Selenomethionine induces polyamine biosynthesis in regenerating rat liver tissue

Summary. Our study was undertaken to elucidate the effects of selenomethionine (SeMet) on polyamine metabolism in regenerating rat liver tissue, as useful model of rapidly growing normal tissue. We have examined the levels of spermine, spermidine and putrescine in liver tissue. At the same time we have evaluated the activities of polyamine oxidase (PAO) and diamine oxidase (DAO), the catabolic enzymes of polyamine metabolism. The obtained results suggest that polyamine levels in regenerating liver tissue, at 7^th day after two-thirds partial hepatectomy, were higher in comparison with control group. The administration of selenomethionine to hepatectomized animals during seven days, in a single daily dose of 2.5 μg/100 g body weight, increases the amount of spermine and spermidine; the level of putrescine does not change under the influence of SeMet in regenerating rat liver tissue. PAO activity is lower in regenerating hepatic tissue than in control group. Supplementation of hepatectomized animals with SeMet significantly decreases the activity of this enzyme. DAO activity was significantly higher in hepatectomized and in operated animals treated with SeMet compared to the sham-operated and control ones. The differential sensitivity observed in our model of highly proliferating normal tissue to SeMet, compared with the reported anticancer activity of this molecule is discussed.     

Oxygen dependence of tyrosine hydroxylase

Summary. The effects of dioxygen on tyrosine hydroxylase (TH) activity was studied, measuring the formation of DOPA from tyrosine, ³H₂O from 3,5-³H-tyrosine, or by direct oxygraphic determination of oxygen consumption. A high enzyme activity was observed during the initial 1–2 min of the reactions, followed by a decline in activity, possibly related to a turnover dependent substoichiometrical oxidation of enzyme bound Fe(II) to the inactive Fe(III) state. During the initial reaction phase, apparent K _m-values of 29–45 μM for dioxygen were determined for all human TH isoforms, i.e. 2–40 times higher than previously reported for TH isolated from animal tissues. After 8 min incubation, the K _m (O₂)-values had declined to an average of 20 ± 4 μM. Thus, TH activity may be severely limited by oxygen availability even at moderate hypoxic conditions, and the enzyme is rapidly and turnover dependent inactivated at the experimental conditions commonly employed to measure in vitro activities. Authors’ address: Jan Haavik, Department of Biomedicine, University of Bergen, 5009 Bergen, Norway     

Plasticity of the central nervous system (CNS) following perinatal asphyxia: Does nicotinamide provide neuroprotection?

Summary. We have investigated the idea that nicotinamide, a non-selective inhibitor of the sentinel enzyme Poly(ADP-ribose) polymerase-I (PARP-1), provides neuroprotection against the long-term neurological changes induced by perinatal asphyxia. Perinatal asphyxia was induced in vivo by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Sibling caesarean-delivered pups were used as controls. The effect of perinatal asphyxia on neurocircuitry development was studied in vitro with organotypic cultures from substantia nigra, neostriatum and neocortex, platted on a coverslip 3 days after birth. After approximately one month in vitro (DIV 25), the cultures were treated for immunocytochemistry to characterise neuronal phenotype with markers against the N-methyl-D-aspartate receptor subunit 1 (NR1), the dopamine pacemaker enzyme tyrosine hydroxylase (TH), and nitric oxide synthase (NOS), the enzyme regulating the bioavailability of NO. Nicotinamide (0.8 mmol/kg, i.p.) or saline was administered to asphyctic and caesarean-delivered pups 24, 48 and 72 h after birth. It was found that nicotinamide treatment prevented the effect of perinatal asphyxia on several neuronal parameters, including TH- and NOS-positive neurite atrophy and NOS-positive neuronal loss; supporting the idea that nicotinamide constitutes a therapeutic alternative for the effects produced by sustained energy-failure conditions, as occurring during perinatal asphyxia.     

