Annals of the New York Academy of Sciences, Dec 1, 2005
Basal phagocytosis of neutrophils, a crucial component of non-specific immunity, is an eminently ... more 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.
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
The research activity upon erythropoiesis regulation carried out by the team in the Physiology De... more 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...
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
Ischemic hypoxia of the head induced in dogs by carotid compression produces EPO secretion, upon ... more Ischemic hypoxia of the head induced in dogs by carotid compression produces EPO secretion, upon direct cerebral stimulation, and adaptive carotid-respiratory reflexes via sino-carotid stimulation. During hypoxic hypoxia, there also occurs extrarenal EPO production, as shown in binephrectomized rats compared to controls. Ischemic hypoxia of a single kidney transplanted in the neck area (the other kidney being removed) does not induce EPO secretion. An additional factor, of extra-renal origin, is required for the renal production of EPO in a non-hypoxic organism. Section of the spinal cord at C6 level in rats does not abolish EPO secretion induced by hypobaric hypoxia, a fact that suggests that there is no nervous center to control EPO release into a peripheral organ, but EPO might be produced in the brain itself, possibly crossing the blood-brain barrier to reach the blood flow. Stereotactical attempts to locate nervous centers of erythropoiesis regulation also failed. EPO secretion...
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
The bilateral olfactory bulbectomy (OB) has been proposed as an animal model of depression. The p... more The bilateral olfactory bulbectomy (OB) has been proposed as an animal model of depression. The present study, carried out on NMRI adult male mice kept on a natural LD 12:12 regimen, aimed to assess the influence of OB on the circadian rhythm of blood neutrophils phagocytosis. The results show that OB mice present an about 20% reduction of the basal phagocytic activity at 09.00 hrs, 15.00 hrs. and 21.00 hrs. and a 40% reduction at 03.00 hrs., leading to a flattened circadian phagocytic curve. The results indicate that bilateral olfactory bulbectomy depresses phagocytosis, alters its circadian rhythm and consequently increases susceptibility to infections.
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
Previous researches of our laboratories (1945, 1946, 1947) have shown that direct electrical stim... more Previous researches of our laboratories (1945, 1946, 1947) have shown that direct electrical stimulation of the tubero-mammillary hypothalamic area in dogs enhances the blood neutrophils phagocytic activity and the phagocytosis exhibiting leukocytes percent. After electrolytic damage of the same area, phagocytic activity decreases and phagocytic response is suppressed (1985, 1988). In the present work, we performed in mice extensive chemical lesions of the arcuate nucleus, by means of the neonatal treatment with monosodium glutamate (MSG). The experiment was carried out on 23 new-born mice. 15 mice were injected with MSG (G group), the other 8, serving as control group, received isotonic saline solution (C group). The studied parameters were, in both groups, the weight evolution of the animals, the blood neutrophils and lymphocytes percentual variation and the neutrophils' phagocytic activity, tested in vitro, expressed through the number of bacteria engulfed by 100 neutrophils ...
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
There has been reported, in some diurnal or equivocal species (man, respectively guinea pig), a c... more There has been reported, in some diurnal or equivocal species (man, respectively guinea pig), a circadian rhythm of the phagocytic activity of blood neutrophils, with an acrophase occurring at the end of the light span. The present study, carried out on NMRI adult male mice kept on a LD 12:12 regimen, aimed to assess any circadian variation in the blood neutrophils' phagocytosis level. Basal phagocytic activity was tested against E. coli, every three hours of a 24 h cycle. The results show that phagocytosis in mice's blood neutrophils also presents a rhythmic circadian variation, whose acrophase is delayed with about eight hours compared to that in man, occurring in the second half of the dark period (3:00 h). The occurrence of high circadian phagocytic levels appears to be correlated with the activity type of the species and, of this point of view, mice cannot be used as a model for chronotherapeutical approaches in humans, without keeping in mind the differences between th...
