Day-night differences in membrane-bound pyroglutamyl-2-naphthylamide-hydrolyzing activity in rat hypothalamus, pituitary, and retina

Membrane-bound pyroglutamyl-2-naphthylamide-hydrolyzing enzyme activity was analyzed fluorometrically in the anterior hypothalamus, pituitary, and retina of adult male rats to investigate day-night differences. Six groups (n=6 per group) were assessed—three during the light span and three during the dark span—under a standard 12 h-12 h light-dark cycle (light on from 07:00 to 19:00 h) and controlled temperature environment, with food and water available ad libitum. In the hypothalamus, enzyme activity levels were higher for time points of the dark than the light period. In contrast, the pituitary and retina exhibited the highest levels at the time points of the light period. The pituitary and retina also exhibited significant differences between the clock-hour means of the light period. Day-night differences in membrane-bound pyroglutamyl-2-naphthylamide-hydrolyzing activity may reflect differences in its susceptible endogenous substrates.     

The effects of Tat protein on locomotor activity and circadian gene expressions in the mouse hypothalamus

Transactivator (Tat), a regulatory protein of HIV-1, plays a very important role in HIV-1 infection by promoting the rapid replication of HIV. Research surrounding Tat protein function has mainly focused on inhibition of the immune system, promotion of growth of vascular endothelial cells, and nervous system damage. To date, very little research has addressed the role of Tat in circadian rhythms. Previous studies in our lab have found that the concentration of Tat protein in HIV patients’ blood was positively correlated with patients’ sleep quality and melatonin concentrations. In this study, we applied a Tat expression plasmid in mice. Result demonstrated that the locomotor activities of mice and the concentration of melatonin were significantly increased. Alternatively, the expression of Clock gene was markedly decreased. On the other hand, the expression of the Cry1 gene was significantly increased, while the expression of Bmal1 and Per1 genes exhibited no significant difference.     

Microelectronic detection of activity level and magnetic orientation of yellow European eel, <Emphasis Type="Italic">Anguilla anguilla</Emphasis> L., in a pond

Synopsis We studied the circadian and monthly activity, the distribution patterns, and orientation to the earths magnetic field, of yellow (non-migratory) female eels in a freshwater pond by means of microchips injected into their muscles. Detectors for microchips mounted in tubes were placed in the pond to detect if eels oriented themselves with respect to earths magnetic field. Based on the frequency of tube visits (search for shelter), the data indicated that the presence of eel in the tubes decreases gradually during the study period. We saw more activity during the night in the first months. There was a seasonal component in the orientation mechanism, with a significantly lower preference component in the summer compared to the fall. A preference for tubes oriented in a south-southwest direction (the direction of the Sargasso Sea) in fall suggests an orientation to the earths magnetic field.     

Pace‐of‐life: Relationships among locomotor activity,life history,and circadian rhythm in the assassin bug,Amphibolus venator

The pace‐of‐life syndrome (POLS) hypothesis means that animal behavior is correlated with life history strategies. Studies have reported that the free‐running period of the circadian rhythm (length of the period) is correlated with life history strategies in some animals. Although the length of the circadian rhythm may be associated with the POLS hypothesis, few studies have investigated the relationships among animal behavior, life history traits, and circadian rhythm. We tested the POLS hypothesis in the assassin bug, Amphibolus venator, which shows individual variation in locomotor activity. We found higher repeatability of differences in locomotor activity between individuals. Moreover, we found a trade‐off between locomotor activity and developmental period such that active individuals developed faster. However, locomotor activity was not correlated with the length of the circadian rhythm in A. venator. Therefore, this study suggests that the length of the circadian rhythm in A. venator does not support the POLS hypothesis.     

Day–Night Differences in Membrane‐Bound Pyroglutamyl‐2‐Naphthylamide‐Hydrolyzing Activity in Rat Hypothalamus,Pituitary, and Retina

Membrane‐bound pyroglutamyl‐2‐naphthylamide‐hydrolyzing enzyme activity was analyzed fluorometrically in the anterior hypothalamus, pituitary, and retina of adult male rats to investigate day–night differences. Six groups (n=6 per group) were assessed—three during the light span and three during the dark span—under a standard 12 h–12 h light–dark cycle (light on from 07:00 to 19:00 h) and controlled temperature environment, with food and water available ad libitum. In the hypothalamus, enzyme activity levels were higher for time points of the dark than the light period. In contrast, the pituitary and retina exhibited the highest levels at the time points of the light period. The pituitary and retina also exhibited significant differences between the clock‐hour means of the light period. Day–night differences in membrane‐bound pyroglutamyl‐2‐naphthylamide‐hydrolyzing activity may reflect differences in its susceptible endogenous substrates.     