Involvement of D-Asp in P450 aromatase activity and estrogen receptors in boar testis

Summary. Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17β-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17β-estradiol synthesis in the testis of those mammals which produce significant amounts of estrogens as well as testosterone. The boar testis contains D-Asp (40 ± 3.6 nmol/g tissue) which, according to immunohistological techniques, is localized mainly in Leydig cells, and, to a lesser extent, in sustentacular (Sertoli), peritubular and some germ cells. The enzyme P450aromatase is present in Leydig cells and few germ cells. In vitro experiments showed that the addition of D-Asp to testicular tissue extracts induced a significant increase of aromatase activity, as evaluated by testosterone conversion into 17β-estradiol. The enzyme’s K_m was not affected by D-Asp (about 25 nM in both control and D-Asp added tests). On the basis of these results we suggest that, as in the ovary, D-Asp is involved in the local control of aromatase activity of boar testis and, therefore, it intervenes in the 17β-estradiol production. In the testis, the D-Asp targets are presumably the Leydig cells, which having also a nuclear estrogen receptor are, in turn, one of the putative targets of the 17β-estradiol that they produce (autocrine effect).     

Protein tyrosine nitration in hydrophilic and hydrophobic environments

Summary. In this review we address current concepts on the biological occurrence, levels and consequences of protein tyrosine nitration in biological systems. We focused on mechanistic aspects, emphasizing on the free radical mechanisms of protein 3-nitrotyrosine formation and critically analyzed the restrictions for obtaining large tyrosine nitration yields in vivo, mainly due to the presence of strong reducing systems (e.g. glutathione) that can potently inhibit at different levels the nitration process. Evidence is provided to show that the existence of metal-catalyzed processes, the assistance of nitric oxide-dependent nitration steps and the facilitation by hydrophobic environments, provide individually and/or in combination, feasible scenarios for nitration in complex biological milieux. Recent studies using hydrophobic tyrosine analogs and tyrosine-containing peptides have revealed that factors controlling nitration in hydrophobic environments such as biomembranes and lipoproteins can differ to those in aqueous compartments. In particular, exclusion of key soluble reductants from the lipid phase will more easily allow nitration and lipid-derived radicals are suggested as important mediators of the one-electron oxidation of tyrosine to tyrosyl radical in proteins associated to hydrophobic environments. Development and testing of hydrophilic and hydrophobic probes that can compete with endogenous constituents for the nitrating intermediates provide tools to unravel nitration mechanisms in vitro and in vivo; additionally, they could also serve to play cellular and tissue protective functions against the toxic effects of protein tyrosine nitration.     

An influence of ligands of metabotropic glutamate receptor subtypes on parkinsonian-like symptoms and the striatopallidal pathway in rats

Summary. Several data indicate that inhibition of glutamatergic transmission may be important to alleviate of parkinsonian symptoms. Therefore, the aim of the present paper is to review recent studies on the search for putative antiparkinsonian-like effects of mGluR ligands and their brain targets. In order to inhibit glutamatergic transmission, the group I mGluRs (mGluR1 and mGluR5) were blocked, and group II (mGluR2/3) or III (mGluR4/7/8) mGluRs were activated. Systemic or intrastriatal administration of group I mGluR antagonists (mGluR5 – MPEP, MTEP; mGluR1 – AIDA) was found to inhibit parkinsonian-like symptoms (catalepsy, muscle rigidity) in rats. MPEP administered systemically and mGluR1 antagonists (AIDA, CPCCOEt, LY367385) injected intrastriatally reversed also the haloperidol-increased proenkephalin (PENK) mRNA expression in the striatopallidal pathway. Similarly, ACPT-1, a group III mGluR agonist, administered into the striatum, globus pallidus or substantia nigra inhibited the catalepsy. Intrastriatal injection of this compound reduced the striatal PENK expression induced by haloperidol. In contrast, a group II mGluR agonist (2R,4R-APDC) administered intrastriatally reduced neither PENK expression nor the above-mentioned parkinsonian-like symptoms. Moreover, a mixed mGluR8 agonist/AMPA antagonist, (R,S)-3,4-DCPG, administered systemically evoked catalepsy and enhanced both the catalepsy and PENK expression induced by haloperidol. The results reviewed in this article seem to indicate that group I mGluR antagonists or some agonists of group III may possess antiparkinsonian properties, and point at the striatopallidal pathway as a potential target of therapeutic intervention.     

