Prenatal Entrainment of Rat Testicular Malate Dehydrogenase Activity Circadian Rhythm

In mammals the photoperiodic synchronization of circadian system starts before birth. During fetal and neonatal period mothers relay the photoperiodic information to their litter. The maternal pineal melatonin 24 h cycle acts as a synchronizing signal. We have studied the effect of pineal maternal sympathetic denervation and administration of melatonin to mothers denervated during gestation on the prenatal synchronization of testicular malate dehydrogenase (MDH) activity circadian rhythm of the offspring 25 days after birth. When mothers were denervated at the 7th, 10th or 11th day of gestation, pups showed disruption of testicular MDH activity circadian rhythms. In contrast, no disruptive effect was observed when the mothers were denervated on the 12th or 14th day of gestation. When denervated mothers (7th day of gestation) were treated with a daily dose of melatonin from the 11th to the 14th day of gestation, pups showed a MDH activity circadian rhythm. The hormone failed to impose a daily phase when administered from the 9th to the 12th day of gestation. Results suggest that prenatal synchronization in the rat occurs very early in the development, before suprachiasmatic nuclei morphologic arrangement and functional activity begin.     

Malate Dehydrogenase Activity of Rat Testis: Circadian Rhythm and its Ontogeny

Malate dehydrogenase activity and soluble protein content in testes from rats exposed to a 14:00 h light:10:00 h dark photoperiod, have been determined every two or four hours over a 24 hour period in 5, 15, 25 and 120 day-old rats. By using the Cosinor method, the ontogeny of an unimodal rhythm was studied for MDH activity and soluble protein content in testis. In 5 and 15 day-old rats, the MDH acrophases were recorded around 19:00 h and 17:00 h, respectively. Rats aged 25 and 110 days showed the MDH acrophases during the dark period. An inversion of the MDH circadian rhythms was detected in 25 day-old compared to those of 5 and 15 day-old rats. An inversion of the protein circadian rhythm was also detected at 15 days compared to that at 5 days. These inversions persist in the adult rats. The amplitude of the MDH and protein rhythms reached the lowest value in adulthood. The mean daily value of testicular MDH increased between day 5 and 15, decreasing at day 35 and remaining unchanged until adulthood. The variation of malate dehydrogenase activity, soluble protein content levels, and the circadian rhythm parameters during the maturation process may be related to gonad development.     

Malate Dehydrogenase Activity of Rat Testis: Circadian Rhythm and its Ontogeny

Malate dehydrogenase activity and soluble protein content in testes from rats exposed to a 14:00 h light:10:00 h dark photoperiod, have been determined every two or four hours over a 24 hour period in 5, 15, 25 and 120 day-old rats. By using the Cosinor method, the ontogeny of an unimodal rhythm was studied for MDH activity and soluble protein content in testis. In 5 and 15 day-old rats, the MDH acrophases were recorded around 19:00 h and 17:00 h, respectively. Rats aged 25 and 110 days showed the MDH acrophases during the dark period. An inversion of the MDH circadian rhythms was detected in 25 day-old compared to those of 5 and 15 day-old rats. An inversion of the protein circadian rhythm was also detected at 15 days compared to that at 5 days. These inversions persist in the adult rats. The amplitude of the MDH and protein rhythms reached the lowest value in adulthood. The mean daily value of testicular MDH increased between day 5 and 15, decreasing at day 35 and remaining unchanged until adulthood. The variation of malate dehydrogenase activity, soluble protein content levels, and the circadian rhythm parameters during the maturation process may be related to gonad development.     

Prenatal and Neonatal Diazepam Administration Synchronizes Testicular Malate Dehydrogenase Circadian Rhythms in Young Rats

Rat or hamster pups exposed to constant light or darkness since birth exhibit many circadian rhythms synchronized with those of the mother. During early development, a number of cues derived from the maternal circadian system synchronize the fetal and neonatal circadian clock. Maternal pineal sympathetic denervation during early pregnancy disrupts maternal synchronization of parotid α-amylase and testicular malate dehydrogenase circadian rhythms in rat pups. Maternal pineal sympathetic denervation was used to study potential agents able to synchronize the fetal or neonatal circadian clock. Melatonin injection to denervated pregnant mothers prevents the pineal sympathetic denervation effect on those circadian rhythms. We now studied the synchronizing effect of a benzodiazepine compound, diazepam. This GABA_A agonist synchronized testicular malate dehydrogenase (MDH) activity of pups when it was injected to sympathetic denervated pregnant dams (a daily dose at 07:00 or 19:00 h from the 14 th to the 20 th day of gestation) or orally administered to the pups (a daily dose at 19:00 h from the 10 th to 24 th day of life). Co-injection of diazepam and GABA_A antagonist, flumazenil, blocked the synchronizing effect of diazepam. The results demonstrate that diazepam has a synchronizing effect on the development of the circadian clock in rats and suggest that modulation of maternal GABA_A could participate in mammalian maternal synchronization.     

