In vitro effect of peas, Pisum pisum, and chickpeas, Cicer arietinum, on the immune system of gilthead seabream, Sparus aurata.

The future for a sustainable aquaculture relies on the formulation of feed including alternatives to fish meal and fish oil that do not impair fish growth and that improve fish health status. Grain legumes such as field peas and chickpeas offer good sources of proteins, carbohydrates, fibers, vitamins, and minerals. The effect of peas and chickpeas on the immune system of seabream was assessed in vitro in order to detect any potential immunosuppressing problem. Peas was determined to be a better fishmeal alternative than chickpeas as they induced higher respiratory burst measured by the nitro blue tetrazolium assay and primed the Phorbol 12-myristate 13-acetate (PMA)-stimulated intracellular respiratory burst whereas chickpeas neither directly stimulated respiratory burst nor primed it. However, when the intra- and extracellular respiratory burst activities were taken into account, high concentrations of peas inhibited the zymosan- and PMA-triggered chemiluminescence. This apparent reduction of the production of reactive oxygen species may reflect in fact the antioxidant activity of legumes. This, together with the absence of effect on the phagocytosis activity, suggested that peas are not immunosuppressing gilthead seabream. Further in vivo studies preferably comporting a bacterial challenge will have to ascertain the absence of immunosuppressing effect of these legumes.

Patterns of gastric evacuation, digesta characteristics and pH changes along the gastrointestinal tract of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.).

A comparative study of gastric evacuation rates (GERs) and digesta content, moisture and pH values along the gastrointestinal tract was performed between gilthead sea bream and European sea bass. In order to distinguish species-specific differences from diet-elicited effects, all parameters were determined under either a fishmeal diet or a carob seed germ meal diet that contained high levels of total and soluble non-starch polysaccharides. GERs were significantly different between species and they were not affected by diet. Similarly, species-specific patterns were revealed in the distribution of digesta and water content along the gastrointestinal tract. In sea bream, stomach digesta and water content decreased with time, whereas in sea bass stomach retained the highest digesta and water content throughout the sampling period. The anterior and distal intestine exhibited the lowest accommodating capacities of digesta and water in either species. Overall, sea bream performed stomach digestion at lower hydration levels and higher pH compared with sea bass. Diet affected stomach moisture in both species and pH of stomach digesta in sea bass and of all intestinal sections in sea bream. The results obtained indicated that water and inorganic ion exchanges through the gut may differentiate between the species and warrant further investigation.

Effects of a natural parasitical infection (Lernanthropus kroyeri) on the immune system of European sea bass, Dicentrarchus labrax L.

The immune response of European sea bass, Dicentrarchus labrax, to a natural infection by the copepod parasite Lernanthropus kroyeri was evaluated for the first time in vivo. The results clearly demonstrated the triggering of the fish immune system by the parasite. Lysozyme activity and alternative complement pathway were involved in the early action against the parasitical infection, whilst classical complement and respiratory burst (RB) activity took over in the later stages of infection. It was hypothesized that the levels of alternative and classical complement and RB stimulation indexes may determine the resistance capacity of the fish to the parasite. It is not clear how parasites can survive despite the strong immunological arsenal deployed by the fish. The continual increase of prevalence and severity of parasite infection suggested that the parasite's mechanism of evasion of the immune system was extremely successful. The contrasting decrease in the negative effects of parasites on the fish health (such as gills anaemia) suggested that an equilibrium between the parasites and their hosts was reached in chronic infection. These dynamic interactions between parasites and fish hosts were probably the main determinant of host specificity.

Mechanisms of the action of zoledronic acid on human MG-63 osteosarcoma cells.

The aim of our study was to analyze the action of zoledronic acid on MG-63 human osteosarcoma cells. The proliferation of MG-63 cells was inhibited by either continuous or pulsatile exposures of zoledronic acid in a dose-dependent manner (10-250 microM). Zoledronic acid did not produce evidence of MG-63 cell death when administered at 100 mM for 48 hours, but only after exposure of 96 hours. Zoledronic acid (100 microM) increased the distribution of MG-63 cells in G0/G1 phase, however, it did not increase the adriamycin-induced apoptosis. In addition, zoledronic acid action was partially neutralized by exogenous administration of geranylgeranyl pyrophosphate (GGPP), but not by farnesyl pyrophosphate (FPP). Furthermore, zoledronic acid resulted in the attenuation of the prenylated form of Ras. Zoledronic acid and EDTA increased fluorescence of Fluo-3 loaded MG-63 cells in a similar pattern. This increase was owing to the release of Ca2+ from intracellular stores since zoledronic acid failed to reveal such a change to intracellular Ca2+ when cells were previously treated with 1 mM caffeine. Moreover, zoledronic acid significantly decreased the expression of estrogen receptor alpha (ERalpha) whereas it did not change significantly the expression of estrogen receptor beta (ERbeta) in MG-63 cells. These data suggest that zoledronic acid can control the proliferation and the differentiation of osteosarcoma-like cells.