The effect of taurine on polymorphonuclear leukocyte functions in endotoxemia

Summary. The aim of the present study was to measure MPO activity in PMN leukocytes after endotoxin administration, and to compare the levels of NO₂ ⁻ competing with taurine for reaction with HOCl. Furthermore we aimed to determine TauCl levels, a product of MPO–H₂O₂–Halide system, and to evaluate anti-inflammatory properties of PMN in endotoxemia. In addition, our second objective was to investigate the effect of taurine, an antioxidant amino acid, on anti-bactericidal and anti-inflammatory functions of PMN after administration of endotoxin together with taurine. All experiments were performed with four groups (control, taurine, endotoxemia, and taurine plus endotoxin) of ten guinea pigs. After endotoxin administration (4 mg/kg), MPO activities increased and taurine levels decreased. Therefore levels of TauCl, NO₂ ^•− increased. We observed the effects of taurine as conflicting. When taurine was administrated alone (300 mg/kg), all of these parameters decreased. Consequently, we suggested that taurine is influential in infected subjects but not on healthy ones as an antioxidative amino acid. In addition, we believe that in vivo effects of taurine may differ from those in vitro depending on its dosage.     

Enhancement of intestinal absorption of macromolecules by spermine in rats

Summary. The aim of this study was to investigate the enhancing effect of polyamines on intestinal absorption of fluorescein isothiocyanate-labeled dextran (MW 4400, FD-4) in the in situ loop study and in vivo oral absorption study. Absorption of FD-4 from the jejunum was significantly enhanced by 5 mM spermine without serious membrane damage in the jejunum. An in vivo oral absorption study was also performed, and plasma FD-4 levels increased significantly after co-administration of 30 mM spermine. In the in vitro transport studies with Caco-2 cells, prolonged incubation with spermine resulted in a gradual decrease in transepithelial electrical resistance. This finding suggests that the absorption-enhancing mechanism of spermine partly includes opening the tight junctions of the epithelium via the paracellular route. These results indicate that excess oral ingestion of polyamines may have widespread health effects via the modulation of the intestinal epithelial barrier function.     

Effects of glucocorticoids on polyamine metabolism in liver and spleen of guinea pig during sensitization

Summary. Glucocorticoids are potent anti-inflammatory and immunosuppressive agents. As endogenous inhibitors of cytokine synthesis, glucocorticoids suppress immune activation and uncontrolled overproduction of cytokines, preventing tissue injury. Also, polyamine spermine is endogenous inhibitor of cytokine production (inhibiting IL-1, IL-6 and TNF synthesis). The idea of our work was to examine dexamethasone effects on the metabolism of polyamines, spermine, spermidine and putrescine and polyamine oxidase activity in liver and spleen during sensitization of guinea pigs. Sensitization was done by application of bovine serum albumin with addition of complete Freund’s adjuvant. Our results indicate that polyamine amounts and polyamine oxidase activity increase during immunogenesis in liver and spleen. Dexamethasone application to sensitized and unsensitized guinea pigs causes depletion of polyamines in liver and spleen. Dexamethasone decreases polyamine oxidase activity in liver and spleen of sensitized guinea pigs, increasing at the same time PAO activity in tissues of unsensitized animals.     

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

 During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents. Received: 14 February 2000 / Accepted: 15 March 2000     

High-throughput fluorescence polarization method for identification of FKBP12 ligands

FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His(6)-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K(i) values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.     

DNA cleavage function of seryl-histidine dipeptide and its application

Summary. Previously published evidences highlighted the effect of transglutaminase (TG, EC 2.3.2.13) activation on the reduction of the in vitro adhesive and invasive behaviour of murine B16-F10 melanoma cells, as well as in vivo. Here, we investigated the influence of spermidine (SPD) incorporation by TG into basement membrane components i.e. laminin (LN) or Matrigel (MG), on the adhesion and invasion of B16-F10 melanoma cells by these TG/SPD-modified substrates. The adhesion assays showed that cell binding to the TG/SPD-modified LN was reduced by 30%, when compared to untreated LN, whereas the reduction obtained using TG/SPD-modified MG was 35%. Similarly, tumor cell invasion by the Boyden chamber system through TG/SPD modified LN or MG was respectively reduced by 45%, and by 69%. Evaluation of matrix metalloproteinase (gelatinases MMP-2 and MMP-9) activities by gel-zymography showed that MMP-2 activity was unaffected, while MMP-9 activity was reduced by about 32% using TG/SPD-modified substrate. These results strongly suggest that the observed antiinvasive effect of TG activation in the host may be ascribed to the covalent incorporation of polyamines, which led to the post-translational modification of some components of the cell basement membrane. This modification may interfere with the metastatic property of melanoma cells, affecting the proteolytic activity necessary for their migration and invasion activities. Authors’ address: Simone Beninati, Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica, I-00133 Rome, Italy     