Sympathetic Regulation of Circadian Rhythm of Serotonin N-Acetyltransferase Activity in Pineal Gland of Infant Rat

Abstract: The circadian rhythms of serotonin N -acetyltransferase activity in the pineal glands of infant and adult rats were compared. The nighttime increase of N -acetyltransferase activity in the pineals of infant rats was blocked by removal of superior cervical ganglion or by pretreatment with reserpine, l -propranolol, and cycloheximide. Injection of isoproterenol to infant rats markedly elevated pineal N -acetyltransferase activity. When the pineal glands of infant rats were organ-cultured, N -acetyltransferase activity spontaneously increased 7–12 h after the rats were killed. When infant rats were previously denervated or pretreated with reserpine and their pineals were cultured, this spontaneous elevation of N -acetyltransferase activity was abolished, indicating that the transient increase of the enzyme activity in organ culture was due to a liberation of catecholamine from degenerating nerve endings. Additional illumination until midnight prevented the nighttime increase of N -acetyltransferase activity in intact infant rats but not in blinded infant rats. These observations are taken to indicate that N -acetyltransferase rhythm in immature rat pineals is regulated by the sympathetic nerves in the same manner as in adult rat pineals, that the immature rat pineal does not contain a time-keeping system, and that there is no extraretinal light perception in infant rats as far as N -acetyltransferase rhythm is concerned.     

Circadian Rhythm in Dihydropyrimidine Dehydrogenase Activity and Reduced Glutathione Content in Peripheral Blood of Nasopharyngeal Carcinoma Patients

Dihydropyrimidine dehydrogenase (DPD) is a rate‐limiting enzyme of 5‐fluorouracil (5‐FU) catabolism. Glutathione (GSH) is a tripeptide involved in platinum complex detoxification. This study explored the circadian rhythms of DPD activity and GSH concentration in the peripheral blood of 16 patients with histologically proven nasopharyngeal carcinoma (NPC) in order to guide the establishment of chronotherapeutic schedules for this cancer. DPD activity and GSH concentration were determined by high performance liquid chromatography (HPLC). Both variables displayed significant circadian rhythms (Cosinor analysis: p=0.009 and 0.012, respectively). Peak DPD activity occurred at about 02:30 h; whereas, peak GSH concentration occurred around 12:40 h. The differences between the peak and nadir mean values were 25.5% and 38.7%, respectively. The study showed that the circadian rhythms in DPD activity and GSH concentration in Chinese NPC are similar to those reported for western patients with colorectal cancer, despite the differences in race and kinds of cancer. These findings imply that the chronotherapeutic schedule of 5‐FU and platinum used to treat European colorectal cancer patients probably is applicable to Chinese NPC patients.     

Circadian Rhythm in Muscarinic Receptor Subtypes in Rat Forebrain

The binding of different ligands to muscarinic receptors in the centra) nervous system is regulated by several factors. Among these are the administration of drugs, disease, ontogeny or aging. Studies carried out in rat brains have demonstrated changes in the density of the muscarinic receptors at different times of the day. These changes might be related to variations in the circadian rhythms. In this work we have studied the binding of the [3H]-N-methyl-escopolamine, the agonist carbachol and the antagonist pirenzepine to muscarinic receptors in rat forebrains at 10.00,14.00,18.00,22.00,02.00 and 06.00 hr. We have observed changes in the density of muscarinic receptors but not changes in affinity to the radioligand. The Bmax values obtained by saturation studies were maximum at 14.00 hr and minimum at 02.00 hr (p < 0.05 Mann-Whitney's test). Inhibition studies in the presence of the non-selective agonist carbachol and the selective antagonist pirenzepine, at the same time-points, did not show statistically significant changes in the Bmax values. These data indicate that changes in the Bmax values are only observed in the total population of muscarinic receptors and are not due to modifications in the subtypes of muscarinic receptors nor to the different affinity states of agonist binding.     