www.nel.edu *Dedicated to Germaine Cornélissen on her 54th birthday, November 22, 2003 and to the... more www.nel.edu *Dedicated to Germaine Cornélissen on her 54th birthday, November 22, 2003 and to the memory of her mother Hélène Minne There's more to "seasons" than meets the eye Signatures in us of the alternation of day and night and of the seasons have their non-photic, nonthermal counterparts: a cornucopia of cycles were mapped with their uncertainties during the past 2 years. New aspects emerged for a biological half-week, week and half-year, for 1-, 2-or 5-decadal cycles, along with cycles of even lower frequencies in tree rings, wars and, as far as available to us, in our cosmos. A near-7-day cycle was found in solar (I-42) as in terrestrial (I-23) magnetism. In the perspective of 2,000 or more years, these wobbly, irregular cycles can be aligned with less irregular rhythms, with a lesser extent of uncertainty in terms of the periods involved. Rhythms and, implicitly, cycles are all qualified by "circa" for reasons not only of biological endogenicity when pertinent and demonstrable in the want of a free-run by non-overlap of 95% confidence intervals, Figure 1 (46, I-95, I-96); in addition, there is the "circa" emphasizing an inferential statistical uncertainty of cycle or rhythm characteristics. During the past 2 years, further support emerged for the postulate that for each non-photic as well as photic physical environmental cycle one should look for a biological cycle with near-matching length and vice versa. The biological transyear was found, when a cycle with an about 1.3-year period
European Journal of Lipid Science and Technology, 2013
ABSTRACT A common technique for assaying lipase/esterase activity in vitro is the enzymatic cleav... more ABSTRACT A common technique for assaying lipase/esterase activity in vitro is the enzymatic cleavage of para-nitrophenyl esters, with spectrophotometric monitorization at 405–410 nm of the released p-nitrophenol (pNP). This method has its limitations, since the extinction coefficient of pNP is strongly pH-dependent. Despite this, the method is being frequently used for investigating lipolytic activity over a pH range without any pH-related corrections, which may bring along false results. We show to what extent the results may be altered by this approach and we review alternative strategies allowing the method to become usable both in the acidic range and with varying pH.
Basal phagocytosis of neutrophils, a crucial component of non-specific immunity, is an eminently ... more 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.
We have previously described the molecular cloning and expression in an Escherichia coli system a... more We have previously described the molecular cloning and expression in an Escherichia coli system and the characterization of a thermostable esterase/lipase from Anoxybacillus flavithermus T1. We herein report on the optimization of the expression process. When using isopropyl--D-thiogalactopyranoside (IPTG) for induction, the highest protein yield was obtained at 30°C, with 0.4 mM IPTG and 1 h induction time. Similar results were obtained at 37°C, but with a higher IPTG concentration (5 mM) and after 8h of induction, which makes the former a better option in terms of cost and time-efectiveness. Better results yet were attained with lactose, a very attractive option, given its high availability, low cost and low toxicity to the host cells. Recovery of the active enzyme from the periplasmic space was highest with a lysis buffer which combines osmotic shock with a membrane destabilization effect (Tris-sucrose/EDTA 1 mM/MgSO 4 5 mM). The freeze-thaw treatment yielded similar results, while treatment with 1% organic solvent (chloroform or DMSO), while effective in permeabilization of the cell membrane, exerted a certain inhibitory effect upon the enzyme.
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
The research activity upon erythropoiesis regulation carried out by the team in the Physiology De... more 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...
A common technique for assaying lipase/esterase activity in vitro is the enzymatic cleavage of pa... more A common technique for assaying lipase/esterase activity in vitro is the enzymatic cleavage of para-nitrophenyl esters, with spectrophotometric monitorization at 405-410 nm of the released p-nitrophenol (pNP). This method has its limitations, since the extinction coefficient of pNP is strongly pH-dependent. Despite this, the method is being frequently used for investigating lipolytic activity over a pH range without any pH-related corrections, which may bring along false results. We show to what extent the results may be altered by this approach and we review alternative strategies allowing the method to become usable both in the acidic range and with varying pH.
A good amount of experimental data suggests the existence of a circadian control of the inflammat... more 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...
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
The phagocytic function was proved to be a periodic, circadian process. Its acrophase appears to ... more 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...
