[Orexin A as mediator in the gut-brain dialogue]. / Orexina A como mediadora en el diálogo intestino-cerebro.

INTRODUCTION: The orexinergic system is one of the chemical mediators that modulate the gut-brain axis, given the involvement of hypothalamic orexin A (OXA) in gastrointestinal motility and secretion, and the presence of OXA in enteroendocrine cells of the intestinal mucosa and in primary afferent neurons of the mesenteric plexus, permitting its participation in gut-brain signaling. AIM: The source of OXA and the signal(s) triggering its peripheral release are not fully understood, and it is not known whether it acts on orexigenic receptors in peripheral tissues to meet physiological or pathological demands. The aim of this review is to address these questions in the light of new data indicating that OXA may have functions in the gut-brain axis that go beyond its participation in energy homeostasis. DEVELOPMENT: OXA in the enteric system protects against systemic and central inflammation, and hypothalamic OXA orchestrates numerous peripheral effects to suppress the systemic inflammatory response. For this reason, OXA may act as an immunomodulator in chronic inflammations or autoimmune diseases. OXA is also involved in the stress response, regulating physiological responses to emotional or stressful stimuli. CONCLUSIONS: OXA exerts anti-inflammatory and gastroprotective effects on the intestinal mucosa; however, it may increase the response to external and/or internal stress in individuals with chronic inflammation, exacerbating the gastrointestinal inflammation. Hence, pharmacologic interventions in the orexinergic system have been proposed to treat diseases in which intestinal hypersensitivity is combined with appetite loss, sleep disturbance, stress, and anxiety.

TITLE: Orexina A como mediadora en el diálogo intestino-cerebro.Introducción. Entre los mediadores químicos que modulan el eje intestino-cerebro debe incluirse el sistema orexinérgico, ya que la orexina A (OXA) hipotalámica interviene en la motilidad y en la secreción gastrointestinal. También está presente en las células enteroendocrinas de la mucosa intestinal y en las neuronas aferentes primarias del plexo mientérico, y puede intervenir en la señalización intestino-cerebro. Objetivo. No se conoce con exactitud la fuente ni la señal que originan la liberación de OXA periférica, ni tampoco si actúa en los receptores orexinérgicos de los tejidos periféricos ante demandas fisiológicas o patológicas. Esta revisión intenta analizar estas cuestiones a la luz de nuevos datos que indican que la OXA en el eje intestino-cerebro puede tener funciones más allá de su participación en la homeostasis energética. Desarrollo. La OXA en el sistema entérico protege de la inflamación sistémica y central, y en el hipotálamo orquesta numerosos efectos periféricos para suprimir la respuesta inflamatoria sistémica. Por ello, podría actuar como sustancia inmunomoduladora en inflamaciones crónicas o en enfermedades autoinmunitarias. La OXA también se relaciona con la respuesta de estrés, regulando las respuestas fisiológicas a estímulos emocionales o estresantes. Conclusiones. Aunque la OXA tiene efectos antiinflamatorios y gastroprotectores de la mucosa intestinal, en procesos de inflamación crónica podría incrementar la respuesta a estímulos estresantes, tanto externos como internos, y exacerbar la inflamación gastrointestinal. Por ello, se han propuesto intervenciones farmacológicas sobre el sistema orexinérgico como tratamiento para enfermedades en las que la hipersensibilidad intestinal coexiste con pérdida de apetito, alteraciones del sueño, estrés y ansiedad.

Modeling high pressure reactivity in unsaturated systems: application to dimethylacetylene.

A general model is introduced to study pressure-induced reactivity on unsaturated systems in the condensed state. The model is applied here to dimethylacetylene (DMA) in the solid phase II (C/2m) because it has been proposed that two DMA molecules can react to form tetramethyl-cyclobutadiene (TMCBD). The proposed reaction process has been modeled by studying the structural and electronic changes undergone by two DMA molecules as they approach each other preserving the crystal symmetry of phase II. Both monodeterminantal (MP2 and DFT) and multideterminantal (CASSCF and MRMP2) methodologies were used to check the reliability of our model in predicting the reactivity of the system under compression. In all cases, structural results are in agreement with low-temperature diffraction experiments for the solid phase II. Our model indicates that DMA is expected to form the TMCBD dimer at intermolecular distances close to 2 A. This value is in excellent agreement with previous calculations on the existence of long carbon-carbon bonds.

