Induction of cyst formation by low temperature in the dinoflagellateGonyaulax polyedra Stein: Dependence on circadian phase and requirement of light

Encystment, which at a temperature of 15°C is photoperiodically controlled inGonyaulax polyedra, can also be induced by a decrease of temperature, from 20 to 10 or 8°C in the absence of photoperiodic signals. The cyst-inducing capacity of the decrease in temperature depends on the circadian phase: in constant light, the maximum of sensitivity was found at the beginning of subjective night. In a light/dark cycle, however, cyst formation was reduced during dark phase, indicating that light is required for the process of encystment. A similar light dependence was seen in the effect of the physiologically occurring cyst inducer 5-methoxytryptamine, but not in the encystment response to the protonophores monensin and nigericin.     

Circadian variation of diazepam acute toxicity in mice

Summary The LD₅₀ of i.p. injected diazepam was determined every 4 h over a 24-h period in albino mice adapted to a 12-h dark/12-h light programmed illumination cycle. Results show that diazepam is more toxic during the light phase of the cycle than during the dark phase and demonstrate circadian variation in the toxicity of the compound in mice.     

Anandamide extends platelets survival through CB1-dependent Akt signaling

Platelets are stored at 22°C, since incubation at 37°C results in loss of viability. Nonetheless, in our body (37°C), platelets survive for 8–10 days. This discrepancy has been explained in terms of deprivation of viability factors or accumulation of apoptotic factors during storage. We report that the endocannabinoid anandamide (AEA) may be one of the agents allowing platelet survival. In fact, at 37°C, human platelets enhance the expression of pro-apoptotic proteins (caspases, Bax, Bak) and decrease the expression of Bcl-xL, thus changing the Bcl-xL/Bak ratio, a key platelet biological clock. AEA or its non-hydrolyzable analogue, methanandamide, extend platelet life span, without reversing the changes in Bcl-xL/Bak ratio induced by heat stress. Instead, AEA binding to type-1 cannabinoid receptor activates Akt, which regulates, through phosphorylation of Bad, the interactions among different Bcl-2 family members. These findings could have implications for platelet collection and, potentially, for their clinical use.     

Growth ofEchinococcus multilocularis in gerbils exposed to different environmnntal temperature

Summary Gerbils kept at high (35°C) temperature harbored a significantly largerEchinococcus multilocularis cyst biomass than those kept at room (21°C) and low (5°C) temperature. Parasite induced adrenomegaly was observed in all temperature groups.Acknowledgments. I am indebted to Miss Marianne Hardy and Mr Terry Durham for their excellent technical assistance.     

A cold-active salmon goose-type lysozyme with high heat tolerance

The Atlantic salmon (Salmo salar) goose-type lysozyme gene was isolated and revealed alternative splicing within exon 2 affecting the signal peptide-encoding region. The lysozyme was produced in Escherichia coli, and the recombinant enzyme showed a high specific lytic activity that was stimulated by low or moderate concentrations of mono- or divalent cations. Relative lytic activities of 70 and 100% were measured at 4°C and 22°C, respectively, and there was no detectable activity at 60°C. However, 30% activity was retained after heating the enzyme for 3 h at 90°C. This unique combination of thermal properties was surprising since the salmon goose-type lysozyme contains no cysteines for protein structure stabilization through disulphide bond formation. The results point to a rapid reversal of inactivation, probably due to instant protein refolding. Received 14 August 2007; received after revision 07 September 2007; accepted 12 September 2007     

Stability analysis of the bacteriophage ϕKMV lysin gp36C and its putative role during infection

The kinetic, thermodynamic and structural stability of gp36C, the virion-associated peptidoglycan hydrolase domain of bacteriophage ϕKMV, is analyzed. Recombinant gp36C is highly thermoresistant (k = 0.595 h⁻¹ at 95°C), but not thermostable (T_m = 50.2°C, ΔH_cal = 6.86 × 10⁴ cal mol⁻¹). However, aggregation influences kinetic stability in an unusual manner since aggregation is more pronounced at 55°C than at higher temperatures. Furthermore, gp36C reversibly unfolds in a two-state endothermic transition, and circular dichroism analysis shows that gp36C almost completely refolds after a 3-h heat treatment at 85°C. These properties are in agreement with gp36C being part of the extensible tail which is ejected in an unfolded state during phage infection. Received 24 April 2006; received after revision 26 May 2006; accepted 10 June 2006     

Response of the starling circadian system to transitions between light and darkness

Summary The effects of exposure to sudden transitions from dark to light (D/L) and light to dark (L/D) were determined on the free running circadian feding rhythm of European stalings (Sturnus vulgaris). D/L transitions (step-up) produced phase advances throughout the circadian cycle. In contrast L/D transitions (step-down) produced both advances and delays. The L/D transition phase-response curve has a contour and shape similar to the phase-response curves previously obtained in birds with light pulses.     

