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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.42, #2-3)

Role of polyamines, their analogs and transglutaminases in biological and clinical perspectives by Enzo Agostinelli (pp. 397-409).

Polyamine catabolism: target for antiproliferative therapies in animals and stress tolerance strategies in plants by Paraskevi Tavladoraki; Alessandra Cona; Rodolfo Federico; Giampiero Tempera; Nikenza Viceconte; Stefania Saccoccio; Valentina Battaglia; Antonio Toninello; Enzo Agostinelli (pp. 411-426).

Metabolism of polyamines spermidine and spermine, and their diamine precursor, putrescine, has been a target for antineoplastic therapy since these naturally occurring alkyl amines were found essential for normal mammalian cell growth. Intracellular polyamine concentrations are maintained at a cell type-specific set point through the coordinated and highly regulated interplay between biosynthesis, transport, and catabolism. A correlation between regulation of cell proliferation and polyamine metabolism is described. In particular, polyamine catabolism involves copper-containing amine oxidases and FAD-dependent polyamine oxidases. Several studies showed an important role of these enzymes in several developmental and disease-related processes in both animals and plants through a control on polyamine homeostasis in response to normal cellular signals, drug treatment, environmental and/or cellular stressors. The production of toxic aldehydes and reactive oxygen species, H₂O₂ in particular, by these oxidases using extracellular and intracellular polyamines as substrates, suggests a mechanism by which the oxidases can be exploited as antineoplastic drug targets. This minireview summarizes recent advances on the physiological roles of polyamine catabolism in animals and plants in an attempt to highlight differences and similarities that may contribute to determine in detail the underlined mechanisms involved. This information could be useful in evaluating the possibility of this metabolic pathway as a target for new antiproliferative therapies in animals and stress tolerance strategies in plants.

Keywords: Polyamines; Polyamine oxidase; Amine oxidase; Tumor cells; Reactive oxygen species; Plants

The activation of hepatic and muscle polyamine catabolism improves glucose homeostasis by Taina Koponen; Marc Cerrada-Gimenez; Eija Pirinen; Esa Hohtola; Jussi Paananen; Susanna Vuohelainen; Maija Tusa; Sini Pirnes-Karhu; Sami Heikkinen; Antti Virkamäki; Anne Uimari; Leena Alhonen; Markku Laakso (pp. 427-440).

The mitochondrial biogenesis and energy expenditure regulator, PGC-1α, has been previously reported to be induced in the white adipose tissue (WAT) and liver of mice overexpressing spermidine/spermine N ¹-acetyltransferase (SSAT). The activation of PGC-1α in these mouse lines leads to increased number of mitochondria, improved glucose homeostasis, reduced WAT mass and elevated basal metabolic rate. The constant activation of polyamine catabolism produces a futile cycle that greatly reduces the ATP pools and induces 5′-AMP-activated protein kinase (AMPK), which in turn activates PGC-1α in WAT. In this study, we have investigated the effects of activated polyamine catabolism on the glucose and energy metabolisms when targeted to specific tissues. For that we used a mouse line overexpressing SSAT under the endogenous SSAT promoter, an inducible SSAT overexpressing mouse model using the metallothionein I promoter (MT-SSAT), and a mouse model with WAT-specific SSAT overexpression (aP2-SSAT). The results demonstrated that WAT-specific SSAT overexpression was sufficient to increase the number of mitochondria, reduce WAT mass and protect the mice from high-fat diet-induced obesity. However, the improvement in the glucose homeostasis is achieved only when polyamine catabolism is enhanced at the same time in the liver and skeletal muscle. Our results suggest that the tissue-specific targeting of activated polyamine catabolism may reveal new possibilities for the development of drugs boosting mitochondrial metabolism and eventually for treatment of obesity and type 2 diabetes.

Keywords: Polyamine catabolism; Transgenic mouse; White adipose tissue; Energy metabolism; AMPK; PGC-1α; Mitochondria

Spermine oxidase: ten years after by Manuela Cervelli; Roberto Amendola; Fabio Polticelli; Paolo Mariottini (pp. 441-450).

Spermine oxidase (SMO) was discovered much more recently than other enzymes involved in polyamine metabolism; this review summarizes 10 years of researches on this enzyme. Spermine oxidase (SMO) is a FAD-dependent enzyme that specifically oxidizes spermine (Spm) and plays a dominant role in the highly regulated mammalian polyamines catabolism. SMO participates in drug response, apoptosis, response to stressful stimuli and etiology of several pathological conditions, including cancer. SMO is a highly inducible enzyme, its deregulation can alter polyamine homeostasis, and dysregulation of polyamine catabolism is often associated with several disease states. The oxidative products of SMO activity are spermidine, and the reactive oxygen species H₂O₂ and the aldehyde 3-aminopropanal each with the potential to produce cellular damages and pathologies. The SMO substrate Spm is a tetramine that plays mandatory roles in several cell functions, such as DNA synthesis, cellular proliferation, modulation of ion channels function, cellular signaling, nitric oxide synthesis and inhibition of immune responses. The goal of this review is to cover the main biochemical, cellular and physiological processes in which SMO is involved.

