Modelling pH evolution and lactic acid production in the growth medium of a lactic acid bacterium: application to set a biological TTI.

Time temperature integrators or indicators (TTIs) are effective tools making the continuous monitoring of the time temperature history of chilled products possible throughout the cold chain. Their correct setting is of critical importance to ensure food quality. The objective of this study was to develop a model to facilitate accurate settings of the CRYOLOG biological TTI, TRACEO. Experimental designs were used to investigate and model the effects of the temperature, the TTI inoculum size, pH, and water activity on its response time. The modelling process went through several steps addressing growth, acidification and inhibition phenomena in dynamic conditions. The model showed satisfactory results and validations in industrial conditions gave clear evidence that such a model is a valuable tool, not only to predict accurate response times of TRACEO, but also to propose precise settings to manufacture the appropriate TTI to trace a particular food according to a given time temperature scenario.

Intracranial aneurysms: clinical value of 3D digital subtraction angiography in the therapeutic decision and endovascular treatment.

PURPOSE: To evaluate three-dimensional (3D) digital subtraction angiography (DSA) as a supplement to two-dimensional (2D) DSA in the endovascular treatment (EVT) of intracranial aneurysms. MATERIALS AND METHODS: In 22 ruptured aneurysms, neck visualization, aneurysm shape, and EVT feasibility were analyzed at 2D DSA (anteroposterior, lateral, and rotational views) and at maximum intensity projection (MIP) and surface shaded display (SSD) 3D DSA. The possibility of obtaining a working view for EVT at 3D DSA and the relevance of measurements in choosing the first coil also were assessed. RESULTS: Two-dimensional DSA images clearly depicted the aneurysm neck in four of 22 aneurysms; MIP images, in 10; and SSD images, in 21, but SSD led to overestimation of the neck size in one aneurysm. Aneurysm shape was precisely demonstrated in five of 22 aneurysms at 2D DSA, in eight at MIP, and in all cases at SSD. In two of 22 aneurysms, EVT seemed to be nonfeasible at 2D DSA; however, SSD demonstrated feasibility and EVT was successfully performed. In one aneurysm, only SSD demonstrated the extension of the neck to a parent vessel, which was proved at surgery. Working views for EVT were deduced from 3D DSA findings in 20 of 21 aneurysms. The choice of the first coil was correct in 19 of 21 aneurysms. CONCLUSION: Three-dimensional DSA is valuable for evaluating the potential for EVT, finding a working view, and performing accurate measurements.

3D angiography. Clinical interest. First applications in interventional neuroradiology.

3D angiography is a true technical revolution that allows improvement in the quality and safety of diagnostic and endovascular treatment procedures. 3D angiography images are obtained by reconstruction of a rotational angiography acquisition done on a C-arm (GE Medical Systems) spinning at 40 degrees per second. The carotid or vertebral selective injection of a total of 15 ml of non-ionic contrast media at 3 ml/sec over 5 seconds allows the selection of the "arterial phase". Four hundred sixty 3D angiographic studies were performed from December 1996 to September 1998 on 260 patients and have been analyzed in MIP (Maximum Intensity Projection) and SSD (Shaded Surface Display) views. The exploration of intracranial aneurysms is simplified and only requires, for each vascular axis, a biplane PA and Lateral run followed by a single rotational angiography run. The 3D angiography image is available on the workstation's screen (Advantage Workstation 3.1, GE Medical Systems) in less than 10 minutes after the acquisition of the rotational run. It therefore allows one to analyze, during the intervention, the aneurysm's angioarchitecture, in particular the neck, and select the best therapeutic technique. When endovascular treatment is the best indication, 3D angiography allows one to define the optimal angle of view and accurately select the microcoils dimensions. 3D angiography replaces the multiple oblique views that used to be required to analyze the complex aneurysms and therefore allows a reduction of the total contrast medium quantity, the patient X-ray dose and the length of the intervention time which is a safety factor. Also, in particular for complex cases, it brings additional elements complementing the results of standard 2D DSA and rotational angiograms. In the cervical vascular pathology, 3D angiography allows for a better assessment of the stenosis level and of dissection lesions. Our current research activities focus on the matching without stereotactic frame between 3D X-ray angiography and volumetric MR acquisition, which should allow us to improve the treatment of intracerebral arterio-venous malformations (AVMs).

