The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne).

We have characterized a cold-induced, boiling stable antifreeze protein. This highly active ice recrystallization inhibition protein shows a much lower thermal hysteresis effect and displays binding behavior that is uncharacteristic of any AFP from fish or insects. Ice-binding studies show it binds to the (1 0 1 0) plane of ice and FTIR studies reveal that it has an unusual type of highly beta-sheeted secondary structure. Ice-binding studies of both glycosylated and nonglycosylated expressed forms indicate that it adsorbs to ice through the protein backbone. These results are discussed in light of the currently proposed mechanisms of AFP action.

Gastrin-releasing peptide phase-shifts suprachiasmatic nuclei neuronal rhythms in vitro.

The main mammalian circadian pacemaker is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Gastrin-releasing peptide (GRP) and its receptor (BB(2)) are synthesized by rodent SCN neurons, but the role of GRP in circadian rhythm processes is unknown. In this study, we examined the phase-resetting actions of GRP on the electrical activity rhythms of hamster and rat SCN neurons in vitro. In both rat and hamster SCN slices, GRP treatment during the day did not alter the time of peak SCN firing. In contrast, GRP application early in the subjective night phase-delayed, whereas similar treatment later in the subjective night phase-advanced the firing rate rhythm in rat and hamster SCN slices. These phase shifts were completely blocked by the selective BB(2) receptor antagonist, [d-Phe(6), Des-Met(14)]-bombesin 6-14 ethylamide. We also investigated the temporal changes in the expression of genes for the BB(1) and BB(2) receptors in the rat SCN using a quantitative competitive RT-PCR protocol. The expression of the genes for both receptors was easily detected, but their expression did not vary over the diurnal cycle. These data show that GRP phase-dependently phase resets the rodent SCN circadian pacemaker in vitro apparently via the BB(2) receptor. Because this pattern of phase shifting resembles that of light on rodent behavioral rhythms, these results support the contention that GRP participates in the photic entrainment of the rodent SCN circadian pacemaker.

Expression of mt(1) melatonin receptor subtype mRNA in the entrained rat suprachiasmatic nucleus: a quantitative RT-PCR study across the diurnal cycle.

Melatonin acts on specific receptors in the suprachiasmatic nuclei (SCN) to phase-dependently regulate the phase of the circadian clock. How the gating of melatonin's effect is restricted to particular times of day is not known, but may be related to temporal differences in receptor availability. In the present study, we used a competitive reverse transcription-polymerase chain reaction (RT-PCR) method to determine if the expression of mt(1) melatonin receptor subtype mRNA in rat SCN varied across the 12:12 light-dark (LD) cycle. Measurement of core body temperature using radiotelemetry confirmed that the male Wistar rats used exhibited a robust diurnal rhythm. mt(1) receptor mRNA was readily detected in reduced SCN slices at all times of day. However, there was no significant variation in the amount of mt(1) mRNA with time of day. Expression of MT(2) melatonin receptor subtype mRNA in reduced SCN slices was confirmed by nested PCR. These results indicate that changes in the level of mt(1) mRNA do not underlie the diurnal and/or circadian variation in the response of the SCN circadian clock to the phase-resetting effects of melatonin.

Sleep dysfunction in Rett syndrome: a trial of exogenous melatonin treatment.

Nine girls with Rett syndrome (mean age, 10.1 years) were monitored 24 hours a day over a period of 10 weeks using wrist actigraphy. Baseline sleep-wake patterns were assessed for 1 week. Subsequently, patients underwent a 4-week melatonin treatment period in a double-blind, placebo-controlled, crossover protocol that employed a 1-week washout between treatment trials. Melatonin doses ranged from 2.5 to 7.5 mg, based upon individual body weight. Baseline sleep quality was poor compared with healthy children. At baseline the group demonstrated a low sleep efficiency (mean [+/- SE], 68.0+/-3.9%), long sleep-onset latency (42.1+/-12.0 minutes), and a short and fragmented total sleep time (7.5+/-0.3 hours; 15+/-2 awakenings per night). Melatonin significantly decreased sleep-onset latency to (mean +/- SE) 19.1+/-5.3 minutes (P<0.05) during the first 3 weeks of treatment. While the variability of individual responsiveness was high, melatonin appeared to improve total sleep time and sleep efficiency in the patients with the worse baseline sleep quality. Finally, a 4-week administration of melatonin appears to be a safe treatment as no adverse side effects were detected, yet long-term effects of chronic melatonin use in pediatric patients are unknown at this time.