Effect of growth temperature and pH on the aminopeptidase activity of Pseudomonas putida, P. fluorescens and Flavobacterium odoratum; the 4-nitroaniline test is reliable.

No significant difference (p > 0.05) was observed in the specific aminopeptidase activity (SAA) developed by Pseudomonas fluorescens, P. putida and Flavobacterium odoratum either growing at pH 5.0-6.5 or at 7 and 12 degrees C. Nevertheless, a significant difference was found when comparing the SAA of these organisms. The SAA of F. odoratum was lower than those of pseudomonads. The 4-nitroaniline test is reliable to estimate the G(-) load of fresh food products.

Evaluation of the foetal time to death in mice after application of direct and indirect euthanasia methods.

Directive 2010/63/EU on the protection of animals used for scientific purposes requires that the killing of mammal foetuses during the last third of their gestational period should be accomplished through effective and humane methods. The fact that murine foetuses are resistant to hypoxia-mediated euthanasia renders the current euthanasia methods ineffective or humane for the foetuses when these methods are applied to pregnant female mice. We have assessed the time to death of foetuses after performing either indirect (dam euthanasia) or direct (via intraplacental injection--a new approach to euthanasia) euthanasia methods in order to determine a euthanasia method that is appropriate, ethical and efficient for the killing of mouse foetuses. The respective times to death of foetuses after performing the three most commonly used euthanasia methods (namely cervical dislocation, CO2inhalation and intraperitoneal sodium pentobarbital administration) were recorded. Absence of foetal heartbeat was monitored via ultrasound. We consider that the most effective and humane method of foetal euthanasia was the one able to achieve foetal death within the shortest possible period of time. Among the indirect euthanasia methods assessed, the administration of a sodium pentobarbital overdose to pregnant female mice was found to be the fastest for foetuses, with an average post-treatment foetal death of approximately 29.8 min. As for the direct euthanasia method assessed, foetal time to death after intraplacental injection of sodium pentobarbital was approximately 14 min. Significant differences among the different mouse strains employed were found. Based on the results obtained in our study, we consider that the administration of a sodium pentobarbital overdose by intraplacental injection to be an effective euthanasia method for murine foetuses.

Changes in adult rat liver mitochondrial populations at different energy states analyzed by flow cytometry.

The present work studies the changes in green fluorescence intensity after Rh-123 staining of the low (LFP) and the high fluorescence populations (HFP) in isolated mitochondria from rat liver. The results show that the HFP represents a mitochondrial compartment less sensitive to changes in energy states. In addition, it is concluded that the use of Rh-123 to monitor changes in mitochondrial membrane potential should be undertaken with caution because, under certain circumstances, there is no correlation between the Rh-123 intensity of fluorescence due to its uptake by mitochondria and previously reported changes in the mitochondrial membrane potential.

Variations of muscle mitochondrial creatine kinase activity in mitochondrial diseases.

Mitochondrial creatine kinase (mtCK) activity has been measured in the mitochondria isolated from the muscle of 69 patients suspected of mitochondrial diseases. The isolated mitochondria did not contain significant amounts of the muscle isoform of creatine kinase, as checked by an immunoassay performed after electrophoretic separation of the various isoforms. Hence, the enzyme assay reliably represented the mtCK activity. Therefore, a simple measurement of CK activity in isolated mitochondria permitted the measurement of mtCK activity. An absence of mtCK activity in muscle was never observed. The lowest activities were not associated to defined mitochondrial diseases linked to defects of respiratory chain complexes or to defects of citric cycle enzymes. On the contrary, mtCK activity was significantly increased in the muscle of patients exhibiting ragged red fibers. This increase was generally associated to an increase of citrate synthase activity. Since ragged-red fibers and elevated mtCK activities were generally not found in children younger than 3 years, even in cases of characteristic oxidative phosphorylation deficiency, it is suggested that the increase in mtCK activity as well as the appearance of ragged-red fibers are not the first events which occur during the evolution of mitochondrial diseases but would rather be long-term secondary processes which slowly develop in deficient mitochondria.

Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification.