Regulation of argininosuccinate synthetase level by corticosteroid and pancreatic hormones during perinatal period.

The urea cycle takes place in the hepatocyte of ureothelic animals. The conversion of ammonia into urea involves five reactions. The first 2 take place in the matrix of the mitochondria, the last 2 occur in the cytosol. Argininosuccinate synthetase (AS) is the third reaction of the urea cycle. It catalyses the condensation of citrulline and aspartate into argininosuccinate. We have previously reported that rat AS activity was present in the cytosol and the outer membrane of the mitochondria. We have shown that, at the activity level, the colocation of AS was changing during fetal and neonatal development and was under the control of corticosteroid and pancreatic hormones. However, an unresolved issue was whether both AS had the same specific activity and that their location was changing during ontogenesis or that the specific activities of mitochondrial and cytosolic enzymes were different and/or modified during this period. In the present report, we compared the compartmentalization of AS activity and protein level in the fetus, the new-born and the adult rat and the role of corticosteroid and pancreatic hormones. Specific activities of both AS remained unchanged during ontogenesis. Glucocorticoids induced an increase in mitochondrial AS while glucagon appeared to induce a concomitant decrease in the level of mitochondrial AS and an increase in cytosolic AS.

Demonstration of argininosuccinate synthetase activity associated with mitochondrial membrane: characterization and hormonal regulation.

Argininosuccinate synthetase (AS) is the third enzyme in ureogenesis, it catalyses the reaction of condensation of citrulline and aspartate into argininosuccinate. In the present report, we described the first characterization of AS within the outer membrane of rat liver mitochondria. Mitochondria-associated AS displayed the same kinetic characteristics as the cytoplasmic enzyme, but was found to be thermostable while cytoplasmic AS was not. The evolution of the co-location of AS was analyzed during ontogenesis. Total AS activity increased throughout rat fetal development. Simultaneously, the subcellular distribution of the enzyme has changed. AS activity was mainly mitochondrial in fetal and new-born liver liver and cytoplasmic in adult rat liver. The variation in subcellular distribution of AS may be due to the dramatic changes in hormonal levels that occur during this period. The role of corticosteroid and pancreatic hormones was studied. During fetal period, corticosteroid hormones induced an increase in mitochondria-associated AS activity. This was prevented by insulin. Glucagon did not modify total AS activity but reduced mitochondrial AS activity, meanwhile, a comparable increase in cytoplasmic AS activity was observed. One may hypothesize that glucagon may participate in the transfer of mitochondrial enzyme into the cytosol.

Development and hormonal control of thioredoxin and the thioredoxin-reductase system in the rat liver during the perinatal period.

The development and hormonal regulation of thioredoxin and of the thioredoxin-reductase system were investigated during the perinatal period in rat liver. An immunological procedure was developed in order to quantify thioredoxin in fetal and neonatal hepatocytes. Both immunoreactive thioredoxin and thioredoxin-reductase activity appeared on day 16.5 of pregnancy. The level of immunoreactive thioredoxin increased during the late fetal period, and its level was the same 24 h after birth. Moreover, its development was not subjected to hormonal regulation by corticosteroids and glucagon. In contrast, thioredoxin-reductase activity increased 3 times during the late fetal period and presented a marked increase 24 h after birth. In the absence of glucocorticoids there was no increase in the level of thioredoxin reductase, while administration of hydrocortisone acetate and glucagon to fetuses prematurely evoked its activity. This study suggests that if thioredoxin acts physiologically, this activity is related to the state of reduction of the molecule rather than to the total concentration in the liver.

Expression of argininosuccinate lyase mRNA in foetal hepatocytes. Regulation by glucocorticoids and insulin.