Effect of cycloheximide on tryptophan binding to rat hepatic nuclei

Summary. This study evaluated whether cycloheximide, an inhibitor of protein synthesis, would affect the binding of L-tryptophan to rat hepatic nuclei or nuclear envelopes. Previous reports have indicated that the binding of L-tryptophan to hepatic nuclear envelope protein was saturable, stereospecific, and of high affinity. Also, the administration of L-tryptophan rapidly stimulated hepatic protein synthesis. In this study, we determined that the addition of cycloheximide in vitro inhibited ³H-tryptophan binding to hepatic nuclei or nuclear envelopes. Heat-treated cycloheximide failed to have this inhibitory binding effect. In vivo treatment of rats with cycloheximide diminished in vitro ³H-tryptophan binding to hepatic nuclei of treated rats compared to controls. Puromycin, another inhibitor of hepatic protein synthesis, when added in vitro did not affect ³H-tryptophan binding to hepatic nuclei but did diminish in vitro binding after in vivo treatment. Thus, cycloheximide added in vitro diminished ³H-tryptophan binding to hepatic nuclei probably by its structural effect on the receptor while cycloheximide administered in vivo may also act in part by inhibiting protein synthesis. Received March 22, 1999, Accepted May 31, 1999     

Kinetic and proteomic analyses of <Emphasis Type="Italic">S</Emphasis>-nitrosoglutathione-treated hexokinase A: consequences for cancer energy metabolism

Summary. Mammalian hexokinase (HXK) is found at the outer mitochondrial membrane, exposed to mitochondrial oxygen- and nitrogen-radicals. Given the important role of this enzyme in metabolic pathways and diseases, the effect of S-nitrosoglutathione (GSNO) on HXK A structure and activity was studied. To focus on the catalytic domain, yeast HXK A was used because it has a significant homology to the mammalian domain that contains both the regulatory and catalytic sites. Biologically relevant [GSNO]/[HXK] caused a significant decrease in V_max with glucose (but not with fructose), along with oxidation of 5 Met and nitration of 4 Tyr. Preincubation of HXK with glucose abrogated the effect of GSNO whereas fructose was ineffective. These results are interpreted by considering the tight binding of glucose to the enzyme as opposed to that of fructose. The segment comprised from amino acids 304 to 306 contained the most modifications. Given that this sequence is highly conserved in HXK from various species, a decline in activity is expected when a high-affinity substrate is presented. Considering that changes in primary structure are envisioned at high [GSNO]/[HXK] ratios, like those present under normal conditions, it could be hypothesized that the high concentration of hexokinase present in fast growing tumors may serve not only to sustain high glycolysis rates, but also to minimize protein damage that might result in activity decline, compromising energy metabolism.     

Lysine catabolism, an effective versatile regulator of lysine level in plants

Lysine is a nutritionally important essential amino acid, whose synthesis in plants is strongly regulated by the rate of its synthesis. Yet, lysine level in plants is also finely controlled by a super-regulated catabolic pathway that catabolizes lysine into glutamate and acetyl Co-A. The first two enzymes of lysine catabolism are synthesized from a single LKR/SDH gene. Expression of this gene is subject to compound developmental, hormonal and stress-associated regulation. Moreover, the LKR/SDH gene of different plant species encodes up to three distinct polypeptides: (i) a bifunctional enzyme containing the linked lysine-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) whose LKR activity is regulated by its linked SDH enzyme; (ii) a monofunctional SDH encoded by an internal promoter, which is a part of the coding DNA region of the LKR/SDH gene; and (iii) a monofunctional, highly potent LKR that is formed by polyadenylation within an intron. LKR activity in the bifunctional LKR/SDH polypeptide is also post-translationally regulated by phosphorylation by casein kinase-2 (CK2), but the consequence of this regulation is still unknown. Why is lysine metabolism super-regulated by synthesis and catabolism? A hypothesis addressing this important question is presented, suggesting that lysine may serve as a regulator of plant growth and interaction with the environment.     

Production of hypotaurine, taurine and sulfate in rats and mice injected with L-cysteinesulfinate

Summary. We studied in vivo production of taurine, hypotaurine and sulfate following subcutaneous administration of L-cysteinesulfinate (CSA) to rats and mice. When 5.0 mmol/kg of body weight of CSA was injected to rats, increased urinary excretions of taurine, hypotaurine and sulfate in 24 h urine were 617, 52 and 1,767 μmol/kg, respectively. From these results together with our previous data, sulfate production was calculated to be 1.6 times greater than taurine production. Increased contents (μmol/g of wet tissue) over the control of taurine and hypotaurine in mouse tissues at 60 min after the injection of 5.0 mmol/kg body weight of CSA were: liver, 3.5 and 9.9; kidney, 0.3 and 5.2; heart, 3.7 and 0.2; blood plasma, 0.4 and 0.2, respectively. Upon loading of hypotaurine or taurine, tissue contents of these amino acids in liver and kidney increased greatly. Our results indicate that liver is the most active tissue for taurine production, followed by kidney, and that external CSA, hypotaurine and taurine are easily taken up by these tissues.     