Keywords: Spermine oxidase; Spermine; Gene expression; Enzyme activity; Inhibitors; Differentiation; Cancer; Brain

Polyamine flux analysis by determination of heavy isotope incorporation from ¹³C, ¹⁵N-enriched amino acids into polyamines by LC–MS/MS by Marc Cerrada-Gimenez; Merja R. Häkkinen; Jouko Vepsäläinen; Seppo Auriola; Leena Alhonen; Tuomo A. Keinänen (pp. 451-460).

The study of polyamine flux, i.e. the circulating flow of polyamines through the interconnected biosynthetic and catabolic pathways, is of considerable interest because of the established links between the polyamine metabolism and many diseases, such as cancer and diabetes. To study polyamine flux in detail, a novel method based on following the label incorporation from the ¹³C, ¹⁵N-labeled polyamine precursors, arginine, methionine and ornithine, into polyamines by LC–MS/MS was implemented. This methodology was tested on three distinct cell lines with different spermidine/spermine-N ¹-acetyltransferase (SSAT) expression levels, i.e. non-transgenic, transgenic and knockout. These trials allowed the identification of the critical conditions for the successful polyamine flux measurement, such as the functional time frame of label incorporation, until plateau phase with the selected precursor is reached. The novel LC–MS/MS-based method for polyamine flux overcame the limitations of previous existing methodologies, with baseline separation of the different polyamine species and the exact quantification of the incorporated label. Moreover, the obtained results clearly show that the increased SSAT expression is associated with accelerated polyamine flux.

Keywords: Polyamines; Polyamine flux; LC/MS-MS; Fetal fibroblasts; SSAT; MDL 72527

Overexpression of spermidine/spermine N ¹-acetyltransferase or treatment with N ¹-N ¹¹-diethylnorspermine attenuates the severity of zinc-induced pancreatitis in mouse by Anne Uimari; Mari Merentie; Reijo Sironen; Sini Pirnes-Karhu; Sirpa Peräniemi; Leena Alhonen (pp. 461-471).

Depletion of pancreatic intracellular polyamine pools has been observed in acute pancreatitis both in the animal models and in humans. In this study, the wild-type mice, polyamine catabolic enzyme spermidine/spermine N ¹-acetyltransferase overexpressing (SSAT mice) and SSAT-deficient mice were used to characterize the new zinc-induced acute pancreatitis mouse model and study the role of polyamines and polyamine catabolism in this model. Intraperitoneal zinc injection induced acute necrotizing pancreatitis in wild-type mice as well as in SSAT-overexpressing and SSAT-deficient mice. Serum α-amylase activity was significantly increased in all zinc-treated mice compared with the untreated controls. However, the α-amylase activities in SSAT mice were constantly lower than those in the other groups. Histopathological examination of pancreatic tissue revealed edema, acinar cell necrosis and necrotizing inflammation, typical for acute pancreatitis. Compared with the other zinc-treated mice less damage according to the histopathological analysis was observed in the pancreatic tissue of SSAT mice. Levels of intracellular spermidine, and occasionally spermine, were significantly decreased in pancreases of all zinc-treated animals and SSAT enzyme activity was enhanced both in wild-type and SSAT mice. Interestingly, a spermine analog, N ¹, N ¹¹-diethylnorspermine (DENSpm), enhanced the proliferation of pancreatic cells and reduced the severity of zinc-induced pancreatitis in wild-type mice. The results show that in mice a single intraperitoneal zinc injection causes acute necrotizing pancreatitis accompanied by decrease of intracellular polyamine pools. The study supports the important role of polyamines for the integrity and function of the pancreas. In addition, the study suggests that whole body overexpression of SSAT obtained in SSAT mice reduces inflammatory pancreatic cell injury.

Keywords: Spermidine/spermine N ¹-acetyltransferase (SSAT); Pancreatitis; Spermine analog; Polyamines

Lipopolysaccharide-induced anti-inflammatory acute phase response is enhanced in spermidine/spermine N ¹-acetyltransferase (SSAT) overexpressing mice by Sini Pirnes-Karhu; Reijo Sironen; Leena Alhonen; Anne Uimari (pp. 473-484).