Argininosuccinate lyase (ASL), the fourth enzyme of the urea cycle, belongs to a group of liver enzymes appearing in the late foetal period in the rat. Several hormones, including glucocorticosteroids and insulin have been implicated in the control of the development of this enzyme activity. In this study, the cloned cDNA was used to measure the relative abundance of ASL mRNA in the livers of rats at various stages of perinatal development and in cultured foetal hepatocytes during hormonal manipulations. The ASL mRNA was first detectable on day 15.5 of gestation and increased in amount concomitantly with the rise in the enzyme activity, suggesting that the appearance of enzyme activity reflects the turning on of specific gene transcription. When foetal hepatocytes were exposed to dexamethasone, an increase in ASL mRNA was detected, which was completely abolished by addition of actinomycin D, suggesting a transcriptional effect of the steroid. In contrast, administration of cortisol to foetuses in utero had no effect on the mRNA level, suggesting that the steroid action is inhibited in the intra-uterine environment. Insulin might be the inhibiting factor since it completely repressed the dexamethasone-induced accumulation of ASL mRNA in foetal hepatocytes. These data were confirmed in vivo by experiments using streptozotocin, which produces insulin-depleted foetuses and causes the accumulation of ASL mRNA. This regulation of ASL mRNA by glucocorticoids and insulin could account for the modulation of the enzyme activity observed in vivo and in vitro.

Induction of the five urea-cycle enzymes by glucagon in cultured foetal rat hepatocytes.

Foetal hepatocytes obtained from rats at different stages were cultured in order to investigate the inducibility of the five urea-cycle enzymes by glucagon and dibutyryl cyclic AMP (Bt2cAMP). When 18.5-day-old hepatocytes were cultured for 3 days with 10(-7) M glucagon, the activities of carbamoyl phosphate synthetase (CPS), argininosuccinase (ASL) and arginase were increased by 1.4-, 1.8- and 1.9-fold, respectively, as compared to controls. These effects were mimicked by 10(-4) M Bt2cAMP, but the activities of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) were never changed by the addition of these compounds. Hepatocytes cultured at earlier stages were not responsive to glucagon unless dexamethasone was added simultaneously, suggesting that this steroid might induce some steps necessary for glucagon action. Bt2cAMP was effective as early as day 16.5 without requiring the presence of steroids. In addition, the effect of the cyclic nucleotide appeared additive or synergistic with that of dexamethasone. The simultaneous addition of actinomycin D did not affect the glucagon-induced increase in enzyme levels, thus suggesting a post-transcriptional effect of the hormone on the foetal enzyme activities. Insulin itself did not have any effect on the basal level of the enzyme activities and had only a moderate inhibitory effect on glucagon-induced ASL activity. This slight effect of insulin is in contrast with the marked inhibitory effect of dexamethasone on this enzyme activity that we described previously.

Effects of pancreatic hormones and glucocorticosteroids on argininosuccinate synthetase and argininosuccinase activities of rat liver during the perinatal period: in vivo and in vitro studies.

The activity changes of two urea cycle enzymes, argininosuccinate synthetase (ASS) and argininosuccinase (ASL), were followed after corticosteroid and pancreatic hormone treatments in utero and in primary cultured fetal hepatocytes. The ASL activity which was induced by glucagon or by (Bu)2cAMP administration was enhanced by a treatment with streptozotocin for 2 days, although ASS was not changed under these conditions. The activity of both enzymes was enhanced by cortisol administration in utero only in streptozotocin-treated fetuses, suggesting an inhibitory effect of insulin. In cultured fetal hepatocytes, dexamethasone produced a marked increase of the two enzyme activities, which was abolished by the simultaneous addition of insulin. The parallel results obtained with these two experimental models allow one to conclude that the high plasma insulin level in late gestation might repress the development of ASS and ASL activities in utero and antagonize the effect of corticosteroids on these enzyme activities.

[Effects of DTT and thioredoxins on rat argininosuccinate synthetase activity in vitro]. / Effets du DTT et des thiorédoxines sur l'activité de l'arginosuccinate synthétase chez le rat in vitro.

Argininosuccinate synthetase (ASS, EC 6.3.4.5), the third enzyme of urea-cycle, was studied in desactivated extracts of rat liver. The enzyme is activated, in vitro, by Mg2+ ions (5 mM) and dithiothreitol (DTT: 10 mM). After reduction by DTT, thioredoxins isolated from rat liver were able to activate ASS by 370%.

[Effects of dithiothreitol and thioredoxines on the in vitro activity of enzymes of the urea cycle in rats]. / Effets du dithiothréitol et des thiorédoxines sur l'activité, in vitro, des enzymes du cycle de l'urée chez le rat.