The presence and function of melatonin and structurally related indoleamines in a dinoflagellate,and a hypothesis on the evolutionary significance of these tryptophan metabolites in unicellulars

The bioluminescent dinoflagellateGonyaulax polyedra contains various indoleamines, in particular, melatonin and 5-methoxytryptamine, as well as enzymes of their biosynthetic pathway. Melatonin exhibits a high-amplitude circadian rhythm characterized by a dramatic increase shortly after the onset of darkness. The maximum of melatonin is followed by a peak of 5-methoxytryptamine. These 5-methoxylated indoleamines seem to be involved in the mediation of the information darkness.G. polyedra shows a short-day response, which consists in the formation of asexual cysts. Light break experiments demonstrate the photoperiodic nature of this reaction. Cells become sensitive to short days only upon exposure to a lowered temperature (<16°C). Melatonin mimics the short-day effect, but only at decreased temperature. 5-Methoxytryptamine is even a better inducer of cyst formation, acting also at 20°C and in any lighting schedule, including LL. Cyst induction is associated with stimulation of bioluminescence and cytoplasmic acidification. A model on the intracellular pathway of photoperiodic information transduction assumes increased deacetylation of melatonin under cyst-inducing conditions, binding of 5-methoxytryptamine to the membrane of an acidic vacuole, proton transfer to the cytoplasm, and decreased intracellular pH as the stimulus for encystment. Melatonin shows the property of a scavenger of superoxide anions. This reaction, which is efficiently catalyzed by hemin, leads to the formation of a substituted kynuramine (AFMK). Destruction of melatonin by light-induced superoxide anions in the presence of cellular hemin may represent a property which, during evolution, has made this molecule suitable as an indicator of darkness. On the other hand, AFMK, which is formed under illumination, might have become a mediator of the information light. Photoperiodism inGonyaulax shows surprising parallels to that in mammals, but allows the analysis of this phenomenon at an entirely cellular level.     

Differential diurnal variations of anandamide and 2-arachidonoyl-glycerol levels in rat brain

The endogenous ligands of cannabinoid receptors, also known as endocannabinoids, have been implicated in many physiological and pathological processes of the central nervous system. Here we show that the levels of the two major endocannabinoids, anandamide and 2-arachidonoyl-glycerol (2-AG), in four areas of the rat brain, change dramatically between the light and dark phases of the day. While anandamide levels in the nucleus accumbens, pre-frontal cortex, striatum and hippocampus were significantly higher in the dark phase, the opposite was observed with 2-AG, whose levels were significantly higher during the light phase in all four regions. We found that the activity of the fatty acid amide hydrolase, which catalyzes the metabolism of anandamide, was significantly lower during the dark phase, thus providing a possible explaination for the increase in anandamide levels. However, the activities of monoacylglycerol lipase and diacylglycerol lipase, two of the possible enzymes catalyzing the degradation and biosynthesis of 2-AG, respectively, changed significantly only in the striatum. These data suggest that the levels of the two major endocannabinoids might be under the control of endogenous factors known to undergo diurnal variations, and underscore the different roles, suggested by previous studies, of anandamide and 2-AG in neurophysiological processes.Received 9 December 2003; received after revision 15 January 2004; accepted 20 January 2004     

Low ambient oxygen tolerance in some freshwater teleosts

Summary Measurements of the asphyxial oxygen level forRhinomugil corsula, Tilapia mossambica, Puntius sarana andCarassius auratus at 30 and 35°C revealed that at 35°C the lethal oxygen level was higher forT. mossambica andP. sarana and lower forR. corsula, but it remained the same forC. auratus at 30 and 35°C. From a thesis (M.P.M.) approved for the degree of Doctor of Philosophy, Madurai Kamaraj University, madurai, India. We wish to express our thanks to Prof. S. Krishnaswamy for providing facilities and to Dr S.V. Job for suggestions.     