Thermoregulation in sick foals aged less than one week.

Metabolic rate, rectal temperature Tr and respiratory quotient (RQ) were determined in 16 sick foals, aged 0-182 h. The foals were categorized into three groups: premature, dysmature or those suffering from neonatal maladjustment syndrome. The mean metabolic rate of the premature foals was 71 watts per unit area of body surface (W m(-2)), significantly lower than that of the other two groups. The overall mean metabolic rate for the sick foals was 82 W m(-2), about 25% below that of healthy foals of similar age. Air temperature (Ta) was 9.5-26.3 degrees C, and several foals shivered despite the provision of additional 'warmth' and insulation. Values of Tr were below 38 degrees C until after 4 h postpartum. Thereafter, Tr averaged 38.1 degrees C but fluctuated widely. The RQ (mean value 0.83) was not correlated with Ta. The lower critical temperature for the sick foals was estimated at 24 degrees C, on average. Methods to prevent excessive heat loss from sick foals are suggested.

Melatonin action and signal transduction in the rat suprachiasmatic circadian clock: activation of protein kinase C at dusk and dawn.

Nocturnal synthesis of the pineal hormone melatonin (MEL) is regulated by the circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. We examined the hypothesis that MEL can feed back to regulate the SCN using a brain slice preparation from rat. We monitored the SCN ensemble firing rate and found that MEL advanced the time of peak firing rate by more than 3 h at restricted circadian times (CTs) near subjective dusk [CT 10-14 (10-14 h after lights on)] and dawn (CT 23-0) on days 2 and 3 after treatment. The effect of MEL at CT 10 was blocked by pertussis toxin. The protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate, reset the SCN firing rate rhythm with a profile of temporal sensitivity congruent with that of MEL. Two specific PKC inhibitors, calphostin C and chelerythrine chloride, independently blocked MEL-induced phase advances at each sensitive period. Furthermore, MEL administration increased PKC phosphotransferase activity transiently to 200% at CT 10 and CT 23, but not at CT 6. These data demonstrate that 1) MEL can directly modulate the circadian timing of the SCN within two windows of sensitivity corresponding to dusk and dawn; and 2) MEL alters SCN cellular function via a pertussis toxin-sensitive G protein pathway that activates PKC.

Metabolic rate of sleeping infants.

AIM: To measure the sleeping metabolic rate (SMR) of healthy infants in the first year of life. METHODS: The SMR was measured on 73 infants aged 1 to 12 months in a special nursery using indirect calorimetry. One hundred satisfactory observations were made. The room air and radiative temperatures, humidity, and amount of insulation were measured. Parents chose the clothing and bedding that they judged their infant needed. RESULTS: The mean (SD) SMR was 2.4 (0.4) watts (W)/kg or 45 (10) W/m2. The mean SMR of infants aged 1-2 months was 38 compared with 44 W/m2 in infants of 8-12 months; the difference was not significant. There were no obvious differences in SMR between boys and girls. But there were wide differences in SMR between apparently similar infants, range 1.4 to 3.5 W/kg. Most parents selected insulation between 1 and 3 togs, and this was weakly negatively correlated with air temperature. CONCLUSION: These wide variations in SMR mean that it is impossible to give specific guidelines on the amount of clothing and bedding a particular infant will need for thermal comfort in a given room temperature.

Individual variation in sleeping metabolic rates in infants.