Annals of the New York Academy of Sciences, Dec 1, 2005
Basal phagocytosis of neutrophils, a crucial component of non-specific immunity, is an eminently ... more 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.
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
The research activity upon erythropoiesis regulation carried out by the team in the Physiology De... more 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...
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
Ischemic hypoxia of the head induced in dogs by carotid compression produces EPO secretion, upon ... more Ischemic hypoxia of the head induced in dogs by carotid compression produces EPO secretion, upon direct cerebral stimulation, and adaptive carotid-respiratory reflexes via sino-carotid stimulation. During hypoxic hypoxia, there also occurs extrarenal EPO production, as shown in binephrectomized rats compared to controls. Ischemic hypoxia of a single kidney transplanted in the neck area (the other kidney being removed) does not induce EPO secretion. An additional factor, of extra-renal origin, is required for the renal production of EPO in a non-hypoxic organism. Section of the spinal cord at C6 level in rats does not abolish EPO secretion induced by hypobaric hypoxia, a fact that suggests that there is no nervous center to control EPO release into a peripheral organ, but EPO might be produced in the brain itself, possibly crossing the blood-brain barrier to reach the blood flow. Stereotactical attempts to locate nervous centers of erythropoiesis regulation also failed. EPO secretion...
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
The bilateral olfactory bulbectomy (OB) has been proposed as an animal model of depression. The p... more The bilateral olfactory bulbectomy (OB) has been proposed as an animal model of depression. The present study, carried out on NMRI adult male mice kept on a natural LD 12:12 regimen, aimed to assess the influence of OB on the circadian rhythm of blood neutrophils phagocytosis. The results show that OB mice present an about 20% reduction of the basal phagocytic activity at 09.00 hrs, 15.00 hrs. and 21.00 hrs. and a 40% reduction at 03.00 hrs., leading to a flattened circadian phagocytic curve. The results indicate that bilateral olfactory bulbectomy depresses phagocytosis, alters its circadian rhythm and consequently increases susceptibility to infections.
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
Previous researches of our laboratories (1945, 1946, 1947) have shown that direct electrical stim... more Previous researches of our laboratories (1945, 1946, 1947) have shown that direct electrical stimulation of the tubero-mammillary hypothalamic area in dogs enhances the blood neutrophils phagocytic activity and the phagocytosis exhibiting leukocytes percent. After electrolytic damage of the same area, phagocytic activity decreases and phagocytic response is suppressed (1985, 1988). In the present work, we performed in mice extensive chemical lesions of the arcuate nucleus, by means of the neonatal treatment with monosodium glutamate (MSG). The experiment was carried out on 23 new-born mice. 15 mice were injected with MSG (G group), the other 8, serving as control group, received isotonic saline solution (C group). The studied parameters were, in both groups, the weight evolution of the animals, the blood neutrophils and lymphocytes percentual variation and the neutrophils' phagocytic activity, tested in vitro, expressed through the number of bacteria engulfed by 100 neutrophils ...
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
There has been reported, in some diurnal or equivocal species (man, respectively guinea pig), a c... more There has been reported, in some diurnal or equivocal species (man, respectively guinea pig), a circadian rhythm of the phagocytic activity of blood neutrophils, with an acrophase occurring at the end of the light span. The present study, carried out on NMRI adult male mice kept on a LD 12:12 regimen, aimed to assess any circadian variation in the blood neutrophils' phagocytosis level. Basal phagocytic activity was tested against E. coli, every three hours of a 24 h cycle. The results show that phagocytosis in mice's blood neutrophils also presents a rhythmic circadian variation, whose acrophase is delayed with about eight hours compared to that in man, occurring in the second half of the dark period (3:00 h). The occurrence of high circadian phagocytic levels appears to be correlated with the activity type of the species and, of this point of view, mice cannot be used as a model for chronotherapeutical approaches in humans, without keeping in mind the differences between th...