Day length and evening temperature predict circannual variation in activity duration of the colony of the Indian cliff swallow,Hirundo fluvicola

In avian species, circannual rhythms, in a number of biological variables, including locomotor activity, have been studied under both laboratory and natural environmental conditions. However, information on circannual rhythm in daily activity duration of Indian cliff swallow (Hirundo fluvicola) is not available. It is a communal mud nesting, non-migratory species and inhabits low under arch bridges. Although it figures in the IUCN Red List under the least concerned category, it is important to study its behavioral ecology that may be of utmost importance from conservation stand point. In the present investigation, we examined the circannual rhythm in daily activity duration in this species at a communal roosting site under Kharoon river bridge on the Raipur–Bhilai highway (21°15′07.54′′N; 81°32′30.65′′E), Raipur, India, for eight consecutive days, every month from August 2009 to October 2011. On each study day, observations were made in two sessions, morning and evening. We monitored and recorded emergence time of the first bird from the nest in the morning and entry time of the last bird to the nest in the evening. We also recorded the ambient temperature and humidity of the study site simultaneously. Average daily activity duration (the time difference between the first bird’s emergence time and the last bird’s entry time from/to the nest, respectively) was computed for each month. A significant circannual rhythm in activity duration was validated. The peak of activity rhythm occurred on 22nd June with a spread between 19th June and 25th June. The activity duration was the shortest in winter and the longest in summer. The daily activity duration was positively correlated to the day length, sunset time, and morning and evening temperatures; whereas negatively correlated with the sunrise time, and morning and evening humidity. The multiple linear regression models suggest that day length alone explains 96% of the total variance in activity duration, whereas only 0.5% variance was attributable to evening temperature. We conclude that the day length is the strongest predictor of circannual variation in daily activity duration of the colony of Indian cliff swallow; whereas the evening temperature has very little effect. Further, we recommend that comparable studies under natural conditions might be very helpful to explore the effect of environmental cues on other intriguing behavioral decisions made by this and other avian species.     

6-h advances alter circadian activity patterns,fasting glucose,and insulin levels in C57BL6/J mice

Chronobiological disruptions, including shift work, have been linked to a number of disorders such as fatigue and diabetes. Additionally, there is evidence to support that exercise cannot only counteract fatigue and the onset of diabetes, but also alleviate the other negative symptoms associated with shift work. Therefore, the present study investigated the effects of wheel running and monthly 6-h phase advances on the circadian locomotor activity patterns and glucose and insulin levels in C57BL6/J mice. 6-h phase advances produced decreases in fasting glucose and increases in insulin, and wheel-running was able to alleviate the spike in insulin secretion. Additionally, mice experiencing the shift increased their food intake, despite having no change in body mass. Circadian wheel-running activity was also altered in phase-advanced mice. These results provide further evidence that chronobiological disruptions can lead to alterations in physiology and behavior, and that exercise can alleviate some of those symptoms.     

Multigranular exocytosis induced by phospholipase A2-activators,melittin and mastoparan,in rat anterior pituitary cells

Summary Effects of phospholipase A₂-activators, melittin and mastoparan, on rat anterior pituitary cells were studied by use of the electron microscope. Rat anterior pituitaries were incubated in HEPES buffer containing 20 g/ml of melittin or the same dose of mastoparan for 5 min, 10 min and 20 min. Features indicating discharge of granule contents by exocytosis were increased with time, and the simultaneous extrusion of a number of secretory granules, named multigranular exocytosis, was often recognized in addition to single-granule exocytosis at 10 min and 20 min. Most membrane pits, where the multigranular exocytosis as well as the single-granule exocytosis occurred, were coated. Moreover, a large number of vesicles coated or noncoated were distributed near the trans side of the Golgi apparatus of melittin-treated or mastoparan-treated cells after 20 min. These vesicles might be related to membrane internalized from the excess surface membrane derived from the limiting membrane of exocytosed granules. These observations indicate that phospholipase A₂-activators induce hormone release involving membrane fusion between limiting membranes of secretory granules, and between granulelimiting membrane and plasma membrane in rat anterior pituitary cells.This study was supported by grants from the Japan Ministry of Education     