AIM: To measure the variation in sleeping metabolic rate (SMR) of healthy infants in the first year of life. METHODS: Attempts were made to measure SMR in the home of one group of infants at monthly intervals over the first year of life and of another group in the 1 to 12 age range, over three consecutive days. Sixty one infants were recruited and 181 measurements made. In 11 infants six or more satisfactory measurements were made a monthly intervals and in another 11 infants, three consecutive daily measurements were obtained. Parents chose the clothing and bedding that they judged their infant needed to fall asleep. The room air and radiative temperatures, humidity, and amount of insulation were recorded. RESULTS: The mean (SD) SMR was 2.4 (0.4) watts (W)/kg or 45 (10) W/m2. The rate was the same for infants in the age groups 0.25, 0.5, 0.75, and 1 years; individual infants did not show a systematic change with age. There was wide variation. Ninety eight per cent of infants had a SMR between 1.8 and 3.1 W/kg. The mean SMR for the infants measured on three consecutive days varied from 1.7 to 3.1 W/kg, and SMR varied within infants, from 0.06 to 0.86 W/kg. The variation within six to 11 measurements on infants over the first year of life ranged from 0.38 to 1.05 W/kg. CONCLUSION: Infants in the first year of life exhibit wide variations in SMR from day to day and from month to month. As the range is wide, guidance on clothing and bedding for thermal comfort can be given only in broad terms.

Non-24-hour sleep-wake syndrome in a sighted man: circadian rhythm studies and efficacy of melatonin treatment.

The case of a 41-year-old sighted man with non-24-hour sleep-wake syndrome is presented. A 7-week baseline assessment confirmed that the patient expressed endogenous melatonin and sleep-wake rhythms with a period of 25.1 hours. We sought to investigate the underlying pathology and to entrain the patient to a normal sleep-wake schedule. No deficiency in melatonin synthesis was found. Furthermore, normal coupling between the melatonin and sleep propensity rhythms was documented using an "ultrashort" sleep-wake protocol. Environmental light exposure was monitored for 41 days, and the circadian timing was calculated. Sensitivity to photic input was determined with light-induced melatonin-suppression tests. Three intensities (500, 1,000, and 2,500 lux) were examined during three separate trials. The 2,500-lux trial resulted in 78% suppression, but the lesser intensity exposures were without substantial effect. Thus, the patient appeared to be subsensitive to bright light. A 4-week trial of daily melatonin administration (0.5 mg at 2100 hours) stabilized the endogenous melatonin and sleep rhythms to a period of 24.1 hours, albeit at a somewhat delayed phase. A 14-month follow-up interview revealed that the patient continued to take melatonin daily, and his sleep-wake schedule was stable to a near 24-hour schedule.

Circadian actions of melatonin at the suprachiasmatic nucleus.

The biological clock in the suprachiasmatic nucleus (SCN) of the hypothalamus plays a well-defined role in regulating melatonin production by the pineal. Emerging evidence indicates that melatonin itself can feed back upon the SCN and thereby influence circadian functions. Melatonin administration has been shown to entrain activity rhythms in rodents and humans. Melatonin binds specifically within the SCN and alters SCN physiology by both acute and clock-resetting mechanisms. The circadian clock in the SCN appears to temporally restrict its own sensitivity to melatonin, such that physiological sensitivity is greatest in the subjective dusk period.

Intrinsic neuronal rhythms in the suprachiasmatic nuclei and their adjustment.

The central role of the suprachiasmatic nuclei in regulating mammalian circadian rhythms is well established. We study the temporal organization of neuronal properties in the suprachiasmatic nucleus (SCN) using a rat hypothalamic brain slice preparation. Electrical properties of single neurons are monitored by extra-cellular and whole-cell patch recording techniques. The ensemble of neurons in the SCN undergoes circadian changes in spontaneous activity, membrane properties and sensitivity to phase adjustment. At any point in this cycle, diversity is observed in individual neurons' electrical properties, including firing rate, firing pattern and response to injected current. Nevertheless, the SCN generate stable, near 24 h oscillations in ensemble neuronal firing rate for at least three days in vitro. The rhythm is sinusoidal, with peak activity, a marker of phase, appearing near midday. In addition to these electrophysiological changes, the SCN undergoes sequential changes in vitro in sensitivities to adjustment. During subjective day, the SCN progresses through periods of sensitivity to cyclic AMP, serotonin, neuropeptide Y, and then to melatonin at dusk. During the subjective night, sensitivities to glutamate, cyclic GMP and then neuropeptide Y are followed by a second period of sensitivity to melatonin at dawn. Because the SCN, when maintained in vitro, is under constant conditions and isolated from afferents, these changes must be generated within the clock in the SCN. The changing sensitivities reflect underlying temporal domains that are characterized by specific sets of biochemical and molecular relationships which occur in an ordered sequence over the circadian cycle.

Activity of the plasma membrane H(+)-ATPase is a key physiological determinant of thermotolerance in Saccharomyces cerevisiae.