Bacterial lipopolysaccharide (LPS) is an effective activator of the components of innate immunity. It has been shown that polyamines and their metabolic enzymes affect the LPS-induced immune response by modulating both pro- and anti-inflammatory actions. On the other hand, LPS causes changes in cellular polyamine metabolism. In this study, the LPS-induced inflammatory response in spermidine/spermine N ¹-acetyltransferase overexpressing transgenic mice (SSAT mice) was analyzed. In liver and kidneys, LPS enhanced the activity of the polyamine biosynthetic enzyme ornithine decarboxylase and increased the intracellular putrescine content in both SSAT overexpressing and wild-type mice. In survival studies, the enhanced polyamine catabolism and concomitantly altered cellular polyamine pools in SSAT mice did not affect the LPS-induced mortality of these animals. However, in the acute phase of LPS-induced inflammatory response, the serum levels of proinflammatory cytokines interleukin-1β and interferon-γ were significantly reduced and, on the contrary, anti-inflammatory cytokine interleukin-10 was significantly increased in the sera of SSAT mice compared with the wild-type animals. In addition, hepatic acute-phase proteins C-reactive protein, haptoglobin and α₁-acid glycoprotein were expressed in higher amounts in SSAT mice than in the wild-type animals. In summary, the study suggests that SSAT overexpression obtained in SSAT mice enhances the anti-inflammatory actions in the acute phase of LPS-induced immune response.

Keywords: Polyamine; Spermidine/spermine N ¹-acetyltransferase (SSAT); Lipopolysaccharide (LPS); Immune response

Tissue-specific alternative splicing of spermidine/spermine N ¹-acetyltransferase by Mervi T. Hyvönen; Anne Uimari; Jouko Vepsäläinen; Alex R. Khomutov; Tuomo A. Keinänen; Leena Alhonen (pp. 485-493).

The polyamines, spermidine and spermine, are abundant organic cations participating in many important cellular processes. We have previously shown that the rate-limiting enzyme of polyamine catabolism, spermidine/spermine N ¹-acetyltransferase (SSAT), has an alternative mRNA splice variant (SSATX) which undergoes degradation via nonsense-mediated mRNA decay (NMD) pathway, and that the intracellular polyamine level regulates the ratio of the SSATX and SSAT splice variants. The aim of this study was to investigate the effect of SSATX level manipulation on SSAT activity in cell culture, and to examine the in vivo expression levels of SSATX and SSAT mRNA. Silencing SSATX expression with small interfering RNA led to increased SSAT activity. Furthermore, transfection of SSAT-deficient cells with mutated SSAT gene (which produced only trace amount of SSATX) yielded higher SSAT activity than transfection with natural SSAT gene (which produced both SSAT and SSATX). Blocking NMD in vivo by protein synthesis inhibitor cycloheximide resulted in accumulation of SSATX mRNA, and like in cell culture, the increase of SSATX mRNA was prevented by administration of polyamine analog N 1,N 11-diethylnorspermine. Although SSATX/total SSAT mRNA ratio did not correlate with polyamine levels or SSAT activity between different tissues, increasing polyamine levels in a given tissue led to decreased SSATX/total SSAT mRNA ratio and vice versa. Taken together, the regulated unproductive splicing and translation of SSAT has a physiological relevance in modulating SSAT activity. However, in addition to polyamine level there seems to be additional factors regulating tissue-specific alternative splicing of SSAT.

Keywords: Polyamines; Polyamine analogs; Transgenic animals; Regulated unproductive splicing and translation; Nonsense-mediated mRNA decay; Cycloheximide; Alternative splicing

Characterization of transgenic mice with overexpression of spermidine synthase by Chenxu Shi; Patricia A. Welsh; Suzanne Sass-Kuhn; Xiaojing Wang; Diane E. McCloskey; Anthony E. Pegg; David J. Feith (pp. 495-505).

A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to ninefold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5- to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.

Keywords: Polyamine; Aminopropyltransferase; Transgenic mice; S-Adenosylmethionine decarboxylase; Spermidine; Spermine

Overexpression of ornithine decarboxylase decreases ventricular systolic function during induction of cardiac hypertrophy by Emanuele Giordano; Rebecca A. Hillary; Thomas C. Vary; Anthony E. Pegg; Andrew D. Sumner; Claudio M. Caldarera; Xue-Qian Zhang; Jianliang Song; JuFang Wang; Joseph Y. Cheung; Lisa M. Shantz (pp. 507-518).