Programmed cell death: similarities and differences in animals and plants. A flower paradigm

Summary. After an overview of the criteria for the definition of cell death in the animal cell and of its different types of death, a comparative analysis of PCD in the plant cell is reported. The cytological characteristics of the plant cell undergoing PCD are described. The role of plant hormones and growth factors in the regulation of this event is discussed with particular emphasis on PCD activation or prevention by polyamine treatment (doses, timing and developmental stage of the organism) in a Developmental cell death plant model: the Nicotiana tabacum (tobacco) flower corolla. Some of the effects of polyamines might be mediated by transglutaminase catalysis. The activity of this enzyme was examined in different parts of the corolla during its life span showing an acropetal trend parallel to the cell death wave. The location of transglutaminase in some sub-cellular compartments suggests that it exerts different functions in the corolla DCD.     

Taurine and neural cell damage

Summary. The inhibitory amino acid taurine is an osmoregulator and neuromodulator, also exerting neuroprotective actions in neural tissue. We review now the involvement of taurine in neuron-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress, and the presence of free radicals, metabolic poisons and an excess of ammonia. The brain concentration of taurine is increased in several models of ischemic injury in vivo. Cell-damaging conditions which perturb the oxidative metabolism needed for active transport across cell membranes generally reduce taurine uptake in vitro, immature brain tissue being more tolerant to the lack of oxygen. In ischemia nonsaturable diffusion increases considerably. Both basal and K⁺-stimulated release of taurine in the hippocampus in vitro is markedly enhanced under cell-damaging conditions, ischemia, free radicals and metabolic poisons being the most potent. Hypoxia, hypoglycemia, ischemia, free radicals and oxidative stress also increase the initial basal release of taurine in cerebellar granule neurons, while the release is only moderately enhanced in hypoxia and ischemia in cerebral cortical astrocytes. The taurine release induced by ischemia is for the most part Ca²⁺-independent, a Ca²⁺-dependent mechanism being discernible only in hippocampal slices from developing mice. Moreover, a considerable portion of hippocampal taurine release in ischemia is mediated by the reversal of Na⁺-dependent transporters. The enhanced release in adults may comprise a swelling-induced component through Cl⁻ channels, which is not discernible in developing mice. Excitotoxic concentrations of glutamate also potentiate taurine release in mouse hippocampal slices. The ability of ionotropic glutamate receptor agonists to evoke taurine release varies under different cell-damaging conditions, the N-methyl-D-aspartate-evoked release being clearly receptor-mediated in ischemia. Neurotoxic ammonia has been shown to provoke taurine release from different brain preparations, indicating that the ammonia-induced release may modify neuronal excitability in hyperammonic conditions. Taurine released simultaneously with an excess of excitatory amino acids in the hippocampus under ischemic and other neuron-damaging conditions may constitute an important protective mechanism against excitotoxicity, counteracting the harmful effects which lead to neuronal death. The release of taurine may prevent excitation from reaching neurotoxic levels. Received January 25, 2000/Accepted January 31, 2000     

Androgenic sensitivity of polyamine-oxidizing enzyme activities in female rat tissues

Summary. Effects of testosterone (10 μg/100 g body weight) on polyamine-oxidizing enzyme activities in female rat uterus, liver and kidney were demonstrated. Testosterone-treated rats exhibited 2.07 fold (p < 0.002) higher uterine polyamine oxidase (PAO) activity and 1.93 fold (p < 0.02) higher diamine oxidase (DAO) activity, as compared to the controls. In the liver, testosterone caused an elevation in PAO (1.39 fold, p < 0.05), but not in DAO activity, whereas in kidney the hormone stimulated DAO (1.30 fold, p < 0.05), but not PAO activity. The effects observed suggest a possible role for testosterone in the modulation of polyamine levels in the female organs studied and especially in uterus. Received May 12, 1999, Accepted December 16, 1999