Entrainment of Circadian Programs

Circadian rhythms were recently proposed as a measure of physiological state and prognosis in disorders of consciousness (DOC). So far, melatonin regulation was never assessed in vegetative state (VS). Aim of our research was to investigate the nocturnal melatonin levels and light-induced melatonin suppression in a cohort of VS patients. We assessed six consecutive patients (four men, age 33.3?±?9.3 years) with post-traumatic VS and nine age-matched healthy volunteers (five men, age 34.3?±?8.9 years) on two consecutive nights: one baseline and one light exposure night. During baseline, night subjects were in bed in a dim (<5?lux) room from 10?pm to 8?am. Blood samples were collected hourly 00:30–3:30?am (00:30?=?MLT1; 1:30?=?MLT2; 2:30?=?MLT3; and 3:30?=?MLT4). Identical setting was used for melatonin suppression test night, except for the exposure to monochromatic (470?nm) light from 1:30 to 3:30?am. Plasma melatonin levels were evaluated by radioimmunoassay. Magnitude of melatonin suppression was assessed by melatonin suppression score (caMSS) and suppression rate. We searched for group differences in melatonin levels, differences between repeated samples melatonin concentrations during baseline night and light exposure night, and light-induced suppression of melatonin secretion. During baseline night, controls showed an increase of melatonin (MLT4 vs MLT1, p?=?0.037), while no significant changes were observed in VS melatonin levels (p?=?0.172). Baseline night MLT4 was significantly lower in VS vs controls (p?=?0.036). During light-exposure night, controls displayed a significant suppression of melatonin (MLT3 and MLT4 vs MLT2, p?=?0.016 and 0.002, respectively), while VS patients displayed no significant changes. The magnitude of light-induced suppression of melatonin levels was statistically different between groups considering control adjusted caMSS (p?=?0.000), suppression rate (p?=?0.002) and absolute percentage difference (p?=?0.012). These results demonstrate for the first time that VS patients present an alteration in night melatonin secretion and reduced light-induced melatonin suppression. These findings confirm previous studies demonstrating a disruption of the circadian system in DOC and suggest a possible benefit from melatonin supplementation in VS.     

Purification of Chlorella Malate Dehydrogenase

Our prior investigation of Chlorella malate dehydrogenase (MDH) revealed supernatant and particulate isoenzymes which were immunologically, chromatographically, and electro-phoretically distinct. By means of ammonium sulfate fractionation and column chromatography a crystalline preparation of the particulate isoenzyme, specific activity of approximately 2000 has been obtained. It appears homogeneous in the analytical ultracentrifuge, by column chromatography and by electrophoresis. This preparation migrates as a single band of activity when subjected to starch gel electrophoresis and its mobility and activity (on the gel) are unaffected by prior incubation with citric acid, pH 2.0, EDTA, β-mercapto-ethanol, substrates or products; however, new bands appear when preincubated with exogenous proteins (RNase, Ovalbumin, Aldolase).     

4-Aminobutyraldehyde Dehydrogenase Activity in Rat Brain

Abstract: An enzyme with NAD⁺-dependent 4-aminobutyraldehyde dehydrogenase activity was purified about 360-fold from rat brain extract. AMP-Sepharose chromatography was effective in separating the enzyme from other NAD⁺-dependent aldehyde dehydrogenases included in the extract. The K _ms for the substrates NAD⁺ and 4-aminobutyraldehyde were 4.8 × 10⁻⁴ and 8.3 × 10⁻⁵ M , respectively. The pH optimum for the enzyme was about 8.0. The ratio of activities toward 4-aminobutyraldehyde, propionaldehyde, succinate semialdehyde, and benzaldehyde was 1.00:0.17:0.24:0.09:0.03 when the activity toward 4-aminobutyraldehyde was set equal to 1.00. The enzyme activity in subcellular fractions of rat brain was localized in cytosol.     

Effect of Ethanol and Theophylline on Circadian Rhythm of Rat Locomotion

The effect of ethanol and theophylline on the circadian rhythm of rat locomotion was investigated. Male Wistar rats synchronized to 12: 12 h light-dark cycles were divided into four groups for treatment with saline, ethanol, theophylline, and ethanol plus theophylline. Animals in each group were orally administered saline, ethanol (2.0 g/kg body wt), theophylline (10 mg/kg body wt), and ethanol plus theophylline, respectively, six times every 2 h during the 12-h light span. Spontaneous loco-motor activity was continuously monitored by an animal activity recorder at 15-min intervals. Total activity count, circadian rhythm characteristics of activity (amplitude, acrophase, and mesor), power spectral patterns, and slope of fluctuation (a measurement of ultradian periodicity) were calculated. Ethanol administration decreased the total activity count by 60% and phase-delayed the onset of activity rhythm by 9.5 h on the day after treatment. The absolute value of the slope of fluctuation was increased by ethanol administration. The mean recovery time evaluated by rhythm detection was 3.8 days. Theophylline administration increased the light phase activity, but caused no phase delay of the onset time of the locomotor activity rhythm. The decrease in total activity count and phase delay of onset of the activity rhythm caused by ethanol were partially antagonized by theophylline. However, the prolonged effects of ethanol, represented by a late recovery time and an increase in the slope of fluctuation, were not influenced by theophylline.     