Ornithine decarboxylase (ODC), the first enzyme of polyamine metabolism, is rapidly upregulated in response to agents that induce a pathological cardiac hypertrophy. Transgenic mice overexpressing ODC in the heart (MHC-ODC mice) experience a much more dramatic left ventricular hypertrophy in response to β-adrenergic stimulation with isoproterenol (ISO) compared to wild-type (WT) controls. ISO also induced arginase activity in transgenic hearts but not in controls. The current work studies the cooperation between the cardiac polyamines and l-arginine (l-Arg) availability in MHC-ODC mice. Although ISO-induced hypertrophy is well-compensated, MHC-ODC mice administered l-Arg along with ISO showed a rapid onset of systolic dysfunction and died within 48 h. Myocytes isolated from MHC-ODC mice administered l-Arg/ISO exhibited reduced contractility and altered calcium transients, suggesting an alteration in [Ca²⁺] homeostasis, and abbreviated action potential duration, which may contribute to arrhythmogenesis. The already elevated levels of spermidine and spermine were not further altered in MHC-ODC hearts by l-Arg/ISO treatment, suggesting alternative l-Arg utilization pathways lead to dysregulation of intracellular calcium. MHC-ODC mice administered an arginase inhibitor (Nor-NOHA) along with ISO died almost as rapidly as l-Arg/ISO-treated mice, while the iNOS inhibitor S-methyl-isothiourea (SMT) was strongly protective against l-Arg/ISO. These results point to the induction of arginase as a protective response to β-adrenergic stimulation in the setting of high polyamines. Further, NO generated by exogenously supplied l-Arg may contribute to the lethal consequences of l-Arg/ISO treatment. Since considerable variations in human cardiac polyamine and l-Arg content are likely, it is possible that alterations in these factors may influence myocyte contractility.

Keywords: Arginase; Heart hypertrophy; Isoproterenol; Ornithine decarboxylase; Calcium homeostasis

Plant ornithine decarboxylase is not post-transcriptionally feedback regulated by polyamines but can interact with a cytosolic ribosomal protein S15 polypeptide by Crista Illingworth; Anthony J. Michael (pp. 519-527).

The formation of putrescine by ornithine decarboxylase (ODC) is a key regulatory step in polyamine biosynthesis in metazoa and fungi. Excess polyamines post-transcriptionally induce the synthesis of a unique non-competitive protein inhibitor of ODC, termed antizyme. Binding of antizyme to an ODC monomer subunit results in enzymatic inhibition, rapid ubiquitin-independent degradation of ODC by the 26S proteasome and recycling of antizyme. Plants possess an additional route for synthesizing putrescine via arginine decarboxylase (ADC). No homologue of ODC antizyme has been detected in plant genomes but several biochemical studies have reported plant ODC antizyme proteins of 9 and 16 kDa. Here we show that plant cells grown in liquid culture do not exhibit any substantial post-transcriptional, polyamine-responsive feedback regulation of ODC or ADC. However, using the yeast two hybrid system, a plant ODC-binding polypeptide was detected: the C-terminal 84-87 amino acids of cytosolic ribosomal protein (rp) S15. The Arabidopsis rpS15 polypeptide interacted specifically with plant ODC but not with human or Saccharomyces cerevisiae ODCs. Co-expression of either the full length or C-terminal rpS15 polypeptides with a plant ODC in yeast did not reduce ODC enzymatic activity. Only the full length mRNA encoding rpS15 was detected in Arabidopsis cells, suggesting that the C-terminal rpS15 polypeptide is encoded by a low abundance mRNA or the polypeptide is not physiologically relevant in plants. These results confirm the primacy of S-adenosylmethionine decarboxylase as the key regulatory enzyme in plant polyamine biosynthesis.

Keywords: Polyamine; Antizyme; Ornithine decarboxylase; Post-transcriptional; Arabidopsis ; Pea

Knockdown of ornithine decarboxylase antizyme 1 causes loss of uptake regulation leading to increased N ¹, N ¹¹-bis(ethyl)norspermine (BENSpm) accumulation and toxicity in NCI H157 lung cancer cells by Alison V. Fraser; Andrew C. Goodwin; Amy Hacker-Prietz; Elizabeth Sugar; Patrick M. Woster; Robert A. Casero Jr. (pp. 529-538).

Ornithine decarboxylase antizyme 1 (AZ1) is a major regulatory protein responsible for the regulation and degradation of ornithine decarboxylase (ODC). To better understand the role of AZ1 in polyamine metabolism and in modulating the response to anticancer polyamine analogues, a small interfering RNA strategy was used to create a series of stable clones in human H157 non-small cell lung cancer cells that expressed less than 5–10% of basal AZ1 levels. Antizyme 1 knockdown clones accumulated greater amounts of the polyamine analogue N ¹,N ¹¹-bis(ethyl)norspermine (BENSpm) and were more sensitive to analogue treatment. The possibility of a loss of polyamine uptake regulation in the knockdown clones was confirmed by polyamine uptake analysis. These results are consistent with the hypothesis that AZ1 knockdown leads to dysregulation of polyamine uptake, resulting in increased analogue accumulation and toxicity. Importantly, there appears to be little difference between AZ1 knockdown cells and cells with normal levels of AZ1 with respect to ODC regulation, suggesting that another regulatory protein, potentially AZ2, compensates for the loss of AZ1. The results of these studies are important for the understanding of both the regulation of polyamine homeostasis and in understanding the factors that regulate tumor cell sensitivity to the anti-tumor polyamine analogues.