The role of membrane integrity and the membrane ATPase in the mechanism of thermotolerance in Saccharomyces cerevisiae was investigated. The resistance to lethal heat of a mutant strain with reduced expression of the membrane ATPase was significantly less than that of the wild-type parent. However, prior exposure to sub-lethal temperatures resulted in the induction of similar levels of thermotolerance in the mutant compared to the parent strain, suggesting that the mechanism of sub-lethal heat-induced thermotolerance is independent of ATPase activity. Supporting this, exposure to sub-lethal heat stress did not result in increased levels of glucose-induced acid efflux at lethal temperatures and there was little correlation between levels of acid efflux and levels of heat resistance. ATPase activity in crude membrane preparations from sub-lethally heat-stressed cells was similar to that in preparations from unstressed cells. Study of net acid flux during heating revealed that pre-stressed cells were able to protect the proton gradient for longer. This may confer an 'advantage' to these cells that results in increased thermotolerance. This was supported by the observation that prior exposure to sub-lethal heat resulted in a transient protection against the large increase in membrane permeability that occurs at lethal temperatures. However, no protection against the large drop in intracellular pH was detected. Sub-lethal heat-induced protection of membrane integrity also occurred to the same extent in the reduced-expression membrane ATPase mutant, further implying that the mechanism of induced thermotolerance is independent of ATPase activity. To conclude, although the membrane ATPase is essential for basal heat resistance, thermotolerance induced by prior exposure to stress is largely conferred by a mechanism that is independent of the enzyme.

Do the suprachiasmatic nuclei oscillate in old rats as they do in young ones?

The basis of the decline in circadian rhythms with aging was addressed by comparing the patterns of three behavioral rhythms in young and old rats with the in vitro rhythm of neuronal activity in the suprachiasmatic nuclei (SCN), the primary circadian pacemaker. In some old rats, rhythms of body temperature, drinking, and activity retained significant 24-h periodicities in entraining light-dark cycles; in others, one or two of the rhythms became aperiodic. When these rats were 23-27.5 mo old they were killed, and single-unit firing rates in SCN brain slices were recorded continuously for 30 h. There was significant damping of mean peak neuronal firing rates in old rats compared with young. SCN neuronal activities were analyzed with reference to previous entrained behavioral rhythm patterns of individual rats as well. Neuronal activity from rats with prior aperiodic behavioral rhythms was erratic, as expected. Neuronal activity from rats that were still maintaining significant 24-h behavioral rhythmicity at the time they were killed was erratic in most cases but normally rhythmic in others. Thus there was no more congruence between the behavioral rhythms and the brain slice rhythms than there was among the behavioral rhythms alone. These results, the first to demonstrate aberrant SCN firing patterns and a decrease in amplitude in old rats, imply that aging could either disrupt coupling between SCN pacemaker cells or their output, or cause deterioration of the pacemaking properties of SCN cells.

The organization of the suprachiasmatic circadian pacemaker of the rat and its regulation by neurotransmitters and modulators.

The long-term goal of our research is to understand how cells of the suprachiasmatic nucleus (SCN) are organized to form a 24-hr biological clock, and what roles specific neurotransmitters and modulators play in timekeeping and resetting processes. We have been addressing these questions by assessing the pattern of spontaneous neuronal activity, using extracellular and whole-cell patch recording techniques in long-lived SCN brain slices from rats. We have observed that a robust pacemaker persists in the ventrolateral region of microdissected SCN, and have begun to define the electrophysiological properties of neurons in this region. Furthermore, we are investigating changing sensitivities of the SCN to resetting by exogenous neurotransmitters, such as glutamate, serotonin, and neuropeptide Y, across the circadian cycle. Our findings emphasize the complexity of organization and control of mammalian circadian timing.

Melatonin directly resets the rat suprachiasmatic circadian clock in vitro.

The environmental photoperiod regulates the synthesis of melatonin by the pineal gland, which in turn induces daily and seasonal adjustments in behavioral and physiological state. The mechanisms by which melatonin mediates these effects are not known, but accumulating data suggest that melatonin modulates a circadian biological clock, either directly or indirectly via neural inputs. The hypothesis that melatonin acts directly at the level of the suprachiasmatic nucleus (SCN), a central mammalian circadian pacemaker, was tested in a rat brain slice preparation maintained in vitro for 2-3 days. Exposure of the SCN to melatonin for 1 h late in the subjective day or early subjective night induced a significant advance in the SCN electrical activity rhythm; at other times melatonin was without apparent effect. These results demonstrate that melatonin can directly reset this circadian clock during the period surrounding the day-night transition.