Gender-dependent alterations in corticosteroid receptor status and spatial performance following 21 days of restraint stress.

The effects of 21-day exposure to restraint stress on hippocampal corticosteroid receptors and on spatial performance of male and female rats were evaluated. Stressed male animals exhibited a decrease in glucocorticoid receptor immunoreactivity in the CA1 area and the dentate gyrus. At the same time, stressed males tested on Morris water maze showed delayed learning and worse memory scores, compared with the control males. By contrast, stressed females exhibited an increase in glucocorticoid receptor immunoreactivity in CA1, similar learning ability and improved memory scores, compared with control females. In addition, stressed females showed a significant increase in mineralocorticoid receptor immunoreactivity in the CA3 area compared with controls. These data show that 21 days of restraint stress affect hippocampal corticosteroid receptors and spatial performance in a gender-specific manner. The observed changes in corticosteroid receptor levels following stress, may be causatively linked to the stress-induced alterations on spatial learning and memory.

Trace metal levels in freshwater fish, sediment and water.

The trace metal concentrations in water, sediment and aquatic organisms, such as fish, could indicate the level and tendency of the pollution. This is important not only for the protection of the environment, but for evaluation of the quality of fish meat either captured from natural waters or cultured in fishponds. The total trace metal concentrations in samples of fish from different regions of Hungary and from different species have been determined by using an X-ray fluorescence technique (EDXRF). Water, sediment and fish samples from fishpond systems with different feeding and stocking has also been analyzed. In the case of zinc contents, differences have been traced between the cultured and wild common carp. In the case of common carp reared under different feeding conditions, differences were also observed in the zinc concentration. The retention of the trace metals in the fish has been studied by measuring the levels in sediment, water and feed. The different retention can be explained by the different availability of zinc in the applied feeds, which can be related to the presence of different metal species in the feeds.

Regulation of tyrosine aminotransferase gene expression by glucocorticoids in quiescent and regenerating liver.

Following 70% hepatectomy, the induction of tyrosine amino-transferase mRNA by glucocorticoids was marginal at 1.5 h, significantly impaired between 3 and 8 h and, at 16 h post-hepatectomy, reached a value approx. 5-fold the basal level, similar to the level observed in quiescent liver. The fold induction of the mRNA was accounted for by a similar fold activation of transcription of the gene by glucocorticoids in regenerating but not in quiescent liver; in the latter, activation of transcription was marginal in spite of glucocorticoid-induced hypersensitivity to cleavage by DNase I at the glucocorticoid-dependent enhancer of the gene. The possibility that in quiescent liver glucocorticoids act at a transcriptional step beyond initiation, increasing the rate of elongation or overcoming a blockage in elongation, was excluded. However, a similar fold induction was determined for total and nuclear tyrosine aminotransferase mRNA in the presence of glucocorticoids, suggesting that in quiescent liver glucocorticoids promote efficient maturation of the tyrosine aminotransferase primary transcript. Thus a glucocorticoid-induced nuclear post-transcriptional up-regulation apparently compensates for impaired activation of transcription of the tyrosine aminotransferase gene by glucocorticoids in quiescent liver.

Glucocorticoid receptor gene expression in the embryonic rat brain.