Keywords: Antizyme; Ornithine decarboxylase; Polyamines; Spermine; BENSpm

Differential expression of ornithine decarboxylase antizyme inhibitors and antizymes in rodent tissues and human cell lines by Bruno Ramos-Molina; Andrés J. López-Contreras; Asunción Cremades; Rafael Peñafiel (pp. 539-547).

Ornithine decarboxylase antizyme inhibitors, AZIN1 and AZIN2, are regulators and homologous proteins of ornithine decarboxylase (ODC), the rate limiting enzyme in the biosynthesis of polyamines. In this study, we have examined by means of real-time RT-PCR the relative abundance of mRNA of the three ODC paralogs in different rodent tissues, as well as in several cell lines derived from human tumors. With the exception of mouse and rat testes, ODC mRNA was the most expressed gene in all tissues examined (values higher than 60%). AZIN2 was more expressed than AZIN1 in testis, epididymis, brain, adrenal gland and lung, whereas the opposite was found in liver, kidney, heart, intestine and pancreas, as well as in all the cell lines examined. mRNA abundance of the three antizymes (AZ1, AZ2 and AZ3) that interact with ODC and antizyme inhibitors was also analyzed. AZ1 and AZ2 mRNA were ubiquitously expressed, AZ1 mRNA being more abundant than that of AZ2, although the ratio was dependent on the mouse tissue. In carcinoma-derived cells AZ1 was more expressed than AZ2, whereas in neuroblastoma-derived cells AZ2 mRNA was much more abundant than that of AZ1. AZ3 was expressed exclusively in rodent testes, where it was the most abundant of the three antizymes (~80%). This study is the first comparative-quantitative analysis on the expression of antizymes and antizyme inhibitors in different types of mammalian cells.

Keywords: Antizymes; Antizyme inhibitor; AZIN2; Ornithine decarboxylase; Polyamines; RT-PCR

Knockdown of antizyme inhibitor decreases prostate tumor growth in vivo by Rachelle R. Olsen; Ivy Chung; Bruce R. Zetter (pp. 549-558).

The endogenous protein antizyme inhibitor (AZI) is a potential oncogene which promotes cell growth by both inhibiting antizyme (AZ) activity and releasing ornithine decarboxylase (ODC) from AZ-mediated degradation. High levels of ODC and polyamines are associated with numerous types of neoplastic transformation, and the genomic region including AZI is frequently amplified in tumors of the ovary and prostate. To determine whether AZI functionally promotes prostate tumor growth, we made PC3M-LN4 (human) and AT6.1 (rat) cancer cell lines stably expressing shRNA to knockdown antizyme inhibitor 1 (AZI). AZI knockdown was confirmed by western blot, quantitative real-time PCR, and immunofluorescence. To examine the ability of these cells to form tumors in vivo, 1 × 10⁶ cells were injected subcutaneously into nude mice either with (PC3M-LN4) or without (AT6.1) Matrigel. Tumor growth was measured two times per week by caliper. We found that cells in which AZI levels had been knocked down by shRNA formed significantly smaller tumors in vivo in both human and rat prostate cancer cell lines. These results suggest that not only does AZI promote tumor growth, but also that AZI may be a valid therapeutic target for cancer treatment.

Keywords: Antizyme inhibitor; Cell proliferation; Prostate cancer; Tumor growth

Inducible expression of antizyme 1 in prostate cancer cell lines after lentivirus mediated gene transfer by Marko Pietilä; Anita Lampinen; Riikka Pellinen; Leena Alhonen (pp. 559-564).