Seasonal variations in the nyctohemeral rhythm of plasma testosterone in the European badger Meles meles L

In the badger a seasonal sexual rhythm of the plasma testosterone is observed. There is also a nycthemeral rhythm of the testosterone secretion; during the regressed period of the testis this rhythm is bimodal with testosterone peaks during the light phase of the day; during the active period and "activity rhythm" with many transitorys peaks during the dark phase is added.     

Development of melatonin rhythm in the pineal gland and eyes of chick embryo

A melatonin rhythm was observed in the pineals of 18-day-old chick embryos incubated under a light-dark regime of 186 h. A low pineal melatonin content was found during the light phase of the day. Concentrations started to increase 2 h after dark onset and reached maximum levels after 4 h of darkness. The amplitude of the pineal melatonin rhythm increased considerably after 2 days and night-time concentrations in 20-day-old embryos were more than 5 times higher than in 18-day-old ones. Significant day/night differences in melatonin production were found both in pineals and eyes. Exposure of eggs to 1 h of light during the dark period decreased the high melatonin concentrations in the eyes but not in the pineals of the 20-day-old chick embryo. The results suggest that in this precocial bird at least part of the circadian system may already operate during embryonic life.     

The effect of global warming on mortality

There is a significant relationship between ambient temperature and mortality. In healthy individuals with no underlying co-morbid conditions, there is an efficient heat regulation system which enables the body to effectively handle thermal stress. However, in vulnerable groups, especially in elderly over the age of 65 years, infants and individuals with co-morbid cardiovascular and/or respiratory conditions, there is a deficiency in thermoregulation. When temperatures exceed a certain limit, being cold winter spells or heat waves, there is an increase in the number of deaths. In particular, it has been shown that at temperatures above 27 °C, the daily mortality rate increases more rapidly per degree rise compared to when it drops below 27 °C.This is especially of relevance with the current emergency of global warming. Besides the direct effect of temperature rises on human health, global warming will have a negative impact on primary producers and livestock, leading to malnutrition, which will in turn lead to a myriad of health related issues. This is further exacerbated by environmental pollution.Public health measures that countries should follow should include not only health-related information strategies aiming to reduce the exposure to heat for vulnerable individuals and the community, but improved urban planning and reduction in energy consumption, among many others. This will reduce the carbon footprint and help avert global warming, thus reducing mortality.     

Development of melatonin rhythm in the pineal gland and eyes of chick embryo.

A melatonin rhythm was observed in the pineals of 18-day-old chick embryos incubated under a light-dark regime of 18: 6 h. A low pineal melatonin content was found during the light phase of the day. Concentrations started to increase 2 h after dark onset and reached maximum levels after 4 h of darkness. The amplitude of the pineal melatonin rhythm increased considerably after 2 days and night-time concentrations in 20-day-old embryos were more than 5 times higher than in 18-day-old ones. Significant day/night differences in melatonin production were found both in pineals and eyes. Exposure of eggs to 1 h of light during the dark period decreased the high melatonin concentrations in the eyes but not in the pineals of the 20-day-old chick embryo. The results suggest that in this precocial bird at least part of the circadian system may already operate during embryonic life.     

Studies in Babylonian lunar theory: part III. The introduction of the uniform zodiac

This paper is the third of a multi-part examination of the Babylonian mathematical lunar theories known as Systems A and B. Part I (Britton, AHES 61:83–145, 2007) addressed the development of the empirical elements needed to separate the effects of lunar and solar anomaly on the intervals between syzygies, accomplished in the construction of the System A lunar theory early in the fourth century B.C. Part II (Britton, AHES 63:357–431, 2009) examines the accomplishment of this separation by the construction of a successful theory depicting the variations due to lunar anomaly in System A and its subsequent adaptation in System B. The present paper examines the introduction of the uniform zodiac, necessary for any theory depicting variations depending on the position of syzygy. It addresses three questions: (1) In light of all available evidence, what is the magnitude of the constant term in the expression Δλ* = C ? 1.3828°Y, describing the difference between the Babylonian sidereal longitudes and modern tropical longitudes? (2) What considerations governed the placement of the Babylonian sidereal zodiac relative to the fixed stars? (3) When was the uniform zodiac introduced? To the first question it finds C = 3.20° ± 0.1°, scarcely different from Huber’s (Centaurus 5:192–208, 1958) estimate of 3.08°, essentially confirming Huber’s result obtained from much less data. For the second it shows that accommodating the three asterisms comprising Taurus limited the placement of the zodiac to within 3°, while the prominence of half sign multiples among the measured intervals between prominent Normal Stars led irresistibly to the choice adopted. Finally, it finds that the zodiac was introduced between ?408 and ?397 and probably within a very few years of ?400.     

