Molecular cloning and characterization of a thermostable esterase/lipase produced by a novel Anoxybacillus flavithermus strain.

A thermophilic strain producing an extracellular esterase/lipase was isolated from a hot spring in Tasnad, Romania, and was identified phenotypically and by 16S rDNA sequencing as Anoxybacillus flavithermus (GenBank ID: JQ267733). The gene encoding the putative carboxyl esterase (GenBank ID: JX494348) was cloned by direct PCR amplification from genomic DNA. The protein, consisting of 246 amino acids and having a predicted molecular weight of 28.03 kDa, is encoded by an ORF of 741 bps. Expression was achieved in Escherichia coli and a recombinant protein with esterolytic activity and estimated molecular weight of 25 kDa was recovered and purified from the periplasmic fraction by IMAC. The purified enzyme, most active at 60-65°C and in the near-neutral range (pH 6.5-8), displayed a half-life at 60°C of about 5 h. Est/Lip displayed a relative tolerance to methanol, DMSO, acetonitrile, and low detergent concentrations (SDS, Triton) increased its thermostability. Highest activity was attained with p-nitrophenyl butyrate, but the enzyme was also able to hydrolyze long chain fatty acid esters, as well as triolein. The primary sequence and predicted tridimensional structure of the enzyme are very similar to those of other Anoxybacillus and Geobacillus carboxyl esterases in a distinct, recently described lipase family. Est/Lip was highly enantioselective, with preference for the (S)-enantiomer of substrates.

Modulatory factors of circadian phagocytic activity.

Basal phagocytosis of neutrophils, a crucial component of non-specific immunity, is an eminently circadian parameter. In mice and rats, rates of phagocytosis peak in the second half of the dark span. A lipopolysaccharide-induced phagocytic response challenged in this period appears the most significant in amplitude and duration. Neonatal administration in rats of the neurotoxic agent monosodium glutamate, which induces massive destruction of the arcuate nucleus, suppresses the phagocytic response and moderately inhibits basal phagocytosis. Physiological phagocytosis in vivo appears to depend on the presence of the nocturnal melatonin surge. Functional pinealectomy, achieved in rats by a 2-week exposure to constant light, lowered the average circadian level of phagocytosis, damped the characteristic rhythm of neutrophil adherence, and decreased the neutrophil count and the amplitude of its circadian oscillation. In an in vivo study, adult rats were given alcohol intraperitoneally (0.1 mL ethanol/kg body weight, 1:10 in saline solution), alone or co-administered with melatonin (1 mg/kg body weight), for 16 days, once a day at 20:00 h. Alcohol-treated animals displayed a drastically depressed and flattened phagocytic curve, a marked decline of the adherence ability, and a reduction of the mean circadian neutrophil and lymphocyte count. Addition of melatonin significantly increased circadian values of phagocytosis, restored adherence, and prevented the numeric depletion of lymphocytes induced by alcohol. In correlation with other experimental evidence, our data speak for a physiological role of melatonin in upkeep of neutrophil phagocytosis and hint towards the existence of several pineal-hypothalamic pathways regulating different components of phagocytosis in vivo.

Stress/strain/life revisited. Quantification by blood pressure chronomics: benetensive, transtensive or maletensive chrono-vasculo-neuro-immuno-modulation.

We propose to initiate the automatic self-assessment of wear and tear as "stress and strain" by the time structures of blood pressure (BP) and heart rate (HR), in order to arrive eventually at an individualized timely and timed routine of life and to early preventive intervention as soon as needed. The routine may involve physiological scheduling of physical and mental activities and meals, and if need be of non-drug or drug treatment for stress amplification, e.g., by exercise, and/or strain (not stress) relief by relaxation. In so doing, we recognize the circulation as a pillar and marker of preventive and active neuroimmunomodulation (NIM), suggesting that some concerns of a vasculo- and broader NIM can be quantified by transdisciplinary chronobiology using its cartography--chronomics--of time structures, i.e., chronomes, from chronos = time and nomos = rule. Thus, we are introducing the chronomics of BP, HR and of other variables in the historical context of pioneers who were indispensable to experimental medicine. We build upon their contributions, but we must point out when, in the past, by necessity rather than choice, the giants provided rationalizing truisms that are no substitute for systematic serial data collection and appropriate computer analysis. A time-unspecified spotcheck as a baseline is much better than no measurement, but very often it is not enough, and it is always insufficient when an estimate of variability constitutes the information needed. For dynamic cycles, there are only reference cycles as a routine, although when maps are available, single timed spotchecks can be invaluable. With reference to their historical context, here we rely only upon data which necessity, rather than philosophy, compels us to collect.

Hypothalamic mechanisms of immunity.