Metabolic changes in thoroughbred and pony foals during the first 24 h post partum.

Metabolic rates, rectal temperatures and respiratory quotients (RQ) were determined during the first 24 h post partum in Thoroughbred and pony foals and in relation to environmental temperature. Both breeds had high metabolic rates (greater than 200 W/m2) during the first hour post partum when they were wet and shivering; by 4 to 24 h values were fairly steady at about 153 and 105 W/m2 for Thoroughbred and pony foals, respectively. At birth, rectal temperature was lower in Thoroughbred foals than in pony foals; values increased during the first hour, and then decreased. At 24 h of age, both breeds had a rectal temperature of 38.4 degrees C. RQ values were high (greater than 0.9) at birth in both breeds, declining to about 0.82 by 4 to 24 h of age. At similar environmental temperatures, the metabolic rates per unit area of Thoroughbred foals were higher than those of pony foals at all ages. The results indicate that breed differences may exist between the thermal responses of newborn Thoroughbred and pony foals.

cGMP induces phase shifts of a mammalian circadian pacemaker at night, in antiphase to cAMP effects.

The suprachiasmatic nuclei (SCN) of mammals contain a circadian clock that synchronizes behavioral and physiological rhythms to the daily cycle of light and darkness. We have been probing the biochemical substrates of this endogenous pacemaker by examining the ability of treatments affecting cyclic nucleotide-dependent pathways to induce changes in the phase of oscillation in electrical activity of rat SCN isolated in brain slices. Our previous work has shown that daytime treatments that stimulate cAMP-dependent pathways induce phase shifts of the SCN pacemaker in vitro but treatments during the subjective night are without effect. In this study we report that the phase of SCN oscillation is reset by treatments that stimulate cGMP-dependent pathways, but only during the subjective night. Thus, the nocturnal period of SCN sensitivity to cGMP is in antiphase to the diurnal period of sensitivity to cAMP. These results suggest that cAMP and cGMP affect the SCN pacemaker through separate biochemical pathways intrinsic to the SCN. These studies provide evidence that changing biochemical substrates within the SCN circadian clock may underlie some aspects of differential temporal sensitivity of mammals to resetting stimuli.

Thermal interaction between animal and microclimate: a comprehensive model.

An equation based on heat transfer theory is developed to predict the rate of heat loss from a homeothermic vertebrate to the environment, specified by the air temperature, humidity, windspeed and radiation receipt. The analysis incorporates the animal's thermoregulatory responses--sweating ability, vasomotor action, and regulation of body-core temperature, metabolic and respiratory rate. The loss of heat and water vapour from cattle is used as an illustration, and particular attention is given to their heat balance in hot environments. The predicted rates of heat loss from cattle indoors at various air temperatures and humidities are consistent with experiments. Outdoors, intercepted solar radiation can reduce substantially heat loss through the body tissue when the air temperature is low. In contrast, at high air temperatures the heat dissipation may not be sensitive to the radiation load, although body-core temperature is. Increased rates of air movement can aggravate strain at low air temperatures, but mitigate strain in a hot environment.

Air movement and heat loss from sheep. I. Boundary layer insulation of a model sheep, with and without fleece.

A model sheep, made from metal cylinders and hemispheres, was heated electrically. Heat loss by forced convection in a wind tunnel was analysed in terms of the dependence of the Nusselt number (Nu) on Reynolds number (Re). For a bare trunk Nu = 0.095 Re0.684, but with fleece covering the trunk to a depth of 3.5 cm, Nu = 0.0112 Re0.875 when the mean radiative temperature of the the coat was taken as the surface temperature. Heat transfer by convection from the whole body, including legs, was described by Nu = 0.029 Re0.80. However, a bulk Nesselt number should not be used to estimate heat loss from a live sheep in a hot environment if the windspeed is below about 4 m s-1 because the relation between mean surface temperature, Nusselt number and convective heat flux is not unique.