The prostate has the highest level of polyamines among all tissues, and it is the only tissue in which polyamines are purposely synthesized for export. It has been suggested that the high local polyamine concentrations suppress cell growth of primary prostatic carcinomas and that this growth control is lost when cancer cells metastasize. It has also been shown that the sensitivity to polyamine-induced growth arrest correlates with antizyme induction in prostate carcinoma cell lines. In this study, we evaluated the sensitivity of poorly metastatic (LNCaP) and highly metastatic (DU145) prostate cancer cell lines to conditional antizyme 1 over-expression. Antizyme 1 induction resulted in a marked loss of ODC activity and polyamine uptake in both cell lines. However, the proliferation of LNCaP cells was repressed by antizyme 1 induction, whereas the proliferation of DU 145 cells was not affected. Neither cell line showed any reduction in polyamine pools after manipulation nor did polyamine addition into the medium save the LNCaP cells from the growth retardation. The growth inhibition of LNCaP cells was accompanied by accumulation of cells in the G1 phase and depletion of cyclin E1 protein. These results confirm that different prostate cancer cell lines show diverse sensitivities to antizyme 1 which may not be directly polyamine related. The high gene transfer capacity of the used lentiviral vector makes the present approach a useful tool to study the therapeutic potential of antizyme 1 both in cell cultures and experimental animals.

Keywords: Cell cycle; Gene therapy; Ornithine decarboxylase; Polyamine

Correlation between antizyme 1 and differentiation of vascular smooth muscle cells cultured in honeycomb-like type-I collagen matrix by Itsuko Ishii; Takaaki Suzuki; Hiromi Kaneko; Masashi Uchida; Yukari Suzuki; Kyohei Higashi; Satoko Yagi; Noritaka Ariyoshi; Kazuei Igarashi; Mitsukazu Kitada (pp. 565-575).

Vascular smooth muscle cells (SMC) are able to proliferate when cultured on plates, but become differentiated when maintained in three-dimensional type I collagen matrices (honeycombs). SMC grown in honeycombs contained a low level of polyamines due to the presence of antizyme 1 (AZ1), a negative regulator of ornithine decarboxylase (ODC) and of polyamine uptake. To clarify the role of AZ1 in differentiation of SMC in honeycombs, an ODC gene was stably transfected into SMC (ODC-SMC). Although proliferation of ODC-SMC on plates was accelerated together with an increase in phosphorylated focal adhesion kinase (FAK) and a decrease in α-actin and myosin, maker proteins of differentiation, growth of ODC-SMC ceased in honeycombs similarly to normal SMC with a low level of phosphorylated FAK and a high level of α-actin and myosin. AZ1 expression in ODC-SMC on plates was low, but that in honeycombs was high. Antizyme in ODC-SMC in honeycombs not only decreased the level of ODC but also inhibited polyamine uptake activity. These results taken together suggest that low levels of polyamines caused by AZ1 in SMC in honeycombs inhibit phosphorylation of FAK and enhance expression of α-actin and myosin, resulting in differentiation through inhibition of focal adhesions.

Keywords: Polyamines; Antizyme 1; Ornithine decarboxylase; Polyamine transport; Differentiation; Smooth muscle cells

Regulatory cross-talk of mouse liver polyamine and methionine metabolic pathways: a systemic approach to its physiopathological consequences by F. Correa-Fiz; A. Reyes-Palomares; I. Fajardo; E. Melgarejo; A. Gutiérrez; J. A. García-Ranea; M. A. Medina; F. Sánchez-Jiménez (pp. 577-595).

Both polyamines and methionine derivatives are nitrogen compounds directly related to the regulation of gene expression. In silico predictions and experimental evidence suggest a cross-talk between polyamine and methionine metabolism in mammalian tissues. Since liver is the major organ that controls nitrogen metabolism of the whole organism, it is the best tissue to further test this hypothesis in vivo. In this work, we studied the effects of the chronic administration of a methionine-supplemented diet (0.5% Met in drinking water for 5 months) on the liver of mice (designated as MET-mice). Metabolic and proteomic approaches were performed and the data obtained were subjected to biocomputational analysis. Results showed that a supplemental methionine intake can indeed regulate biogenic amine metabolism in an in vivo model by multiple mechanisms including metabolic regulation and specific gene demethylation. Furthermore, putative systemic effects were investigated by molecular and cellular biology methods. Among other results, altered expression levels of multiple inflammation and cell proliferation/death balance markers were found and macrophage activation was observed. Overall, the results presented here will be of interest across a variety of biomedical disciplines, including nutrition, orphan diseases, immunology and oncology.

Keywords: Systems biology; Hyperhomocysteinaemia; Methionine; Polyamines; Liver; Inflammation

A combined model of hepatic polyamine and sulfur amino acid metabolism to analyze S-adenosyl methionine availability by Armando Reyes-Palomares; Raúl Montañez; Francisca Sánchez-Jiménez; Miguel Ángel Medina (pp. 597-610).

