Light and feeding entrainment of the molecular circadian clock in a marine teleost (Sparus aurata).

Daily light and feeding cycles act as powerful synchronizers of circadian rhythmicity. Ultimately, these external cues entrain the expression of clock genes, which generate daily rhythmic behavioral and physiological responses in vertebrates. In the present study, we investigated clock genes in a marine teleost (gilthead sea bream). Partial cDNA sequences of key elements from both positive (Bmal1, Clock) and negative (Per2, Cry1) regulatory loops were cloned before studying how feeding time affects the daily rhythms of locomotor activity and clock gene expression in the central (brain) and peripheral (liver) oscillators. To this end, all fish were kept under a light-dark (LD) cycle and were divided into three experimental groups, depending on the time of their daily meal: mid-light (ML), mid-darkness (MD), or at random (RD) times. Finally, the existence of circadian control on gene expression was investigated in the absence of external cues (DD + RD). The behavioral results showed that seabream fed at ML or RD displayed a diurnal activity pattern (>91% of activity during the day), whereas fish fed at MD were nocturnal (89% of activity during the night). Moreover, seabream subjected to regular feeding cycles (ML and MD groups) showed food-anticipatory activity (FAA). Regardless of the mealtime, the daily rhythm of clock gene expression in the brain peaked close to the light-dark transition in the case of Bmal1 and Clock, and at the beginning of the light phase in the case of Per2 and Cry1, showing the existence of phase delay between the positive and negative elements of the molecular clock. In the liver, however, the acrophases of the daily rhythms differed depending on the feeding regime: the maximum expression of Bmal1 and Clock in the ML and RD groups was in antiphase to the expression pattern observed in the fish fed at MD. Under constant conditions (DD + RD), Per2 and Cry1 showed circadian rhythmicity in the brain, whereas Bmal1, Clock, and Per2 did in the liver. Our results indicate that the seabream clock gene expression is endogenously controlled and in liver it is strongly entrained by food signals, rather than by the LD cycle, and that scheduled feeding can shift the phase of the daily rhythm of clock gene expression in a peripheral organ (liver) without changing the phase of these rhythms in a central oscillator (brain), suggesting uncoupling of the light-entrainable oscillator (LEO) from the food-entrainable oscillator (FEO). These findings provide the basis and new tools for improving our knowledge of the circadian system and entraining pathways of this fish species, which is of great interest for the Mediterranean aquaculture.

Does the supine position worsen respiratory function in elderly subjects?

The aim of our study was to test whether the supine position or the sitting position worsens static, forced expiratory flows and measurements of lung mechanics in a group of aged subjects living in a nursing home who were clinically stable and without clinical evidence of cardiorespiratory diseases. Seventeen subjects (mean age 80 +/- 7 years; 16 f) were studied under baseline conditions. Spirometric, breathing pattern and mechanics data by means of an esophageal balloon were measured in sitting and supine positions. Analysis of sitting results showed aged subjects to have a slight flow limitation in peripheral airways, an increase in expiratory airways resistance and mild hyperinflation index (PEEPi = 2.2 +/- 1.9 cm H2O). Pressure time index did not reach the fatigue level in hardly any patient. Maximal inspiratory pressure values (42 +/- 15 cm H2O) were reduced by about 50% in comparison with our normal laboratory standards. Arterial blood gas analysis revealed no pathological data in any subject. When supine, subjects revealed a significant decrease in forced expiratory volume at the first second (p < 0.005), in forced vital capacity (p < 0.01) and in peak expiratory flow (p < 0.05). Moreover, mechanics and breathing pattern data showed a significant decrease in tidal volume (Vt) and dynamic lung compliance (Cld) (p < 0.05) and an increase in respiratory rate/Vt ratio (p < 0.05). Our data confirm the results of previous reports about Cld decrease in supine posture in young normal people. Although our aged subjects showed a significant decrease in forced expiratory volumes and Vt when the supine position was adopted, static mechanics data did not appear modified by the gravitational effect of this posture.

Quantitative gas-liquid chromatographic determination of medroxyprogesterone acetate in human plasma.

PIP: MPA (medroxyprogesterone acetate), a synthetic hormone derived from progesterone, has been shown to have a strong progestational effect in both man and animals after oral administration. This is a report on a specific GLC (gas-liquid chromatographic) method for determining MPA in human plasma. Additional reagents used were: acetone, acetonitrile, benzene, cyclohexane, and heptafluorobutyric anhydride. Each of the following steps in the laboratory procedure is explained: 1) GLC; 2) mass spectrometry; 3) standard external calibration graphs; 4) plasma preparation; and 5) internal calibration graphs. After extraction of the plasma with cyclohexane and formation of the 3-enol heptafluorobutyrate ester of the drug, MPA was determined by GLC on an OV-17 column with an electron-capture detector. All results are graphed. Unfortunately, purification was impossible because MPA can be easily extracted at any pH and has a chemical structure similar to that of cholesterol.^ieng

In vivo studied on the metabolism of dibenzo [c,f]-[1,2] diazepine.

The in vivo metabolism of dibenzo [c,f]-[1,2] diazepine (I) was studied after i.p. administration to rats, in order to observe the influence of the heteroatom on the metabolic pathway. Metabolites were separated by chromatography and their structures elucidated by mass spectrometry, by both gas chromatography coupled with mass spectrometry and the direct inlet system. Six metabolites were identified, some of them by direct comparison with authentic samples.

In vitro studies on the metabolism of dibenzo[c,f]-[1,2]diazepine.

Dibenzo [c,f]-[1,2] diazepine is a new compound with a tricyclic structure and a double bond between two nitrogen atoms in positions 1 and 2 of the molecule. Its in vitro metabolism has been studied, incubating the compound with rat liver microsomes. Two metabolites have been identified: dibenzo [c,f] - [1,2]diazepine-1-N-oxide and a monohydroxyderivative in the aromatic ring.

Quantitative analysis of amineptine (S-1694) in biological samples by gas chromatography--mass fragmentography.

A sensitive, specific method for the quantitative analysis of amineptine in rat blood and brain is described. After extraction and purification amineptine is detected by mass fragmentography, monitoring the fragment ion at m/e 192. The method allows the quantitative analysis of as little as 50 ng of amineptine per ml blood and 200 ng per g for brain.