The early ontogenetic pattern of glucocorticoid receptor (GR) gene expression was studied in the rat brain through embryonic days (E) 12 to 17. Using a [35S]-labelled GR antisense RNA probe for in situ hybridization, we first detected GR mRNA in E13 embryos. The strongest signal was in Rathke's pouch, but the hypothalamic, and to a lesser degree the pontine and rhinencephalic neuroepithelium were also moderately labelled. Significant levels of GR mRNA were also detected in the choroid plexus and the epithelia lining the ventricles on E13. Receptor gene expression was further extended by E15 to the neuroepithelium and the differentiating field of several neuronal structure primordia, including the basal ganglia, rhinencephalon, hippocampus, pons and cerebellum. On E17, GR gene expression was in addition detected in the amygdala, subiculum and olfactory bulb and cortex. The integrity of the mRNA transcripts revealed by in situ hybridization was assessed by Northern blot analysis of total RNA from embryonic brain and pituitary. A major approximately 7-kb transcript was detected throughout embryonic development. An adult-like GR protein was shown by immunoblotting analysis to be expressed in brain and pituitary extracts already by E13. Based on our results, we postulate a receptor-mediated regulatory role for glucocorticoids in the embryonic development of the rat brain.

Temporary loss of glucocorticoid receptor-mediated regulation of gene expression in heat-shocked cells.

The effect of heat shock on the transcriptional activity of glucocorticoid receptor was assessed using HeLa cells stably transfected with the chloramphenicol acetyltransferase (CAT) gene the transcription of which is controlled by two glucocorticoid-responsive elements placed directly upstream of a core promoter. Heat shock inactivated the high-affinity glucocorticoid binding capacity of the cells and nullified the rate of accumulation of CAT mRNA in the presence of hormone. Hormonal responsiveness was restored on return to normal temperature concomitantly with recovery of high-affinity glucocorticoid binding capacity. Heat inactivation of the receptor was coincident with loss of its solubility and apparently unrelated to receptor degradation.

Glucocorticoid regulation of glycerolphosphate dehydrogenase expression in the developing rat brain.

Glucocorticoid regulation of glycerolphosphate dehydrogenase (GPDH) activity and gene expression in the developing rat brain appears complex throughout the postnatal developmental period and attains the adult pattern after the first month of life. GPDH enzyme activity is higher in the limbic system than in the cerebral cortex of intact young animals. Adrenalectomy of young rats, before the first month of life, does not affect GPDH enzyme activity in the brain areas mentioned above, while in the adult animals it results in a statistically significant decrease in activity. Furthermore, "adult type" glucocorticoid responsivity of GPDH enzyme activity is attained in the developing limbic system earlier--by day 40 of life--than in the cerebral cortex. During the first month of life, GPDH basal mRNA levels are increased in the absence of glucocorticoids, in both the limbic system and the cortex, in contrast to the effect of adrenalectomy in the adults, where GPDH mRNA levels are decreased in the absence of the adrenals. The observed pattern of glucocorticoid regulation of GPDH during development in the rat is discussed in relation to the possible existence of various levels of regulation of GPDH gene and enzyme activity.

The efficiency of nuclear processing of the tyrosine aminotransferase mRNA transcript increases after partial hepatectomy.

Following a two-thirds partial hepatectomy, an approximately fivefold increase in the levels of nuclear and total mRNA for tyrosine aminotransferase was observed at 1 h and 1.5 h, respectively, and a return to the levels of the quiescent state, i.e. the levels found in non-operated livers from adrenalectomized rats, was established 16 h post-hepatectomy. The increase in mRNA levels was not accounted for by a comparable change in the rate of transcription of the gene which, at 0.5 h post-hepatectomy, reached a maximum value that amounted to only 1.4-fold the value for quiescent liver. Subsequent changes in the transcription rate largely accounted for the changes in mRNA levels observed later on. Although tyrosine aminotransferase mRNA levels were equal in quiescent and 16-h-regenerating liver, the rate of transcription of the gene in quiescent liver was threefold higher than the rate in 16-h-regenerating liver. The maintenance of a higher rate of gene transcription in quiescent liver, as compared to regenerating liver, was shown to depend on ongoing protein synthesis. The possibility that the high rate of gene transcription was due to blockage or pausing during transcript elongation in quiescent liver was excluded. The inference is that the pronounced increase in tyrosine aminotransferase mRNA levels within 1 h of partial hepatectomy is largely due to a rapid increase in the efficiency of nuclear processing of the primary transcript.

The lipid composition of selected tissues from a Mediterranean monk seal, Monachus monachus.