In order to determine whether disruption of mitochondrial function could trigger apoptosis in murine haematopoietic cells, we used the potassium ionophore valinomycin. Valinomycin induces apoptosis in the murine pre-B cell line BAF3, which cannot be inhibited by interleukin-3 addition or Bcl-2 over-expression. Valinomycin triggers rapid loss of mitochondrial membrane potential. This precedes cytoplasmic acidification, which leads to cysteine-active-site protease activation, DNA fragmentation and cell death. Bongkrekic acid, an inhibitor of the mitochondrial permeability transition, prevents acidification and subsequent induction of apoptosis by valinomycin.

Thyroid hormones regulate the onset of osmotic activity of rat liver mitochondria after birth.

The effect of thyroid hormone deprivation on the osmotic activity of liver mitochondria from early newborn rats was studied. Experimentally induced hypothyroidism prevented the increase in the osmotic activity of mitochondria observed immediately after birth. Osmotic activity was restored by T4 and T3 treatment to hypothyroid newborns but not when this treatment was supplemented with cycloheximide. Under the same circumstances, streptomycin had no effect. Hypothyroidism abolished the change in the slope of the osmotic curve (plot of inverse absorbance of mitochondrial suspensions incubated in sucrose solutions vs. inverse sucrose concentration) observed in mitochondria from euthyroid newborns at 110-120 mOsm sucrose, suggesting that hypothyroidism prevents the formation of tight physical connections between mitochondrial outer and inner membranes. Thyroid hormone deprivation increased the passive permeability of the mitochondrial inner membrane to protons, resulting in a decreased respiratory control ratio. Hypothyroidism prevented the sharp decrease in the affinity of mitochondria for ATP observed in euthyroid newborns immediately after birth. These results corroborate our previous suggestion (Endocrinology, 1995, 136:4448) that, during the early neonatal period, thyroid hormones control the synthesis of some nucleus-coded protein(s) involved in the assembly of F0,F1-ATPase.

Hypothyroidism prevents postnatal changes in rat liver mitochondrial populations defined by rhodamine-123 staining.

The effect of hypothyroidism on the percentages of low fluorescence population (LFP) and high fluorescence population (HFP) rhodamine-123-stained mitochondria, respiratory parameters, and ATPase activity were studied in liver mitochondria from early newborn rats. Hypothyroidism prevented the decrease in the percentage of HFP and the subsequent increase in LFP that occurs immediately after birth. This effect coincides with the impairment of mitochondrial respiratory function, as shown by the low respiratory control ratio and the low activity of F0,F1-ATPase found in hypothyroid newborns. All of these changes were reversed by the administration of thyroid hormones. ATP in vitro promotes the conversion of HFP into LFP and increases the respiratory control ratio in hypothyroid newborns, although this effect was not observed after thyroid hormone treatment. The effect of thyroid hormones on both the postnatal changes in mitochondrial populations and in F0,F1-ATPase activity was prevented by cycloheximide, but not by streptomycin. Thus, the observed effects of thyroid hormones on neonatal mitochondria must be accomplished by the induction of the synthesis of some nuclei-coded protein, possibly involved in F0,F1-ATPase assembly.

Identification by flow cytometry of two distinct rhodamine-123-stained mitochondrial populations in rat liver.

Isolated rat liver mitochondria were split into three fractions of increasing density when applied to a Percoll gradient. NADH-ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome-c oxidase but not F1-ATPase activities increased with density as well as respiratory rate in state 3 and the respiratory control index. Flow cytometry of mitochondrial density fractions stained with rhodamine-123 revealed the occurrence in each density fraction of two distinct mitochondrial populations with different fluorescence intensity. The high fluorescence population was minor and its proportion decreased with density. The extent of high fluorescence population staining depended on the deenergized state of the mitochondria suggesting that this population represents an immature form of the mitochondria which may develop into a fully functional organelle by the incorporation of structural and/or functional proteins.

Postnatal changes in rhodamine-123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by ATP.

The incubation of term fetus mitochondria with ATP mimicked in vitro the increase in the respiratory control index and in the percentage of the rhodamine-123-low fluorescence population that occurred in vivo immediately after birth, suggesting that both phenomena are closely associated. The administration of streptomycin inhibited the increase in the percentage of the low fluorescence population that occurred immediately after birth, while the administration of cycloheximide even reversed these changes. These results suggest that the in vivo interconversion between mitochondrial forms depends on both cytosolic and mitochondrial protein synthesis.

