NADP-glutamate dehydrogenase activity in nonstarter lactic acid bacteria: effects of temperature, pH and NaCl on enzyme activity and expression.

AIMS: To screen the glutamate dehydrogenase (GDH) activity of nonstarter lactic acid bacteria (NSLAB) and to determine the effects of temperature, pH and NaCl values used for cheese ripening on enzyme activity and expression of GDH gene. METHODS AND RESULTS: A subcellular fractionation protocol and specific enzyme assays were used. The effect of temperature, pH and NaCl on enzyme activity was evaluated. The expression of GDH gene was monitored by real-time PCR. One selected strain was also used as adjunct starter for cheese making to evaluate the catabolism of free amino acids and the production of volatile organic compounds (VOC) during cheese ripening. The cytoplasm fraction of all strains showed in vitro NADP-dependent GDH activity. NADP-GDH activity was markedly strain dependent and varied according to the interactions between temperature, pH and NaCl. Lactobacillus plantarum DPPMA49 showed the highest NADP-GDH activity under temperature, pH and NaCl values found during cheese ripening. RT-PCR analysis revealed that GDH expression of Lact. plantarum DPPMA49 was down-expressed by low temperature (<13°C) and over-expressed by NaCl (1·87-5·62%). According to NADP-GDH activity, the highest level of VOC (alcohols, aldehydes, miscellaneous and carboxylic acids) was found in cheeses made with DPPMA49. CONCLUSIONS: The results of this study may be considered as an example of the influence of temperature, pH and NaCl on enzyme activity and expression of functional genes, such as GDH, in cheese-related bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: It focuses on the phenotypic and molecular characterization of the NADP-GDH in lactobacilli under cheese-ripening conditions. The findings of this study contribute to the knowledge about enzymes involved in the catabolism of amino acids, to be used as an important selection trait for cheese strains.

Respiratory effects of preoptic-anterior hypothalamic electrical stimulation during sleep in cats.

The respiratory effects of preoptic-anterior hypothalamic (PO-AH) and vagal repetitive electrical stimulation (RES) were studied in unrestrained cats during sleep. PO-AH RES influenced breathing consistently during synchronized sleep but only occasionally during desynchronized sleep. Induction of desynchronized sleep by PO-AH RES was also observed. The respiratory effects of PO-AH RES on one side were enhanced by concomitant PO-AH warming on the other side during synchronized sleep but not during desynchronized sleep. Breathing was affected by vagal RES during both sleep stages, although an increase in threshold and in variability of respiratory responses was apparent during the latter sleep stage. The results show that PO-AH unresponsiveness during desynchronized sleep is nonspecific.

Selective brain cooling is impaired in REM sleep.

There are systemic and selective mechanisms for brain cooling in mammals. The difference between the temperatures of the vertebral and the carotid blood perfusing the brain is determined by selective heat loss and is, therefore, a quantitative indicator of the intensity of selective brain cooling. Across the wake-sleep cycle systemic and selective brain cooling are affected by state-dependent autonomic changes. In REM sleep selective brain cooling is impaired.

Behavioral state-dependent thermal feedback influencing the hypothalamic thermostat.

The experimental evidence on the behavioral state-dependent compartmentalization of temperature in the central nervous system of three homeothermic species has been reviewed to address the question of how selective brain cooling influences hypothalamic temperature regulation.

Cold-defense function of brown adipose tissue during sleep.

Rats with chronically implanted electroencephalograph scalp electrodes and thermistors were exposed to 24 and 4 degrees C ambient temperatures during the light hours before and after acclimation to 4 degrees C ambient temperature for 9 days. During synchronized sleep, deep interscapular temperature was higher at 4 degrees C than at 24 degrees C both before and after acclimation to cold. After ablation of brown adipose tissue, deep interscapular temperature was lower at 4 degrees C than at 24 degrees C during synchronized sleep. In the presence of brown adipose tissue, deep interscapular temperature decreased sharply during desynchronized sleep at 4 degrees C both before and after acclimation to cold. This decrease was subsequent to and correlated with an increase in the temperature of the nasal mucosa. The decrease in deep interscapular temperature during desynchronized sleep at 4 degrees C ambient temperature was markedly reduced by ablation of the interscapular brown adipose tissue.

Septal influences on the activity of hippocampal neurones during sleep.

Septal influences on CA1 and DG neurones were studied in sleeping cats before and after septal lesions suppressing the theta rhythm. Septal influences tonically gauge the activity level of a discrete group of CA1 and DG neurones, regardless of sleep stages. The observed splitting of each CA1 and DG population into two groups of units showing opposite changes in firing rate from SS to DS may be related to extraseptal inputs.

[Depression of responsivity of the preoptic region to repetitive electric stimulation during desynchronized sleep]. / Depressione della responsività della regione preottica alla stimolazione elettrica iterativa durante il sonno desincronizzato.

Respiratory effects of preoptic and vagal repetitive electrical stimulation were studied in unrestrained cats during sleep. Preoptic stimulation influenced breathing during synchronized sleep, but not during desynchronized sleep. Breathing was affected by vagal stimulation during both synchronized and desynchronized sleep, although an increase in threshold was apparent during the latter stage of sleep. The results show that the depression of preoptic responsiveness during desynchronized sleep is non-specific.

Absence of thermoregulatory vasomotor responses during fast wave sleep in cats.

The skin temperature of the pinna was recorded in unrestrained cats sleeping at different ambient temperatures. The results show that during fast wave sleep vasomotion is inconsistent with homeothermic regulation as ear skin temperature increases and decreases at low and high ambient temperatures, respectively. The effects of preoptic heating during slow wave and fast wave sleep reveal that a depression of the responsiveness of hypothalamic thermoceptive structures underlies the absence of thermoregulatory vasomotor responses during fast wave sleep.