Many molecular details remain to be uncovered concerning the regulation of polyamine metabolism. A previous model of mammalian polyamine metabolism showed that S-adenosyl methionine availability could play a key role in polyamine homeostasis. To get a deeper insight in this prediction, we have built a combined model by integration of the previously published polyamine model and one-carbon and glutathione metabolism model, published by different research groups. The combined model is robust and it is able to achieve physiological steady-state values, as well as to reproduce the predictions of the individual models. Furthermore, a transition between two versions of our model with new regulatory factors added properly simulates the switch in methionine adenosyl transferase isozymes occurring when the liver enters in proliferative conditions. The combined model is useful to support the previous prediction on the role of S-adenosyl methionine availability in polyamine homeostasis. Furthermore, it could be easily adapted to get deeper insights on the connections of polyamines with energy metabolism.

Keywords: Metabolic modeling; Systems biology; Polyamines; S-adenosyl methionine; Methionine cycle; Folate cycle

Regulation of polyamine metabolism by translational control by Oscar Perez-Leal; Salim Merali (pp. 611-617).

Polyamines are low molecular weight, positively charged compounds that are ubiquitous in all living cells. They play a crucial role in many biochemical processes including regulation of transcription and translation, modulation of enzyme activities, regulation of ion channels and apoptosis. A strict balance between synthesis, catabolism and excretion tightly controls the cellular concentration of polyamines. The concentrations of rate-limiting enzymes in the polyamine synthesis and degradation pathways are regulated at different levels, including transcription, translation and degradation. Polyamines can modulate the translation of most of the enzymes required for their synthesis and catabolism through feedback mechanisms that are unique for each enzyme. Translational control is associated with cis-acting and trans-acting factors that can be influenced by the concentration of polyamines through mechanisms that are not completely understood. In this review, we present an overview of the translational control mechanisms of the proteins in the polyamine pathway, including ornithine decarboxylase (ODC), ODC antizyme, S-adenosylmethionine decarboxylase and spermidine/spermine N ¹ acetyltransferase, highlighting the areas where more research is needed. A better understanding of the translational control of these enzymes would offer the possibility of a novel pharmacological intervention against cancer and other diseases.

Keywords: Translational control; Translational repressor; Polyamines; Spermidine; Spermine

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells by Ikumi Ishii; Yoshihiko Ikeguchi; Hiroshi Mano; Masahiro Wada; Anthony E. Pegg; Akira Shirahata (pp. 619-626).

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.

Keywords: Adipogenesis; Polyamine metabolism; Spermine; Spermidine; Polyamine oxidation; 3T3-L1

Arginine and polyamines in Helicobacter pylori-induced immune dysregulation and gastric carcinogenesis by Rupesh Chaturvedi; Thibaut de Sablet; Lori A. Coburn; Alain P. Gobert; Keith T. Wilson (pp. 627-640).

l-arginine (l-Arg) is metabolized by nitric oxide synthase and arginase enzymes. The gastric pathogen Helicobacter pylori causes peptic ulcer disease and gastric cancer. We have shown that alterations in l-Arg availability and metabolism into polyamines contribute significantly to the dysregulation of the host immune response to this infection. Nitric oxide (NO) derived from inducible NO synthase (iNOS) can kill H. pylori. There are multiple mechanisms leading to failure of this process, including competition for l-Arg substrate by H. pylori arginase, and induction of host macrophage arginase II (Arg2) and ornithine decarboxylase (ODC). Generation of spermine by ODC inhibits iNOS translation and NO-mediated H. pylori killing. Expression of ODC is dependent on formation of a unique AP-1 complex, leading to upregulation of c-Myc as a transcriptional enhancer. Macrophage apoptosis is mediated by oxidation of spermine via the enzyme spermine oxidase (SMO) that generates hydrogen peroxide (H₂O₂), and thus oxidative stress-induced mitochondrial membrane polarization. Our studies have demonstrated that apoptosis occurs through a pERK → pc-Fos/c-Jun → c-Myc → ODC → SMO pathway. In gastric epithelial cells, activation of oxidative stress by H. pylori is dependent on SMO induction and results in both apoptosis and DNA damage, such that inhibition or knockdown of SMO markedly attenuates these events. In summary, l-Arg metabolism by the arginase–ODC pathway and the activation of SMO leads to H. pylori-induced DNA damage and immune dysregulation through polyamine-mediated oxidative stress and impairment of antimicrobial NO synthesis. Our studies indicate novel targets for therapeutic intervention in H. pylori-associated diseases, including gastritis, ulcer disease, and gastric cancer.

Keywords: H. pylori ; Polyamines; iNOS; Gastritis

Distamycin A and derivatives as synergic drugs in cisplatin-sensitive and -resistant ovarian cancer cells by Gaetano Marverti; Giambattista Guaitoli; Alessio Ligabue; Chiara Frassineti; Maria Giuseppina Monti; Paolo Lombardi; Maria Paola Costi (pp. 641-653).