The lipid composition of blubber, brain, muscle and heart from a Mediterranean monk seal Monachus monachus (an endangered species) were examined to allow comparisons with more common species of seals. Only neutral lipids (mainly triacylglycerols) were detectable in the blubber lipids, whereas polar lipids predominated in the heart and in the brain. Neutral and polar lipids comprised almost equal proportions in both liver and muscle. Choline glycerophospholipids (CGP) were the major polar lipids, followed by ethanolamine glycerophospholipids (EGP) in the liver, heart and muscle. Cerebrosides accounted for 28.8% of the brain lipids. All lipid classes of the liver contained high levels (31-47%) of polyunsaturated fatty acids (PUFA), with the exception of phosphatidylserine. The total proportion of n-6 PUFA exceeded that of n-3 PUFA in all lipid classes of the liver, due mainly to the high levels of 20:4n-6. The highest level of 20:4n-6 occurred in phosphatidylinositol, where it comprised 32.4% of the total fatty acids. The CGP and EGP of the brain contained lower levels of PUFA than those of the liver, muscle and heart. Alkenyl ethers accounted for 35.8% of the total long-chain moieties in brain EGP. The fatty acid composition of blubber triacylglycerols differed from those of the lipid classes from other tissues in that it had a very low ratio of n-6 to n-3 PUFA (0.3) as a result of a lower content of 20:4n-6.

Subunit composition of the untransformed glucocorticoid receptor in the cytosol and in the cell.

We have used bifunctional reagents to examine the subunit composition of the non-DNA-binding form of the rat and human glucocorticoid receptor. Treatment of intact cells and cell extracts with a reversible cross-linker, followed by electrophoretic analysis of immunoadsorbed receptor revealed that three proteins of apparent approximate molecular masses, 90, 53 and 14 kDa are associated with the receptor. The first of these was identified immunochemically as a 90-kDa heat-shock protein (hsp90). The complex isolated from HeLa cells contained 2.2 mol hsp90/mol steroid-binding subunit. Cross-linking of the receptor complex in the cytosol completely prevented salt-induced dissociation of the subunits. The cross-linked receptor was electrophoretically resolved into two oligomeric complexes of apparent molecular mass 288 kDa and 347 kDa, reflecting the association of the 53-kDa protein with a fraction of the receptor. Since no higher oligomeric complexes could be generated by cross-linking cell extracts under different conditions, we conclude that most of the untransformed cytosolic receptor is devoid of additional components.

Glucocorticoid receptor structure as probed by endogenous proteases.

Transformation of the glucocorticoid-receptor complex by heating the cytosol in the presence of calcium is accompanied by formation of a series of truncated complexes, of which DI and DIIc are the major members. Formation of DIIc (but not of DI) is inhibited by leupeptin, and the intact transformed complex DIIa appears instead. Estimation of the molecular weights and Stokes' radii of all major complexes revealed that forms DI and DIIc have the same Mr, 48 kDa, but differ in shape, and appear to be digestion products generated by cleavage at the same site. Proteolysis of glucocorticoid receptor, covalently labelled with [3H]dexamethasone mesylate in rat thymus and brain cytosol, corroborated these findings and further implied that DI is the product of digestion of the non-transformed form of the receptor. Covalently labelled receptor fragments, related to the products formed when cytosol is heated, are detected in the nuclei of thymocytes, implying that the same proteolytic cleavages sites are involved in receptor turnover. Cleavage sites in the non-transformed covalently labelled receptor were identified in the "stepladder" of fragments of Mr, 85, 65, 49, 35, 27-30 kDa, generated in the absence of calcium, with an additional 78 kDa fragment in its presence. In the transformed conformation, two of the cleavage sites giving rise to the 65 and 35 kDa fragments, appear to be protected. It is speculated that the change in the proteolytic susceptibility of the cleavage site for the 35 kDa fragment relates to the "unmasking" of enhancer-activating and/or DNA-binding receptor functions previously postulated.

Aminotransferase activity in the liver and white muscle of Mugil capito fed diets containing different levels of protein and carbohydrate.

Four groups of Mugil capito were fed diets with protein and carbohydrate contents changing reciprocally for 121 days. Food consumption changed proportionally to the carbohydrate content of the diet, while final fish growth was similar for all groups of fish. Liver transaminase levels changed significantly with the protein content of the diet. The changes in body transaminase levels were lower in magnitude. The results obtained are compared to those obtained from other fish species and possible reasons for the differences observed are discussed.

Glucocorticoid receptors in developing rat brain and liver.