Retention of concurrent taste aversion learning after electrolytic lesioning of the interpositus-dentate region of the cerebellum.

Lesions in the interpositus-dentate region of the cerebellum impair short-term, or concurrent, TAL. In this type of learning, animals must discriminate between two flavor stimuli presented at the same time, one of which is associated with an aversive product. The task is learned by the control animals, and within this group the animals that acquire it adequately enough (15/22, 70% criterion) retain the learned taste discrimination when they are subjected to it again after being lesioned in the interpositus-dentate region. These results suggest that the deep nuclei are essential in the concurrent TAL acquisition process, but not in its retention.

Bilateral lesions in the cerebellar interpositus-dentate region impair taste aversion learning in rats.

Taste Aversion Learning (TAL) has been induced through two different behavioral procedures: a short-term o concurrent (two-daily flavors) and a long-term (one-daily flavor) procedure. For the first, two gustatory/olfactory stimuli are presented separately but at the same time on a daily basis. One of the flavors is paired with simultaneous intragastric administration of hypertonic NaCl and the other is paired with physiological saline. In the long-term procedure, the two stimuli are presented on alternate days, one of them followed by intragastric injection of the aversive stimulus, and the other by saline. The subjects for both types of tests were animals that had been lesioned in the interpositus-dentate region of the cerebellum. The experiments show that the lesions disrupt short-term TAL, but have no effect on long-term TAL. The results are discussed in terms of the role of the cerebellum in relation to TAL and the different anatomical substrates of both learning modalities.

[Validation of half-night polysomnography for sleep apnea/hypoapnea syndrome]. / Validación de estudios polisomnográficos de mitad de la noche en el síndrome de apneas/hipoapneas durante el sueño.

UNLABELLED: A diagnosis of sleep apnea/hypopnea syndrome (SAHS) is based on clinical signs and nighttime polysomnograms. Brief polysomnography has been proposed as an alternative to all-night recording. OBJECTIVES: The aim of this study was to determine whether a polysomnograms obtained during the first half of the night is sufficient for establishing a diagnosis of SAHS and to determine the correlation between polysomnographic variables recorded during the first four hours (half the study time) with those recorded over the full eight hours (full study time), as well as to determine diagnostic agreement. DESIGN: Thirty-five patients suspected of having SAHS were studied prospectively. Baseline polysomnograms were scored blindly by two independent observers following standard methods. A diagnosis of SAHS was made according to guidelines of the Spanish Society of Pneumology and Chest Surgery. During the first half of the night and up to the end of each recording period we gathered neurophysiological and respiratory variables and diagnostic impressions. RESULTS: The correlation between variables (sleep stage, overall AHI, REM-AHI, non-REM-AHI and sleep efficiency) recorded in the first half of the night and throughout the night was significant (p < 0.05) by both Pearson's correlation coefficient (r) and by the intraclass correlation coefficient (ICC). In 33 of 35 patients (94.3%) diagnostic agreement was achieved (95% CI 80.84-99.30); when SAHS was severe, agreement was 100%. CONCLUSION: Based on these results, we conclude that for patients with a diagnosis of severe SAHS during the first half of the night, data recorded during the second half can be considered supplementary.

Electrolytic lesions of the pedunculopontine nucleus disrupt concurrent learned aversion induced by NaCl.

Bilateral electrolytic lesions in the pedunculopontine nucleus (PPN) impair acquisition of short-term, or concurrent, Taste Aversion Learning (TAL) in rats. This type of TAL is characterized by the daily presentation of two different flavor stimuli at the same time, one associated with simultaneous intragastric administration of an aversive product (hypertonic NaCl) and the other with physiological saline. Sham-lesioned control animals learn this taste discrimination task, but both lesioned animals and control animals learn a long-term, or delayed, TAL task in which each gustatory stimulus is presented individually every other day and the intragastric products, LiCl (0.15 M) and physiological saline, are administered after a 15-min delay. These results are analyzed in the context of the cerebellar circuits involved in learning and in relation to the two TAL modalities described above.