The five urea cycle enzymes were studied in desactivated extracts of rat liver. After reduction by dithiothreitol (DTT) and in presence of Mg2+ ions, thioredoxines isolated from rat liver were able to activate carbamyl phosphate synthetase-I (CPS-I) and argininosuccinate synthetase (ASS) respectively by 468% and by 370%. Thioredoxines were purified from adult rat liver and an antiserum was raised to these proteins. After immunologic quantitation, their level in adult rat was 0.103 mg/g liver.

Changes in ornithine transcarbamylase activity and protein level during perinatal period in rat liver. Effects of actinomycin D.

Ornithine transcarbamylase activity and immunoreactive enzyme level are compared during perinatal period and in adult rat. Ornithine transcarbamylase activity regularly rises during late fetal period and presents a marked increase 24 hours after birth. Immunoreactive enzyme level does not correlate with this developmental pattern. Ornithine transcarbamylase level increases from 0.06 mg on day 19.5 of pregnancy to 0.417 mg/g liver on day 21.5 and remains constant after birth (0.418 mg/g liver). These results suggest that inactive mitochondrial ornithine transcarbamylase accumulates before birth and that the postnatal increase in enzyme activity is mainly associated with an activation. Furthermore, the paradoxical effect of actinomycin D on ornithine transcarbamylase activity is associated with an increase in enzyme level (about 25%).

Role of dexamethasone and insulin on the development of the five urea-cycle enzymes in cultured rat foetal hepatocytes.

The activity changes of the urea-cycle enzymes were monitored in cultured foetal hepatocytes after dexamethasone and insulin treatments. Addition of dexamethasone induced the development of carbamoyl-phosphate synthetase, argininosuccinate synthetase, argininosuccinase and arginase activities as soon as day 16.5 of gestation. When insulin was added together with dexamethasone, it markedly inhibited the steroid-induced increase in carbamoyl-phosphate synthetase, argininosuccinate synthetase and argininosuccinase activities.

Effects of corticosteroids and pancreatic hormones on carbamyl phosphate synthetase-I and ornithine transcarbamylase activities in fetal rat liver.

The effects of corticosteroids and pancreatic hormones on two mitochondrial enzymes of ureagenesis, carbamyl phosphate synthetase-I (CPS-I) and ornithine transcarbamylase (OTC), were investigated and compared in fetal rat liver. Supplementing hydrocortisone acetate (50 micrograms) to 18.5-day-old fetuses significantly increased CPS-I activity (by 36%) and decreased OTC activity (by 23%). An actinomycin D supply (2 micrograms) to 18.5-day-old fetuses prematurely increased OTC activity and decreased fetal insulin level (by 42%). This treatment had no effect on CPS-I activity. Glucagon supply (25 micrograms) during the late fetal period increased both activities within 2 h, while dibutyryl-cAMP enhanced OTC activity 17 h later. These results suggested that the fetal development of CPS-I activity was under the control of corticosteroids and glucagon. In contrast, corticosteroid hormones produced an inhibitory effect on OTC activity. This might be explained by the permissive effect of corticosteroids on insulin action, since insulin might act as a repressor in utero of enzyme development. Thus, the paradoxical effect of actinomycin D on OTC activity was probably due to the decrease in fetal insulinemia.

Role of insulin on the control of postnatal increase in ornithine transcarbamylase activity in rat liver.

Repeatedly supplementing glucose after birth abolishes postnatal increase in ornithine transcarbamylase activity and increases the plasma insulin level. A single administration of actinomycin D does not affect this activity. When glucose and actinomycin D are administered in association at birth or 13 hours after birth, actinomycin D overcomes the inhibitory effect of glucose on ornithine transcarbamylase activity and decreases insulinemia at the level found in normal neonate. Insulin supply 20 hours after birth to neonates, which previously received glucose and actinomycin D, decreases the enzyme activity and increases plasma insulin level. It is concluded that the postnatal increase in ornithine transcarbamylase activity is bound to the normal postnatal decrease in insulinemia. Furthermore, the paradoxical effect of actinomycin D on enzyme activity might be due to an inhibition of insulin synthesis or a decrease in insulin release.