Dexamethasone receptors were measured by conventional equilibrium steroid binding studies in rat liver and brain cytosol, during late prenatal and postnatal development, Receptor binding could be detected in both cytosol preparations as early as the 17th day of gestation. Receptor levels in the cytosol from intact animals reached adult values by the 1st day after birth in both tissues. Using adrenalectomized animals an increase which reached adult values was observed during the first postnatal week for liver and the second postnatal week for brain. At physiological concentrations of endogenous glucocorticoids depletion of receptor from the cytosol of intact animals was minimal at postnatal day 1 and reached adult levels by day 7. Chromatographic analysis in DEAE-Sephadex A50 minicolumns of unactivated and activated receptor constituents revealed the same pattern as that of adult animals. Glucocorticoid receptor complex from developing liver and brain was shown to be capable of binding to isolated adult liver nuclei after in vitro activation. However full capacity, for nuclear binding was observed in vivo, after injection of inducing doses of [3H]dexamethasone: By the end of the first week after birth adult nuclear binding capacity was observed in experiments in vivo while values peaked during the second week, in both tissues studied.

Activation and changes in the sedimentation properties of rat liver glucocorticoid receptor.

The glucocorticoid receptor in rat liver cytosol was studied by sucrose gradient sedimentation, DEAE-Sephadex A-50 column chromatography and DNA-cellulose binding in order to assign specific hydrodynamic properties to both the unactivated and the activated glucocorticoid--receptor complex with [3H]-dexamethasone. Activation was effected by heat, NaCl (0.4 M) or KSCN (0.1 M) treatment. The state of activation was judged by both DNA-cellulose binding and DEAE-Sephadex A-50 anion exchange chromatography. In isotonic phosphate buffer, unactivated and activated glucocorticoid--receptor complex sedimented as a 5 S and a 4 S peak, respectively. This 5 S-4 S transformation was blocked by sodium molybdate. In hypotonic phosphate buffer, both the unactivated and the activated glucocorticoid--receptor complex assumed higher s values due to aggregation. The activated complex (4 S) yielded aggregates of 5-6 S in a reversible manner, neither complex being affected by sodium molybdate. The unactivated complex was shown to assume two distinct aggregation states of 6 S and 8-9 S, which yielded a 10-11 S heavy aggregate upon addition of molybdate. This effect on the unactivated glucocorticoid--receptor complex was readily reversed by removing the molybdate. Aggregation at low ionic strength was promoted by a low mol. wt. component(s), separated from cytosol by gel filtration through Sephadex G-10. The state of aggregation had no pronounced effect on the DNA binding properties of the activated forms or on the sensitivity of the unactivated forms to molybdate.

The distribution and properties of the glucocorticoid receptor from rat brain and pituitary.

The distribution and properties of cytoplasmic binding sites for the synthetic glucocorticoid dexamethasone and the natural glucocorticoid corticosterone in the brain and the pituitary were studied in detail. Cortisol-17 beta acid, a derivative which does not bind to the glucocorticoid receptor but is a competitor of corticosterone binding to plasma, was used to overcome plasma interference. In vitro competition assays in the presence of excess cortisol acid reveal that dexamethasone is as effective a competitor for [3H]corticosterone binding as corticosterone itself. Scatchard analysis of equilibrium experiments with both steroids, using cytosol from various brain areas and from the pituitary yielded linear plots, suggesting one class of binding sites. The quantitative distribution of the sites follows the pattern: cortex greater than hippocampus greater than or equal to pituitary greater than hypothalamus greater than brain stem white matter. Furthermore, kinetic analysis of corticosterone dissociation showed a first order reaction, thus indicating the presence of one type of receptor in all brain areas examined. Rat brain cytosolic receptors for corticosterone and dexamethasone elute from DEAE-Sephadex A-50 anion exchange columns at 0.3 M NaCl in the presence of stabilizing sodium molybdate and at 0.15 M NaCl and/or in the buffer wash when heat-activated, thus exhibiting the characteristic activation pattern of rat liver cytosolic glucocorticoid receptor. The ratio of the buffer wash to the 0.15 M NaCl form is low for dexamethasone and very high for corticosterone. Receptor complexes from various brain parts showed the same activation pattern. In our experiments, brain corticosterone and dexamethasone receptors stabilized by sodium molybdate are indistinguishable by a number of techniques, thus indicating that it is unnecessary to evoke specific binding sites for each glucocorticoid.