www.nel.edu *Dedicated to Germaine Cornélissen on her 54th birthday, November 22, 2003 and to the... more www.nel.edu *Dedicated to Germaine Cornélissen on her 54th birthday, November 22, 2003 and to the memory of her mother Hélène Minne There's more to "seasons" than meets the eye Signatures in us of the alternation of day and night and of the seasons have their non-photic, nonthermal counterparts: a cornucopia of cycles were mapped with their uncertainties during the past 2 years. New aspects emerged for a biological half-week, week and half-year, for 1-, 2-or 5-decadal cycles, along with cycles of even lower frequencies in tree rings, wars and, as far as available to us, in our cosmos. A near-7-day cycle was found in solar (I-42) as in terrestrial (I-23) magnetism. In the perspective of 2,000 or more years, these wobbly, irregular cycles can be aligned with less irregular rhythms, with a lesser extent of uncertainty in terms of the periods involved. Rhythms and, implicitly, cycles are all qualified by "circa" for reasons not only of biological endogenicity when pertinent and demonstrable in the want of a free-run by non-overlap of 95% confidence intervals, Figure 1 (46, I-95, I-96); in addition, there is the "circa" emphasizing an inferential statistical uncertainty of cycle or rhythm characteristics. During the past 2 years, further support emerged for the postulate that for each non-photic as well as photic physical environmental cycle one should look for a biological cycle with near-matching length and vice versa. The biological transyear was found, when a cycle with an about 1.3-year period
European Journal of Lipid Science and Technology, 2013
ABSTRACT A common technique for assaying lipase/esterase activity in vitro is the enzymatic cleav... more ABSTRACT A common technique for assaying lipase/esterase activity in vitro is the enzymatic cleavage of para-nitrophenyl esters, with spectrophotometric monitorization at 405–410 nm of the released p-nitrophenol (pNP). This method has its limitations, since the extinction coefficient of pNP is strongly pH-dependent. Despite this, the method is being frequently used for investigating lipolytic activity over a pH range without any pH-related corrections, which may bring along false results. We show to what extent the results may be altered by this approach and we review alternative strategies allowing the method to become usable both in the acidic range and with varying pH.
Basal phagocytosis of neutrophils, a crucial component of non-specific immunity, is an eminently ... more 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.
We have previously described the molecular cloning and expression in an Escherichia coli system a... more We have previously described the molecular cloning and expression in an Escherichia coli system and the characterization of a thermostable esterase/lipase from Anoxybacillus flavithermus T1. We herein report on the optimization of the expression process. When using isopropyl--D-thiogalactopyranoside (IPTG) for induction, the highest protein yield was obtained at 30°C, with 0.4 mM IPTG and 1 h induction time. Similar results were obtained at 37°C, but with a higher IPTG concentration (5 mM) and after 8h of induction, which makes the former a better option in terms of cost and time-efectiveness. Better results yet were attained with lactose, a very attractive option, given its high availability, low cost and low toxicity to the host cells. Recovery of the active enzyme from the periplasmic space was highest with a lysis buffer which combines osmotic shock with a membrane destabilization effect (Tris-sucrose/EDTA 1 mM/MgSO 4 5 mM). The freeze-thaw treatment yielded similar results, while treatment with 1% organic solvent (chloroform or DMSO), while effective in permeabilization of the cell membrane, exerted a certain inhibitory effect upon the enzyme.
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
The research activity upon erythropoiesis regulation carried out by the team in the Physiology De... more 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...
A common technique for assaying lipase/esterase activity in vitro is the enzymatic cleavage of pa... more A common technique for assaying lipase/esterase activity in vitro is the enzymatic cleavage of para-nitrophenyl esters, with spectrophotometric monitorization at 405-410 nm of the released p-nitrophenol (pNP). This method has its limitations, since the extinction coefficient of pNP is strongly pH-dependent. Despite this, the method is being frequently used for investigating lipolytic activity over a pH range without any pH-related corrections, which may bring along false results. We show to what extent the results may be altered by this approach and we review alternative strategies allowing the method to become usable both in the acidic range and with varying pH.
A good amount of experimental data suggests the existence of a circadian control of the inflammat... more 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...
Romanian journal of physiology : physiological sciences / [Academia de Stiinte Medicale]
The phagocytic function was proved to be a periodic, circadian process. Its acrophase appears to ... more 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...
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