Significance of alterations in the metabolomics of sulfur-containing amino acids during liver regeneration

It has been known that liver regeneration is accompanied with a profound change in the metabolomics of sulfur-containing substances in liver. However, its physiological significance in the liver regenerative process is still unclear. Our previous work showed that buthioninesulfoximine and phorone, both widely used to deplete intracellular glutathione (GSH) in biological experiments, induced contrasting changes in the sulfur-containing amino acid metabolism in liver. In this study we employed these GSH-depleting agents to evaluate the role of sulfur-containing substances in the early phase of liver regeneration. Male rats treated with buthioninesulfoximine or phorone were subjected to two-thirds partial hepatectomy (PHx). At the doses used, the magnitude of GSH depletion after PHx was comparable, but buthioninesulfoximine administration inhibited the progression of liver regeneration as determined by liver weight increase, elevation of serum alanine aminotransferase activity, and cyclin D1 and proliferating cell nuclear antigen (PCNA) protein expressions, whereas liver recovery was significantly accelerated in the phorone-treated rats, suggesting that the role of GSH in this process is minimal. Hepatic concentrations of methionine, S-adenosylmethionine, cysteine, taurine and GSH were all elevated by PHx. Methionine adenosyltransferase activity was also induced in the remnant liver. Buthioninesulfoximine administration depressed the elevation of S-adenosylmethionine, but increased the catabolism of cysteine to taurine. In contrast, S-adenosylmethionine elevation was augmented whereas cysteine, hypotaurine and taurine were decreased in the phorone-treated rats. PHx elevated hepatic putrescine and spermidine, but lowered spermine concentrations. Buthioninesulfoximine administration increased putrescine further, but decreased spermidine and spermine concentrations. On the contrary, both spermidine and spermine concentrations were elevated in the rats treated with phorone. The results suggest that the availability of S-adenosylmethionine plays a critical role in the progression of liver regeneration via enhancement of polyamine synthesis. These findings raise the possibility that regulating hepatic transsulfuration reactions may be capable of modifying the recovery process after liver injury.     

Exogenous spermidine elevating cadmium tolerance in Salix matsudana involves cadmium detoxification and antioxidant defense

In this study, exogenous spermidine role on Salix matsudana tolerance to cadmium was evaluated. Spermidine and cadmium presented antagonistic effects on the biomass, copper and zinc concentrations in S. matsudana. cadmium mainly distributed in the cell wall of subcellular fraction; 46.97%–60.43% of cadmium existed in a sodium chloride-extracted form. Cadmium contents in roots, leaves, and twigs ranged from 2002.67 to 3961.00, 111.59 to 229.72, and 102.56 to 221.27?mg/kg, respectively. Spermidine application elevated cadmium concentrations in the roots, cuttings, and cell wall and the ratio of deionized water-extracted cadmium, but decreased cadmium levels in the twigs and leaves and the fractions of cadmium extracted by ethanol and sodium chloride, respectively. Putrescine and malondialdehyde were important indicators of cadmium-induced oxidative damage. Exogenous spermidine alleviated the accumulation of superoxide anion, hydrogen peroxide, malondialdehyde via promoting the levels of spermidine, soluble protein, superoxide dismutase, reductive ascorbate, glutathione reductase, and glutathione peroxidase in S. matsudana leaves under the corresponding cadmium stress. The results indicated that S. matsudana was a candidate for cadmium rhizoremediation and extraction in leaves; the spermidine application enhanced the cadmium tolerance of S. matsudana through promoting cadmium accumulation in roots, cell wall, and less bioactive chemical forms and the antioxidative ability.     

Molecular and functional characterization of a spermidine transporter (TcPAT12) from Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas' disease, is the only eukaryotic cell which lacks the ability to synthesize polyamines de novo. In this work, we describe for the first time the molecular and biochemical properties of a high-affinity spermidine transporter from T. cruzi. The transporter gene TcPAT12 was functionally expressed in Xenopus laevis oocytes, showing high levels of spermidine uptake. Similar apparent affinity constants for spermidine uptake were obtained when comparing T. cruzi epimastigotes and heterologous expressed TcPAT12 in X. laevis. In addition, TcPAT12 also transports putrescine and the amino acid l-arginine at lower rates than spermidine.     

Electrochemically fabricated polypyrrole nanofiber-modified electrode as a new electrochemical DNA biosensor

A new biosensor employing immobilized DNA on a nano-structured conductive polymer fixed onto a platinum electrode is presented. Upon optimization of synthesis parameters, polypyrrole nanofibers, 30-90 nm in diameter, were synthesized in an aqueous media by the electropolymerization of pyrrole using normal pulse voltammetry (NPV). The nanofiber film was investigated by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Double-stranded DNA was physisorbed onto the PPy nanofiber films. Various parameters, including the pH and DNA concentration, were optimized. The DNA immobilized on the nanofiber films was characterized using differential pulse voltammetry (DPV) and Fourier-transform infrared (FTIR) spectroscopy. Using DPV to study the interaction of spermidine with DNA, a binding constant (K) value of 4.08 x 10(5)+/-0.05 M(-1) was obtained. For the determination of spermidine, the proposed method exhibited a good dynamic range, correlation coefficient (0.05-1.0 microM and 0.9983, respectively) and a low detection limit (0.02 microM), although Ca(2+) ions were found to electrostatically bind to DNA and weaken the spermidine-DNA interaction.     