Precocious induction of arginase in primary cultures of fetal rat hepatocytes.

Fetal rat hepatocytes were isolated and cultured in primary culture to investigate activity changes of arginase under defined conditions. In hormone-free medium, cultured cells maintained the enzyme activity at levels equal to that of freshly isolated cells for at least 4 d. Arginase activity could be induced by dexamethasone in hepatocytes isolated from 16.5-d-old fetuses although cells were competent to respond to glucagon only at the stage of 18.5 d. The combination of the two hormones induced greater levels of arginase activity than the individual compounds. These findings indicate that glucocorticoid and glucagon receptors appear early and sequentially before birth and reveal that cultured fetal hepatocytes provide a suitable system for the investigation of the role of hormones in the initiation of enzyme synthesis.

[In vitro activation by dithiothreitol and thioredoxins of carbamyl phosphate synthetase-I in rat liver]. / Activation in vitro par le dithiothreitol et les thiorédoxines de la carbamyl phosphate synthétase-I dans le foie de rat.

Carbamyl phosphate synthetase-I (CPS-I; EC 6.3.4.16), a mitochondrial enzyme of the urea-cycle, was studied in deactivated extracts of rat liver. It has been found to be activated in vitro by dithiothreitol (DTT) and Mg2+ ions. After reduction by DTT, thioredoxins, isolated from rat liver, were able to activate CPS-I by 468%.

Hormonal regulation of two urea-cycle enzymes in cultured foetal hepatocytes.

Foetal-rat hepatocytes were cultured in primary monolayer culture, and activity changes of argininosuccinate synthetase (ASS, EC 6.3.4.5) and argininosuccinase (ASL, EC 4.3.2.1) were followed under defined hormone conditions. In hormone-free medium, cultured cells maintained the enzyme activities at values equal to those of freshly isolated cells for at least 3 days. Continuous addition of dexamethasone produced the development of the two enzyme activities, but only after the first 20h of culture. Under these conditions, urea production by the foetal hepatocytes was concomitantly increased in the culture medium. Pretreatment with dexamethasone for 20h was sufficient to produce the development of ASL activity within the 2 following days. Introduced alone, glucagon induced an increase of ASL activity, but did not affect the ASS activity. The most powerful stimulation of ASS and ASL could be observed in cultured hepatocytes if glucagon and dexamethasone were added simultaneously or sequentially. These results indicated that the development of the receptor complex for the induction of urea-cycle enzymes appears early before birth and established that glucocorticoids amplify the glucagon stimulation of these enzyme activities during foetal life.

[Urea cycle enzyme activities in fetal rat hepatocytes in primary cultures]. / Activités enzymatiques du cycle de l'urée dans les hépatocytes de foetus de rat en culture primaire.

The development of urea synthetizing enzymes, namely argininosuccinate synthetase, argininosuccinase and arginase was studied in fetal Rat liver cells in culture. The enzyme activities were measured in 18.5 days old isolated hepatocytes and could not develop without addition of dexamethasone in the culture medium.

Regulation of ureagenesis in neonatal rat liver: influence of cycloheximide in vivo and in vitro.

In order to establish whether the postnatal rise in five urea cycle enzyme activities is associated with protein synthesis, the effects of cycloheximide on urea cycle enzyme activities, citrulline and urea productions in vivo and in vitro were studied. A single injection of cycloheximide (10 microgram) 9 h after birth significantly prevented the rise in enzyme activities and citrulline concentration which normally occurred after birth, while the urea level remained unchanged. These data suggest that the postnatal rise in urea cycle enzyme activities might be associated with a protein synthesis. The rate of citrulline synthesis by liver slices was reduced (about 50%) in cycloheximide-treated neonatal liver, while the rate of urea production was not significantly decreased. The results obtained in vivo are in good agreement with in vitro experiments: citrullinogenesis was only affected by cycloheximide treatment. When cycloheximide 10 mM was added to the incubation medium, the ability of control neonatal liver slices to produce citrulline and urea was reduced to 42 and 11%, respectively. Since argininosuccinic acid addition in the medium did not produce a rise in urea synthesis, it is concluded that argininosuccinase is most responsive after incubation in cycloheximide and therefore becomes the rate-limiting step of the urea synthesis.