Increased cyclic GMP content in confluent and serum-restricted heart cell cultures exposed to polyamines

Cyclic AMP (cAMP) and cyclic GMP (cGMP) have been implicated as intracellular signals in the transition from a resting to a growing state. This suggestion comes from observations showing that the addition of growth promoting factors to quiescent cell cultures causes a rapid and transient decrease in cAMP and an increase in cGMP contents [9, 11] and that exogenous cAMP or cGMP congeners reduce or stimulate cell growth respectively [6, 13]. In view of this antagonistic effect elicited by the two nucleotides, it has been suggested that a fall in cAMP/cGMP ratio might be the triggering event for the initiation of cell proliferation [6]. Since polyamines correlate positively with active cell division [7], a possible involvement of these biogenic polycations in the regulation of cellular cyclic nucleotide contents is worthwhile investigating. Our previous reports have shown indeed that in different cultured cell types, spermine, spermidine and putrescine, at relatively low doses, are able to reduce cAMP content [3] by increasing cAMP-dependent phosphodiesterase activity (cAMP-PDE) [4] and to counteract the action of different cAMP-mediated effectors [3]. Besides endogenous polyamines seem to be involved in the cAMP-mediated induction of cAMP-PDE, as observed in heart cell cultures [4]. This report shows that the addition of each individual polyamine to confluent and serum-restricted heart cell cultures, while lowering cAMP content, induces an early and rapid increase of cGMP content by reducing the rate of its degradation.     

Continuous administration of heparin in patients with deep vein thrombosis can increase plasma levels of diamine oxidase

Besides its anticoagulant activity, the sulfated polysaccharide heparin has numerous other biological effects. Especially the antiinflammatory and immunoregulatory properties of heparin may be associated with its ability to release the histamine-degrading enzyme diamine oxidase (DAO) from tissue-bound sites into the circulation. Whereas DAO activity is at the limits of detection in normal human plasma, the application of heparin leads to a significant increase of plasma DAO activity. However, previously, only the effect of bolus injection of unfractionated heparin (UFH) had been studied. To investigate DAO release during continuous heparin infusion, 28 patients with deep vein thrombosis (DVT) undergoing heparin therapy were analyzed. Whereas continuous heparin infusion did not lead to any increase of plasma DAO activity in 12 patients (43%), 6 patients (21%) showed a single elevated and 10 patients (36%) permanently elevated plasma DAO activity. The groups of patients exhibiting different DAO release responses did not differ in age, sex, body weight, concomitant diseases, heparin infusion rates, coagulation indices, location and extension of thrombosis, or clinical outcome. However, the rate of idiopathic DVT was significantly higher in the group of patients releasing DAO. This study shows, for the first time, that continuous heparin infusion can lead to DAO release and that individuals exhibit considerable differences in their release response. Although the significance of heparin-induced DAO release needs further clarification, our results indicate that postheparin plasma DAO activity could be an interesting parameter correlated with idiopathic DVT.     

Overexpression of spermidine/spermine N1-acetyltransferase elevates the threshold to pentylenetetrazol-induced seizure activity in transgenic mice

Activation of polyamine catabolism in transgenic mice through an overexpression of spermidine/spermine N(1)-acetyltransferase (SSAT) results in a massive overaccumulation of the diamine putrescine in most tissues including brain. Putrescine pool in transgenic animals was strikingly expanded in every six brain regions analyzed at present. Pons (23-fold), cerebellum (37-fold), cerebrum (34-fold), and hippocampus (16-fold) showed the greatest increases in putrescine levels. Moreover, the molar ratio of putrescine to spermidine was increased in the different brain regions of the transgenic animals on an average of nearly 40-fold. Upon an exposure of the animals to pentylenetetrazol (PTZ) infusions, a compound known to induce epilepsy-like seizure activity, the SSAT transgenic mice showed significantly elevated seizure threshold to both clonic and tonic convulsions in comparison with their syngenic littermates. This difference, however, disappeared when the animals were treated with ifenprodil prior to PTZ infusions. The latter compound acts as an antagonist of N-methyl-D-aspartate receptor by binding to the polyamine site of the receptor. Overexpression of SSAT likewise appeared to protect the transgenic animals from PTZ-induced neuron loss in the hippocampus. As putrescine is known to serve as a precursor to gamma-aminobutyric acid (GABA), we carried out (1)H NMR analyses the results of which revealed that the levels of the inhibitory amino acid GABA and its excitatory counterpart glutamate were indistinguishable in syngenic and transgenic animals in all brain regions analyzed. The present results suggest that the frequently observed enhanced accumulation of putrescine in response to brain insults belongs to neuroprotective measures rather than being a cause of the subsequent injury.     

A sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase

Background: Most of the polyamines circulating in blood are spermidine (Spd) and spermine (Spm) with only trace amounts of putrescine (Put), and they are mainly localized in erythrocytes. We developed a simple and sensitive colorimetric assay for polyamines in erythrocytes using oat seedling polyamine oxidase (OSPO). The method is based on the unique substrate specificity of OSPO, which is active toward Spd and Spm, but not toward diamines such as Put and cadaverine and monoamines such as histamine. Methods: The polyamines, which were purified from packed erythrocytes by weak cation-exchange chromatography, were incubated with OSPO at 37 °C for 15 min. In the presence of the H₂O₂ produced by this polyamine oxidase reaction and a new chromogen, N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), peroxidase (POD) catalyzes the formation of N-[4-[[4-(dimethylamino)phenyl]imino]-2,5-cyclohexadien-1-ylidene]-N-methylmethanaminium chloride (Bindschedler's Green) having an absorption maximum at 727 nm. Results: The detection limit was 0.2 μmol/l for packed erythrocytes. The within-run and between-run precisions (coefficient of variation, CVs) were 5.6–15.2% and 6.5–16.4%, respectively. Analytical recoveries were 93.3–97.4%. Polyamine values obtained by this assay correlated well with those obtained by an HPLC (y=0.948x+1.912; r=0.944; n=46). Conclusions: This colorimetric assay is simple and highly sensitive and practical for clinical use.     

Polyamines and polyamine-metabolizing enzymes in schizophrenia: Current knowledge and concepts of therapy

Polyamines play preeminent roles in a variety of cellular functions in the central nervous system and other organs. A large body of evidence suggests that the polyamine pathway is prominently involved in the etiology and pathology of schizophrenia. Alterations in the expression and activity of polyamine metabolizing enzymes, as well as changes in the levels of the individual polyamines, their precursors and derivatives, have been measured in schizophrenia and animal models of the disease. Additionally, neuroleptic treatment has been shown to influence polyamine concentrations in brain and blood of individuals with schizophrenia. Thus, the polyamine system may appear to be a promising target for neuropharmacological treatment of schizophrenia. However, for a number of practical reasons there is currently only limited hope for a polyamine-based schizophrenia therapy.     

Roles of nitric oxide and polyamines in brain tumor growth

Nitric oxide (NO) and polyamines: putrescine, spermidine and spermine, are key arginine metabolites in mammalian tissues that play critical roles i.a. in regulation of vascular tone (NO), and cell cycle regulation (polyamines). In the brain, both classes of molecules additionally have neuromodulatory and neuroprotective potential, and NO also a neurotoxic potential. Here we review evidence that brain tumors use the NO- and polyamine-synthesizing machineries to the benefit of their differentiation and growth from healthy glia and neurons. With a few exceptions, brain tumors show increased activities of one or all of the three arginine (Arg) to NO-converting nitric oxide synthase (NOS) isoforms (iNOS, eNOS, nNOS), but also elevated activities of polyamines-generating and modifying enzymes: arginase I/II, ornithine decarboxylase and spermidine/spermine N¹-acetyltransferase. The degree of stimulation of NO- and polyamine synthesis often correlates with brain tumor malignancy. Excess NO, but also spermine, spermidine and their N¹-acetylated forms, are tumor- and context-dependently involved in angiogenesis, tumor initiation and growth, and resistance to chemo- or radiotherapy. Hypothetically, increased demand for NO and/or polyamines is likely to contribute to Arg auxotrophy of malignant brain tumors, albeit the causal nexus awaits experimental verification.     

Polyamines: Emerging players in bacteria–host interactions

Polyamines are small polycationic molecules found in almost all cells and associated with a wide variety of physiological processes. In recent years it has become increasingly clear that, in addition to core physiological functions, polyamines play a crucial role in bacterial pathogenesis. Considerable evidence has built up that bacteria have evolved mechanisms to turn these molecules to their own advantage and a novel standpoint to look at host–bacterium interactions emerges from the interplay among polyamines, host cells and infecting bacteria. In this review, we highlight how human bacterial pathogens have developed their own resourceful strategies to exploit polyamines or manipulate polyamine-related processes to optimize their fitness within the host. Besides contributing to a better understanding of the complex relationship between a pathogen and its host, acquisitions in this field have a significant potential towards the development of novel antibacterial therapeutic approaches.     