Determination of dansylated polyamines in red blood cells by liquid chromatography-tandem mass spectrometry

The concentration of polyamines in red blood cells (RBCs) is considered to be an index of cell proliferation. This index has been demonstrated to be of clinical importance for the follow-up and treatment of some cancer patients. The concentration of polyamines in RBCs is usually determined by high-performance liquid chromatography (HPLC) with fluorescence detection. In the current work, we present a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of putrescine, spermidine, and spermine, the three major polyamines in RBCs. The polyamines were dansylated and analyzed by an LC gradient of 20-min duration on a C18 column on-line with a tandem mass spectrometer. An internal standard (1,8-diaminooctane) was used for quantification. This method exhibited excellent linearity for the three polyamines with regression coefficients higher than 0.99. The limits of detection for putrescine, spermidine, and spermine were 0.10, 0.75, and 0.50 pmol/ml, respectively. The intrarun precision values for putrescine, spermidine, and spermine all were better than 10%, and the interrun precision values were 13%, 9%, and 20%, respectively. The LC-MS/MS method is sufficiently simple and reliable enough to replace the currently used HPLC method with fluorescence detection in which putrescine is not always detectable.     

Changes in free polyamine concentration induced by salt stress in seedlings of different species

Growth rate, mineral composition and changes in polyamine concentration induced in response to salinity were studied in six crop species: spinach, lettuce, bean, pepper, beetroot and tomato. Salinity decreased growth rate, but sensitivity differed amongst the species: pepper being the most sensitive, followed by bean, tomato, lettuce and spinach, with beetroot being the most tolerant. The increase of Na⁺ and total cation with salinity in shoots was the highest in spinach and beetroot, the most tolerant species, while in pepper it was the lowest. Changes in putrescine (Put) concentration in shoots were related to salinity tolerance (increased in the most sensitive), while changes in spermidine (Spd; decreases) and spermine (Spm; increases) were similar with most species, except for pepper in which salinity strongly increased Put, Spd and Spm. Therefore, total polyamine concentration increased in pepper shoot, while it decreased in the other species. Thus, results show that Put accumulation was a consequence of salt stress in the most sensitive species, while salt tolerant species (beetroot) showed little change in polyamine concentration, and higher concentration in both Na⁺ and total cations. The role of polyamines or cation increased concentration after saline treatment in species with different salt tolerance is discussed.     

Spermine is not essential for growth of Saccharomyces cerevisiae: identification of the SPE4 gene (spermine synthase) and characterization of a spe4 deletion mutant

Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [Hamasaki-Katagiri, N., Tabor, C.W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35–43].     

Pharmacological Characterization of Heterodimeric NMDA Receptors Composed of NR 1a and 2B Subunits: Differences with Receptors Formed from NR 1a and 2A

Abstract: Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both ¹²⁵I-MK-801 binding assays and electrophysiological measures. Binding of ¹²⁵I-MK-801 to cells transfected with NR 1a/2B is saturable with a K _D of 440 p M . The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK-801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, ¹²⁵I-MK-801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA-induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits.     

Changes in free polyamine titers and expression of polyamine biosynthetic genes during growth of peach <Emphasis Type="Italic">in vitro</Emphasis> callus

In the present paper, correlation between free polyamines and growth of peach (Prunus persica cv. Yuzora) in vitro callus was investigated. Growth of the callus was divided into three phases based on measurement of fresh weight. Free polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), could be detected during peach callus growth. Changes in free Put titers followed the callus growth rate, as shown by low and stable levels in the first stage, quick increase at the beginning of the second phase, and slow increase in the last phase, whereas fluctuations of Spd and Spm titers were aberrant from that of Put at early stage. Expressions of five key genes involved in polyamine biosynthesis were characterized, in which only the genes leading to Put synthesis, ADC (arginine decarboxylase) and ODC (ornithine decarboxylase), agreed with callus growth and fluctuation of Put titers. Treatment of the callus with D-arginine, an inhibitor of ADC, led to significant growth inhibition and enormous reduction of endogenous Put, coupled with obvious decrease of mRNA levels of ADC and ODC. Exogenous application of Put partially restored the callus growth, along with resumption of endogenous Put and expression levels of ADC and ODC. Spd and Spm titers experienced minor change in comparison to Put. The data presented here suggested that free Put played an important part in peach callus growth. Putative mechanisms or mode of action underlying the role of Put in peach callus growth and different expression patterns of the genes responsible for polyamine biosynthesis are also discussed.     

Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae

It has been reported that Gap1p on the plasma membrane of Saccharomyces cerevisiae can catalyze the uptake of many kinds of amino acids. In the present study, we found that Gap1p also catalyzed the uptake of putrescine and spermidine, but not spermine. The Km and Vmax values for putrescine and spermidine were 390 and 21 microM, and 4.6 and 0.59 nmol/min/mg protein, respectively. The uptake of putrescine was strongly inhibited by basic amino acids, lysine, arginine, and histidine, whose Ki values were 25-35 microM. Thus, it is deduced that spermidine and basic amino acids have almost the same affinity for Gap1p. When the concentrations of amino acids in the medium were reduced to one-third and 0.5 mM putrescine or 0.1 mM spermidine was added to the medium, accumulation of putrescine or spermidine by Gap1p was observed. Furthermore, when yeast was transformed with the GAP1 gene and cultured in the presence of 60 mM putrescine, cell growth was inhibited through overaccumulation of putrescine. GAP1 mRNA was found to be induced by polyamines. This is the first report of the identification, at a molecular level, of a polyamine uptake protein on the plasma membrane in eukaryotes.