgehog. También es necesaria la presencia de colesterol en la regulación de varias enzimas, sobre todo en las que están en relación con su síntesis. Finalmente, se ha observado que algunos intermediarios de la síntesis del colesterol modulan la velocidad del ciclo celular al interactuar con el DNA nuclear y, al parecer, esta asociación también está relacionada con los procesos cancerígenos.