Translation initiation factor eIF-5A, the hypusine-containing protein, is phosphorylated on serine and tyrosine and O-glycosylated in Trichomonas vaginalis

The eukaryotic translation factor eIF-5A is highly conserved throughout eukaryotes and undergoes an unusual polyamine-dependent post-translational modification called hypusination. Trichomonas vaginalis has two tveif-5a genes (tveif-5a1 and tveif-5a2), each encoding a 19-kDa protein. In this report, we describe the detection of two forms with different isoelectric points (5.2 and 5.5) that correspond to the precursor and mature TveIF-5A, respectively. In addition, we demonstrated that only the mature form of TveIF-5A is phosphorylated and glycosylated via two-dimensional gel electrophoresis-western blot (2DE-WB) assays using anti-phosphoserine and anti-phosphotyrosine antibodies and the SNA, ConA and MAA lectins. Interestingly, when the protozoa were grown in 1,4-diamino-2-butanone (DAB), an inhibitor of putrescine biosynthesis, and transferred to medium containing exogenous putrescine, a new spot with an isoelectric point of 5.3 was observed, presumably corresponding to a phosphorylated intermediate or deoxyhypusine form. Our data indicate that, in T. vaginalis, phosphorylations and glycosylations are necessary to obtain the mature TveIF-5A, and we confirm the identity of the precursor, intermediate and mature forms of TveIF-5A by mass spectrometry analysis.     

On the mechanisms of ageing suppression by dietary restriction-is persistent glycolysis the problem?

The mechanism(s) by which dietary restriction (DR) suppresses ageing and onset of age-related pathologies are discussed in relation to frequency of glycolysis, and the reactivity of glycolytic intermediates. Most glycolytic intermediates are potentially toxic and readily modify (i.e. glycate) proteins and other macromolecules non-enzymically. Attention is drawn to the reactivity of methyglyoxal (MG) which is formed predominantly from the glycolytic intermediates dihydroxyacetone- and glyceraldehyde-3-phosphates. MG rapidly glycates proteins, damages mitochondria and induces a pro-oxidant state, similar to that observed in aged cells. It is suggested that because DR animals' energy metabolism is less glycolytic than in those fed ad libitum, intracellular MG levels are lowered by DR The decreased glycolysis during DR may delay senescence by lowering intracellular MG concentration compared to ad libitum-fed animals. Because of the reactivity MG and glycolytic intermediates, occasional glycolysis could be hormetic where glyoxalase, carnosine synthetase and ornithine decarboxylase are upregulated to control cellular MG concentration. It is suggested that in ad libitum-fed animals persistent glycolysis permanently raises MG levels which progressively overwhelm protective processes, particularly in non-mitotic tissues, to create the senescent state earlier than in DR animals. The possible impact of diet and intracellular glycating agents on age-related mitochondrial dysfunction is also discussed.     

Investigation of biologically active amines in some selected edible mushrooms

Mushrooms are highly valued due to nutritional and functional properties as well as small environmental footprint. However, scarce information is available regarding amines in commercial products. The objective of this study was to investigate the levels of bioactive amines in eight fresh edible commercial mushrooms species. An ion-pair HPLC method with post-column derivatization with o-phthalaldehyde and fluorescence detection was fit for the purpose. Seven out of nine amines were present and levels varied among species. Spermidine was ubiquitous to mushrooms, with highest content in Black Shimeji (12.4 mg/100 g). The levels of spermidine in mushrooms classify them as high polyamines sources, which is valued due to its association with growth, health promotion and antioxidant properties. Agmatine was present in all Pleurotus. Tyramine, tryptamine and phenylethylamine were detected in some species; the levels of cadaverine and putrescine were discrete. A four-principal component model explained 99.4% of the variance and it was able to separate Pleurotus spp. (White shimeji, Hiratake, Black shimeji and Salmon) from Agaricus bisporus (Champignon and Portobello) and Lentinula edodes (Shitake). Hierarchical cluster analysis confirmed the potential of using the occurrence and levels of amines to separate some mushroom species.