Comparison of the effects of TRH and D-Ala2-metenkephalinamide on hippocampal electrical activity and behavior in the unanesthetized rat

Administration of TRH into the lateral ventricle of unanesthetized rats produced increases in the incidence of hippocampal theta (5.9–9.1 Hz) rhythm, locomotor activity and shaking behavior. The increase in theta rhythm produced by TRH was brief (<5 min) and was coincident with a brief, large increase in locomotor activity. Intracerebroventricular injection of either TRH or D-Ala²-metenkephalinamide (D-Ala²-ME) also induced episodes of shaking behavior. Shakes induced by D-Ala²-ME were associated with the occurrence of hippocampal epileptiform activity whereas those caused by TRH occurred in the absence of any recorded abnormalities in hippocampal activity. These results suggest that the increase in hippocampal theta rhythm after TRH is secondary to the increase in locomotor activity and, that in contrast to enkephalins, shaking behavior caused by TRH may not be related to an action on the electrographic activity of the hippocampus.     

Seasonal variations in properties of healthy and diabetic rat heart mitochondria: Mg2+-ATPase activity, content of conjugated dienes and membrane fluidity

Our previous preliminary results pointed to possible seasonal variations in Mg2+-ATPase activity of rat heart mitochondria (MIT). It is not too surprising since seasonal differences were already reported in myocardial function, metabolism and ultrastructure of the intact as well as hemodynamically overloaded rabbit hearts and also in other tissues. The present study is aimed to elucidate whether seasonal differences observed in rat heart MIT Mg2+-ATPase activity will be accompanied with changes in membrane fluidity and in the content of conjugated dienes (CD) in the lipid bilayers of MIT membranes as well as whether the above seasonal differences will also be present in the diabetic heart. Our results revealed that values of Mg2+-ATPase activity in the winter/spring-period (W/S-P) exceeded significantly (p<0.05-0.001) those in the summer/autumn-period (S/A-P). Similar trend was also observed in hearts of animals with acute (8 days) streptozotocin diabetes. With the exception of values of CD in the S/A-P, all values of Mg2+-ATPase activities, membrane fluidity and CD concentrations in diabetic hearts exceeded those observed in the healthy hearts. Our results indicate that seasonal differences may play a decisive role in the evaluation of properties and function of rat heart MIT.     

A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

Diurnal rhythmic expression of the rhythm-related genes, <Emphasis Type="Italic">rPeriod1, rPeriod2,</Emphasis> and <Emphasis Type="Italic"> rClock</Emphasis>, in the rat brain

High densities of the mRNA of three rhythm-related genes, rPeriod1 (rPer1), rPer2, and rClock, which share high homology in Drosophila and mammals, are found in the rat hypothalamic suprachiasmatic nucleus (SCN). The SCN, however, is not the only brain region that expresses these genes. To understand the possible physiological roles of these rhythm-related genes, we examined expression of these genes in different brain regions at various time points in male Sprague--Dawley rats. Using semi quantitativein situ hybridization with ³⁵S-riboprobes to evaluate mRNA levels, the diurnal rhythmicity of rPer1, and rPer2 mRNA levels was found in the SCN, arcuate nucleus, and median eminence/pars tuberalis. Expression patterns of mRNA for rPer1 and rPer2, however, were not similar in these brain regions. The rhythmicity in these brain regions was specific, because it was not observed in the cerebellum or hippocampus. Moreover, diurnal changes in rClock mRNA expression were not detected in any of the brain regions examined. These findings suggest that the different expression patterns observed for rPer1, rPer2, and rClock mRNAs may be attributed to their different physiological roles in these brain regions, and support previous work indicating that circadian rhythms in the brain are widespread.     