The present article considers a synthetical analysis of the results reported by our laboratory in the last twenty years in the field of neuroimmunomodulation. The studies we discuss here continue a previous research activity, a synthesis of which has also been published in this journal (Baciu, 1988). In that paper, we reported data concerning the role of the hypothalamic tubero-mammillary area in triggering of the phagocytic and of the secondary immune specific response. Here, we present an analysis of experimental facts gathered after 1988, and also of some prior to that date, which were not included in the above-mentioned review. They regard localizations, attained with stereotactical methods, of hypothalamic areas involved in maintenance of basal phagocytosis and of its circadian rhythm, of the phagocytic and of the primary and secondary specific response. We attempted to re-analyze these data in an integrative view, and accomplish a coherent image of the hypothalamic mechanisms of the nonspecific and specific immune response. The conclusion we draw is that the nervous system may exert its modulatory action upon the immune response in several ways: i) subsequent to a direct hypothalamic stimulation (electrical or through bacteria or bacterial products) or to a cortico-hypothalamic stimulation; ii) depending on the nature, intensity, duration, and frequency of the appropriate stimulus, it may either enhance the immune response, via neural and humoral pathways, or depress it; iii) via the hypothalamus-pituitary-adrenal axis. Nervous triggering and enhancement of the immune response are essential, their occurrence in the initial stages ensuring its favorable course. The finding that repeated electroconvulsant shocks, employed for hypothalamus stimulation in dogs of different breed, age, weight, and individual history, are followed by extremely variable changes of the phagocytic activity raises the question on the individuality of the immune response.

The sympatho-adrenal response and erythropoietin production in adaptation to hypoxia.

The research activity upon erythropoiesis regulation carried out by the team in the Physiology Department and in the Institute of Medical Research of the Romanian Academy in Cluj-Napoca developed continuously after 1950. Our studies contributed to the isolation, identification and characterization of erythropoietin (Epo) and also to a better understanding of the nervous adaptation mechanisms to hypoxia. At present, it is well known that hypoxia acts upon erythropoiesis through Epo production. Direct central nervous stimulation through hypoxia induces, via a neuro-humoral mechanism, a sympatho-adrenal response and release of Epo. Adaptive polyglobulia as a response to hypoxia increases the capacity of oxygen binding and transport. In this paper we attempted to identify the role of the sympathetic nervous system in adaptation to hypoxia correlated with Epo secretion. Experiments were carried out in three groups of rats, respectively, with cervical, thoracic, and lumbar (without celiac) sympathectomy. The sympathectomized animals were submitted to hypobaric or to hemorrhagic hypoxia, in parallel with control groups. Erythrocytic parameters (red blood cells, reticulocytes, hematocrit, and haemoglobin) were repeatedly assayed during the following 2-4 weeks. The results showed that animals with cervical sympathectomy adapt in a deficient manner to hypoxia; lacking the adaptive sino-carotid reflexes, adaptation occurs through increased Epo secretion, animals with cervical sympathectomy having higher counts of reticulocytes and of red blood cells at the end of experiment than intact animals. Thoracic sympathectomy has little influence upon the erythrocytic response, as the largest part of the respiratory and circulatory sympathetic reactions occur via the cervical sympathetic nerve. Lumbar sympathectomy without removal of the celiac ganglion does not decrease the erythrocytic response as expected; on the contrary, the erythrocytic response is increased as compared to controls.

Neutrophil adherence in rats submitted to light-dark alternance and to constant light.

A good amount of experimental data suggests the existence of a circadian control of the inflammatory process. It was shown that migration of neutrophils in chemotactic gradient, ingestion of particles, vascular permeability etc. are rhythmical circadian functions. Melatonin, the pineal hormone secreted during the darkness phase, has been shown to be involved in the control of inflammation. The present study aims to assess whether neutrophil adherence to nylon fibers exhibits circadian rhythmicity and also if its amplitude and/or chronostructure are altered in a constant light regimen. Wistar rats were submitted to either an artificial light-darkness 12/12 regimen (LD) or to constant light (LL), for 15 days. Adherence of the neutrophils in whole blood was assessed at 10:00, 16:00, 22:00, and 04:00 hrs. In LD. neutrophil adherence appears to be a rhythmic, biphasic function, with the acrophase at 10:00, a secondary peak at 22:00 and trough values in the late dark hours. Constant light induces a depression of the adherence ability by about 10%, except for the 04:00 hrs point, where the value in LL is higher than in LD. The fact that adherence and phagocytic activity do not oscillate in phase suggests that the physiological relevance of neutrophil adherence goes beyond that of a first stage of the phagocytic process.

Circadian phagocytic activity in rats under light-dark and constant light regimens.

The phagocytic function was proved to be a periodic, circadian process. Its acrophase appears to be differently timed in species with different activity type, occurring in the evening in diurnal species and at night in nocturnal ones. The main pineal hormone melatonin, whose secretion occurs strictly at dark, has been shown to play a role in the control of inflammation and to exert a certain stimulatory effect upon phagocytosis in vitro. The aim of the present study was to assess whether the phagocytic activity of neutrophils in the blood of rats exhibits a circadian rhythmicity similar to that of other nocturnal rodents (mice) and also if a constant light regimen alters its amplitude and/or chronostructure. Wistar rats were submitted to either an artificial light-dark 12/12 regimen (LD) or to constant light (LL), for 15 days. In vitro phagocytosis of the neutrophils in whole blood against E.coli was assessed at 10:00, 16:00, 22:00, and 04:00 hours. In LD, phagocytosis appears to be a rhythmical function, with statistically significant differences between the highest value at 04:00 hrs and the lowest at 10:00 hrs. Constant light induces a 30% depression of the phagocytic ability throughout the whole 24 hours cycle, without altering its oscillations. The darkness period appears to play the role of a synchronizer; in its absence the rhythm tends to free-run. It may be stated that rhythmical melatonin secretion is responsible only for maintenance of the phagocytic level, probably via the anterior hypothalamic area and thymus, while it cannot account directly for the nocturnal increase of phagocytosis.