植物苹果酸脱氢酶研究进展

苹果酸是植物体内参与C4循环、景天酸循环等众多代谢途径的关键代谢物。苹果酸含量提高的途径主要来自植物体内合成的提高。苹果酸脱氢酶(MDH)可引起草酰乙酸盐的氧化作用以形成苹果酸盐,增加植物体内苹果酸的含量,从而显著提高植物体的耐酸性以及对铝毒的抗性。本文全面回顾了国内外对苹果酸脱氢酶(MDH)在植物生理学、生物化学、分子生物学、系统分化领域的研究进展,并针对其在植物体耐酸性机制机理研究领域所取得的研究成果进行了追溯。     

Circadian Locomotor Activity Rhythm During Ontogeny in Crayfish Procambarus Clarkii

The characteristics of the circadian rhythm of locomotor activity in the crayfish Procambarus clarkii during ontogeny under constant darkness and light-dark (LD 12:12) conditions were studied in 132 juvenile crayfish, aged 10-140 days, divided in four groups. All animals were individually monitored with a motor activity recording system. Activity was quantitatively and qualitatively analyzed. All ages showed a circadian rhythm, although the probability of its appearance increased with age. Period values oscillated between 25.0 h in group I (2-4-week-old animals) and 24.3 h in group IV (16-20-week-old animals with more than 6 molts), but always with a high standard deviation. Groups II (5-10-week-old animals) and IV showed a statistically significant bimodal nonrandom synchrony of phases. The activity/ rest relationship diminishes as development progresses and is most uniform in group IV. We discuss the possibility that the pacemaker system responsible for this rhythm might be present from the moment of eclosion, but the coupling strength of this system with the effectors might change along development. The results presented in this work seem to indicate that the central pacemakers responsible for the activity and the ERG rhythm are not the same.     

Ethanol-induced Adaptation of Alcohol Dehydrogenase Activity in Rat Brain

ALTHOUGH the presence of alcohol dehydrogenase (ADH) in cerebral tissue has been established¹, a physiological role for such a brain ethanol-oxidizing system has been unclear. The brain may be more biochemically adaptive than was once thought²; thus, it seemed possible that brain ADH may be substrate-induced. We now report that significant elevations of brain ADH activity occur in alcohol-imbibing rats; no changes from control values were found in liver ADH, liver aldehyde dehydrogenase (AldDH), or brain AldDH activities.     

Entrainment to Light of Circadian Activity Rhythms in Tench (Tinca tinca)

The present article analyzes locomotor activity rhythms in Tinca tinca. To that end, three different experiments were conducted on 24 animals (20 g body weight) kept in pairs in 60‐liter aquaria fitted with infrared sensors connected to a computer to continuously record fish movements. The first experiment was designed to study the endogenous circadian clock under free‐running conditions [ultradian 40:40 min LD pulses and constant dark (DD)] and after shifting the LD cycle. Our results demonstrate that tench has a strictly nocturnal activity pattern, an endogenous rhythm being evident in 45.8% of the fish analyzed. The second experiment was conducted to test the influence of different photoperiods (LD 6:18, 12:12, 18:6, and 22:2) on locomotor activity, the results showing that even under an extremely long photoperiod, tench activity is restricted to dark hours. The third experiment examined the effect of light intensity on locomotor activity rhythms. When fish were exposed to decreasing light intensities (from 300:0 lux to 30:0, 3:0, and 0.3:0 lux) while maintaining a constant photoperiod (LD 12:12), the highest percentage of locomotor activity was in all cases associated with the hours of complete darkness (0 lux). In short, our results clearly show that (a) tench is a species with a strictly nocturnal behavior, and (b) daily activity rhythms gradually entrain after shifting the LD cycle and persist under free‐running conditions, pointing to their circadian nature. However, light strongly influences activity rhythms, since (c) the length of the active phase is directly controlled by the photophase, and (d) strictly nocturnal behavior persists even under very dim light conditions (0.3 lux). The above findings deepen our knowledge of tench behavior, which may help to optimize the aquacultural management of this species, for example, by adjusting feeding strategies to their nocturnal behavior.     