A comparison of the effects of PGI2, PGE2 and PGH2 on the cyclic nucleotide levels in rat anterior pituitary glands in vitro

Rat anterior pituitary explants were incubated with PGI₂, PGH₂ and PGE₂ in the presence of theophylline (1mM) and the production of cyclic AMP was measured. PGE₂ was found to be about 20 times more potent than PGI₂ while PGH₂ was slightly more effective than PGI₂. The results suggest that PGI₂ does not play a physiological role in cyclic AMP mediated events in the rat anterior pituitary.     

Effects of phloridzin, metavanadate and oligomycin on membrane bound (Na++ K++ Mg2+)ATPase activity in sugar beet roots

To clarify the reaction mechanism of a (Na⁺+ K⁺+ Mg²⁺)ATPase activity in sugar beet roots ( Beta vulgaris L. cv. Monohill) phloridzin, oligomycin (inhibitors of animal ATPases) and metavanadate (NH₄VO₃) have been used. Kinetic studies showed that: 1) Phloridzin inhibition is uncompetitive with respect to MgATP and not influenced by Na⁺ or K⁺. 2) This inhibition is only found in preparations made in the absence of sucrose. 3) Oligomycin and vanadate inhibit the ATPase in different ways. Omission of sucrose from the preparation medium favours vanadate inhibition but suppresses oligomycin inhibition. 4) The kinetic pattern of the Na⁺ activation of the ATPase differs in preparations made in the absence and presence of sucrose, but that of K⁺ activation is the same. – These results indicate that inclusion as against omission of sucrose from the preparation medium causes a conformational change of the membrane fragments/vesicles, which then expose different surfaces to the surrounding medium.     

Expression of high activity of ornithine decarboxylase and occurrence of unusual chromophobic cells in anterior pituitary gland of a novel growth-retarded strain of mice, grm/grm

Extremely high activity of ornithine decarboxylase (ODC) was detected in the pituitary gland of growth- retarded mice, grm/grm at 2 months after birth. The elevated enzyme activity gradually decreased to the control level in 14 months after birth. In the pituitary gland of the growth-retarded mice, unusual chromophobic cells were also present from the early stages after birth. The chromophobic cells showed conspicuous proliferations and resulted in a distinct hyperplasia of the tissue after 4 months after birth. These findings suggest that OCD is correlated to the progressive transformation of pituitary cells into the chromophobic cells.     

Temperature-dependent relationship between K+ influx,Mg2+-ATPase activity,transmembrane potential and membrane lipid composition in mycoplasma

The temperature-dependent relationship between K⁺ active influx, Mg²⁺-ATPase activity, transmembrane potential (ΔΨ) and the membrane lipid composition has been investigated in mycoplasma PG3. Native organisms were grown in a medium containing 10 μg/ml cholesterol and either oleic plus palmitic (chol (+), O + P) or elaidic (chol (+), E) acids. Adapted cells were grown in a medium free of exogenous cholesterol and supplemented with elaidic acid (chol (?), E).Arrhenius plots of ⁴²K⁺ active influx gave a linear relationship for (chol (+), O + P) cells ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?9}\text{kcal}$). On the other hand, when oleic plus palmitic acids are replaced by elaidic acid, an upward discontinuity appears between 28 and 30°C, which is associated with a large increase in the apparent activation energy of the process ($\text{t > 30°}\text{C}\text{, E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?24}\text{kcal}\text{; t < 30°}\text{C}\text{, E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?40}\text{kcal}$).Finally, a biphasic response with a break at approx. 23°C ($\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?7}\text{kcal}\text{, t > 23°}\text{C}$; $\text{E}{}_{\mathrm{<mtext>A</mtext>}}\text{= ?44}\text{kcal}\text{, t < 23°}\text{C}$) is observed for (chol (?), E) organisms. From the lack of correspondence between these effects on the K⁺ influx and the temperature dependence of both the Mg²⁺-ATPase activity and ΔΨ, it is suggested that changes in the membrane lipid composition affect the K⁺ transport at the level of the K⁺ carrier itself.Differential scanning calorimetry, steady-state fluorescence polarization of diphenylhexatriene and freeze-fracture electron microscopy experiments further suggest that the effect is largely due to modifications of the membrane microviscosity and that the K⁺ carrier is associated with the most fluid lipid species present in the membrane.