Wirkung des Mondlichtes auf die Nachtaktivität von Zugvögeln. — Lotsenversuch an Rotkehlchen (Erithacus rubecula) und Gartenrotschwänzen (Phoenicurus phoenicurus)

Summary 8 Robins (Erithacus rubecula) and 3 Redstarts (Phoenicurus phoenicurus) were maintained in registration cages in autumn 1965. Locomotor activity was registered continuously under natural light conditions, temperature remaining fairly constant. All but one bird developed nocturnal restlessness (Zugunruhe) at least during the migratory season (Figure 1). 8 out of 10 birds showed a higher mean activity at night during full moon phases than during new moon phases; 2 birds showed roughly equal activity during both phases of the cycle (Figure 2). Since the measurements of nocturnal activity at least partially correspond to migratory motivation, it is reasonable to assume that free-living individuals of the species investigated are more active during nights in the full moon phase and possibly fly farther than at dark nights.     

Effect of light on variations in blood testosterone in the ram: demonstration of a photosensitive phase in diurnal rhythm]

6 groups each of 5 adult rams were subjected in light controlled pens to a pretreatment of normal variations in daylength during May and June. Then one group followed the normal daylength and the other five different "skeleton photoperiods" of 8 hrs of light per day consisting of: 7 hrs continuous light (7 L) + 1 hr light (1 L) situated at different times of the dark period (D) according to groups. Blood samples were taken once each week and hourly during one 24 hr period in June, July and September. Peripheral plasma testosterone measured by radio-immunoassay was used as an index of testicular activity. There was increase in the level of testosterone resuting from a rise in the number of peak release during the 24 hrs. In the different light treatments tested, only the treatment (7L+9D+1L)+7D) stimulated testosterone secretion with the pattern of testosterone of this group being similar to that of the control group. This indicates that there exists a photosensitive phase at 16 to 17 hrs after the beginning of the principal light (the subjective dawn).     

Untersuchungen zur Thermodynamik des peripheren Sehprozesses

Summary In a range from 6 to 30°C, the influence of temperature on the relation between the light intensity and the amplitude of the b-wave of the exposure potential has been investigated for the isolated frog retina. Between 10 and 25°C, the gross activation energy of the response to light leads to a temperature coefficient which shows that the formation of the b-wave is mainly checked by diffusion processes. While at 6°C the b-wave has vanished, a complete delayed off-response can still be registered.     

Cellular internalization of proinsulin C-peptide

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37°C for up to 1 h, and was reduced at 4°C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action. Received 31 October 2006; received after revision 27 December 2006; accepted 30 December 2006     

Single-cell recordings from chick pineal glands in vitro reveal ultradian and circadian oscillations

Evidence is clear that each melatonin-producing cell in the chick pineal gland contains a circadian oscillator that continues to function in vitro, resulting in a prominent day/night rhythm of melatonin secretion. The aim of the present investigation was to examine whether the circadian organization of the gland has an electrophysiological correlate. To this end, single-cell recordings were made from isolated chick pineal glands kept in vitro under a light/dark cycle of 12:12 h, identical to that of the donors, or under continuous light or darkness. In all the glands investigated, a very small percentage of cells exhibited sodium-dependent spontaneous spike activity with a mean frequency below 10 Hz. The cells revealed rhythms with periods in the 15- to 60-min range and, additionally, exhibited ultradian and circadian rhythms in firing, with periods of 10.75+/-1.06 h and 26.25+/-1.26 h (mean +/- standard deviation), respectively. Most of the cells exhibited circadian rhythms with higher activity during daytime than at night, showing that the electrical activity and melatonin rhythm were out of phase. Under constant light or darkness, the circadian rhythm persisted. When the light/dark cycle of the donors was phase-advanced by 5 h, the cells revealed complete entrainment. We discuss whether the cells, albeit small in number, could function as a secondary ultradian/circadian oscillator contributing to the ultradian/circadian organization of the gland.