Entrainment Properties of the Locomotor Activity Rhythm of Drosophila melanogaster Under Different Photoperiodic Regimens

In this paper we report the results of an experiment to assess how closely repeated brief light pulses (LPs) mimic the effects of 12:12 h light/dark (LD) cycles (PPc). The locomotor activity rhythm of individual fruit flies from a laboratory population of Drosophila melanogaster was monitored under four different photoperiodic regimens, created using 12 h of light and 12 h of darkness or brief light pulses (LPs). The phase relationship (Ψ) and the stability (precision) of the locomotor activity rhythm during entrainment were estimated in order to compare the state of the circadian clocks under the four different photoperiodic regimens. The flies (n = 72) were subjected to four different LD cycles: (i) 12 h of light and 12 h of darkness (complete photoperiod, PPc); (ii) a single brief LP of 15 min duration presented close to the onset of activity (SLP-1); (iii) a single brief LP of 15 min duration administered close to the offset of activity (SLP-2); and (iv) two brief LPs administered 12 h apart (skeleton photoperiod, PPs). The locomotor activity rhythm of the flies was first monitored under constant darkness (DD) for about 10 days and then under the four different photoperiodic regimens for about 10 days, and finally in DD for the remainder of the experiment. The Ψ of the locomotor activity rhythm and its precision under PPc and PPs did not differ significantly, but they were significantly different from the SLP-1 and SLP-2 conditions. The results provide interesting insights into photoentrainment mechanisms of circadian clocks in D. melanogaster, and suggest that skeleton photoperiods, but not single brief LPs, mimic the actions of complete photoperiods.     

Circadian Rhythm and Seasonal Variations in Basal cAMP Content of Rat Heart Ventricles

The cAMP content in rat heart ventricles was studied at 3-hr intervals during 24hr at different times of the year. A significant circadian rhythm in cAMP content was found. Time of the year reproducibly influenced the 24-hr mean, the amplitude as well as the peak value in cAMP in relation to circadian time.     

Synchronization of the Circadian Activity Rhythm in Hamsters Following Intermittent Schedule Shifts

The time course of resynchronization of the circadian activity rhythm of hamsters was observed following a 10-hr advance or delay in the light-dark cycle (LD 12:12). Twenty-six shift patterns of the lighting schedule were studied; they consisted of continuous (daily), three-step, two-step and one-step shifting. So long as the daily shift of the lighting schedule was 1 hr or less, the locomotor rhythm followed the continuous shift perfectly. As the amount of daily shift increased, the time course of activity onset deviated more from the time of lights off; the tendency was more marked in advancing than in delaying shifts. Responses of the activity rhythm to stepwise shifting were essentially the same as those to a continuous shift. They were, however, characterized by larger individual variations, and it took additional days before entrainment was achieved. By fitting the time course of entrainment to an exponential model with a constant term, estimates of time constant and shift error were derived. The time constant became shorter with increasing amounts of daily shifts up to 2 hr per day, increasing the number of shift steps, and/or reducing the amount of the initial shift of the seies. The shift error estimated was 0.51 ± 0.12 hr, indicating precise resynchronization. Accordingly, a quicker resynchronization may be expected when a multiple step shift with a moderate initial shift are employed. In the case of a 10-hr shift, for example, a shift of 3 hr followed by another 7 hr may be recommended.     

Synchronization of the Circadian Activity Rhythm in Hamsters Following Intermittent Schedule Shifts

The time course of resynchronization of the circadian activity rhythm of hamsters was observed following a 10-hr advance or delay in the light-dark cycle (LD 12:12). Twenty-six shift patterns of the lighting schedule were studied; they consisted of continuous (daily), three-step, two-step and one-step shifting. So long as the daily shift of the lighting schedule was 1 hr or less, the locomotor rhythm followed the continuous shift perfectly. As the amount of daily shift increased, the time course of activity onset deviated more from the time of lights off; the tendency was more marked in advancing than in delaying shifts. Responses of the activity rhythm to stepwise shifting were essentially the same as those to a continuous shift. They were, however, characterized by larger individual variations, and it took additional days before entrainment was achieved. By fitting the time course of entrainment to an exponential model with a constant term, estimates of time constant and shift error were derived. The time constant became shorter with increasing amounts of daily shifts up to 2 hr per day, increasing the number of shift steps, and/or reducing the amount of the initial shift of the seies. The shift error estimated was 0.51 ± 0.12 hr, indicating precise resynchronization. Accordingly, a quicker resynchronization may be expected when a multiple step shift with a moderate initial shift are employed. In the case of a 10-hr shift, for example, a shift of 3 hr followed by another 7 hr may be recommended.