A pontine-hypothalamic temperature difference correlated with cutaneous and respiratory heat loss.

The role of cutaneous and respiratory heat loss for selective brain cooling in different species is discussed and new experimental results from a comparative study are summarized. In three species (cat, rabbit and rat) the difference between pontine and hypothalamic temperatures was studied as a function of head heat exchanger vasomotion appraised by the difference between hypothalamic and ear pinna (cats and rabbits) or nasal mucosa (rats) temperatures during the behavioral states of wakefulness and slow wave sleep at an ambient temperature of 24+/-1 degrees C. The results show that: (i) the pontine-hypothalamic temperature difference is an useful indicator of selective brain cooling since it is positive and inversely correlated with the hypothalamic-ear pinna temperature difference in cats and rabbits and with the hypothalamic-nasal mucosa temperature difference in rats; (ii) respiratory heat loss prevails quantitatively over cutaneous heat loss in maintaining this difference.

[Hypothalamic temperature and thermogenesis of brown fat during desynchronized sleep in rats]. / Temperatura ipotalamica e termogenesi del grasso bruno durante il sonno desincronizzato del ratto.

The relationship between hypothalamic temperature and deep interscapular temperature measured just below the brown fat lobes has been studied during desynchronized sleep at two ambient temperatures (24 degrees C and 4 degrees C) before and after adaptation (9 days) to cold (4 degrees C). The results show that the increase in hypothalamic temperature during this stage of sleep occurs independently of a transfer of heat from interscapular brown fat.

[Deep interscapular temperature and thermogenesis of brown fat during sleep]. / Temperatura interscapolare profonda e termogenesi del grasso bruno durante il sonno.

Deep interscapular temperature measured just below the brown fat lobes was studied in rats during sleep at two ambient temperatures (24 degrees C and 4 degrees C) before and after adaptation (9 days) to cold (4 degrees C). The results show that in the cold ambient deep interscapular temperature decreases during desynchronized sleep independently of adaptation. Such change in temperature is probably the result of the depression in sympathetic vasoconstrictor influences on heat exchangers producing blood and brown fat cooling in sequence during this stage of sleep.

The influence of the light-dark and wake-sleep cycles on preoptic cAMP concentration in the rat.

The concentration of cAMP was measured in the preoptic region of rats sleeping at normal laboratory conditions (12:12 h LD, Ta 22 +/- 0.5 degrees C). The results show that the nucleotide concentration changed both during the circadian LD cycle and during the ultradian wake-sleep cycle.

The effect of propranolol on cAMP concentration in the rat preoptic region during the wake-sleep cycle.

In control conditions preoptic cAMP concentration during wakefulness was significantly higher than during synchronized sleep. No differences in nucleotide concentration were observed in the cerebral cortex. Propranolol decreases brain cAMP concentration. This change was associated with the suppression of the difference observed between wakefulness and synchronized sleep in the preoptic region.

Quantitative analysis of short term deprivation and recovery of desynchronized sleep in cats.

A quantitative analysis of desynchronized sleep (DS) deprivation (exposure to low ambient temperature) and recovery was carried out in unrestrained cats. The results show that: (1) the circadian obligate quota of DS is precisely regulated by a control mechanism paying by the 24th h the DS debt induced by up to 14 h of total DS deprivation, if recovery occurs during the rest phase of circadian photoperiodicity (CP); (2) during static rebound DS hourly duration is increased by 40 +/- 4 sec/h of total deprivation (mean and S.E.) and there is a statistically significant increase in the hourly frequency and in the duration of DS episodes, with respect to control values; (3) the hourly obligate and facultative quotas of DS amount to 409 +/- 82 and 229 +/- 140 sec/h (mean and S.D.), respectively. (4) preoptic heating during deprivation at low ambient temperature decreases the DS static rebound during recovery at control ambient temperature by an amount corresponding to the cumulative duration of DS episodes elicited by the preoptic treatment; (5) preoptic temperature decreases the more markedly the shorter the duration of exposure to low ambient temperature and regains control values the sooner the smaller the DS debt incurred during deprivation; (6) DS debt cumulation is a continuous process which develops at a steady rate during day and night, whereas DS debt payment is a discontinuous process (ultradian rhythm of DS) which develops at a variable rate depending on the DS debt and on the phase of CP.

[Quantitative study of desynchronized sleep recovery after short term total deprivation in the cat]. / Studio quantitativo del recupero del sonno desincronizzato dopo deprivazione totale di breve durata nel gatto.

The results show that the cumulative hourly duration of desynchronized sleep during recovery is increased with respect to the control value of 40 +/- 4 sec per hour of total deprivation.

[Changes in the ultradian rhythm of desynchronized sleep after short term total deprivation in the cat]. / Modificazione del ritmo ultradiano del sonno desincronizzato dopo deprivazione totale di breve durata nel gatto.

The results show a statistically significant increase in the hourly frequency and in the duration of desynchronized sleep episodes during recovery with respect to control values.

[Effect of heating of the preoptic area during deprivation of successive desynchronized sleep recovery in the cat]. / Effetto del riscaldamento della regione preottica durante deprivazione sul successivo recupero di sonno desincronizzato nel gatto.

The results show that preoptic heating during deprivation at low ambient temperature decreases the desynchronized sleep rebound during recovery at control ambient temperature by an amount corresponding to the cumulative duration of desynchronized sleep episodes elicited by the preoptic treatment.