Acquired resistance to cisplatin (cDDP) is a multifactorial process that represents one of the main problems in ovarian cancer therapy. Distamycin A is a minor groove DNA binder whose toxicity has limited its use and prompted the synthesis of derivatives such as NAX001 and NAX002, which have a carbamoyl moiety and different numbers of pyrrolamidine groups. Their interaction with a B-DNA model and with an extended-TATA box model, [Polyd(AT)], was investigated using isothermal titration calorimetry (ITC) to better understand their mechanism of interaction with DNA and therefore better explain their cellular effects. Distamycin A interactions with Dickerson and Poly[d(AT)₆] oligonucleotides show a different thermodynamic with respect to NAX002. The bulkier distamycin A analogue shows a non optimal binding to DNA due to its additional pyrrolamidine group. Cellular assays performed on cDDP-sensitive and -resistant cells showed that these compounds, distamycin A in particular, affect the expression of folate cycle enzymes even at cellular level. The optimal interaction of distamycin A with DNA may account for the down-regulation of both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) and the up-regulation of spermidine/spermine N1-acetyltransferase (SSAT) caused by this compound. These effects seem differently modulated by the cDDP-resistance phenotype. NAX002 which presents a lower affinity to DNA and slightly affected these enzymes, showed a synergic inhibition profile in combination with cDDP. In addition, their combination with cDDP or polyamine analogues increased cell sensitivity to the drugs suggesting that these interactions may have potential for development in the treatment of ovarian carcinoma.

Keywords: Distamycin A; Polyamines; DNA recognition; Ovarian cancer; Cisplatin-resistance; Thymidylate synthase

Polyamine depletion enhances the roscovitine-induced apoptosis through the activation of mitochondria in HCT116 colon carcinoma cells by Elif Damla Arısan; Ajda Çoker; Narçin Palavan-Ünsal (pp. 655-665).

Small molecule inhibitors of cyclin-dependent kinases (CDKs) show high therapeutic potential in various cancer types which are characterized by the accumulation of transformed cells due to impaired apoptotic machinery. Roscovitine, a CDK inhibitor showed to be a potent apoptotic inducer in several cancer cells. Polyamines, putrescine, spermidine and spermine, are biogenic amines involved in many cellular processes, including apoptosis. In this study, we explored the potential role of polyamines in roscovitine-induced apoptosis in HCT116 colon cancer cells. Roscovitine induced apoptosis by activating mitochondrial pathway caspases and modulating the expression of Bcl-2 family members. Depletion of polyamines by treatment with difluoromethylornithine (DFMO) increased roscovitine-induced apoptosis. Transient silencing of ornithine decarboxylase, polyamine biosynthesis enzyme and special target of DFMO also increased roscovitine-induced apoptosis in HCT116 cells. Interestingly, additional putrescine treatment was found pro-apoptotic due to the presence of non-functional ornithine decarboxylase (ODC). Finally, roscovitine altered polyamine catabolic pathway and led to decrease in putrescine and spermidine levels. Therefore, the metabolic regulation of polyamines may dictate the power of roscovitine induced apoptotic responses in HCT116 colon cancer cells.

Keywords: Polyamines; Apoptosis; Roscovitine; DFMO; Colon carcinoma

Role of polyamines in hypertrophy and terminal differentiation of osteoarthritic chondrocytes by A. Facchini; R. M. Borzì; E. Olivotto; D. Platano; S. Pagani; S. Cetrullo; F. Flamigni (pp. 667-678).

Polyamines are naturally occurring, positively charged polycations which are able to control several cellular processes in different cell types, by interacting with negatively charged compounds and structures within the living cell. Functional genomics in rodents targeting key biosynthetic or catabolic enzymes have revealed a series of phenotypic changes, many of them related to human diseases. Several pieces of evidence from the literature point at a role of polyamines in promoting chondrocyte differentiation, a process which is physiological in growth plate maturation or fracture healing, but has pathological consequences in articular chondrocytes, programmed to keep a maturational arrested state. Inappropriate differentiation of articular chondrocytes results in osteoarthritis. Thus, we have studied the effects of exogenously added spermine or spermidine in chondrocyte maturation recapitulated in 3D cultures, to tease out the effects on gene and protein expression of key chondrogenesis regulatory transcription factors, markers and effectors, as well as their posttranscriptional regulation. The results indicate that both polyamines are able to increase the rate and the extent of chondrogenesis, with enhanced collagen 2 deposition and remodeling with downstream generation of collagen 2 bioactive peptides. These were able to promote nuclear localization of RUNX-2, the pivotal transcription factor in chondrocyte hypertrophy and osteoblast generation. Indeed, samples stimulated with polyamines showed an enhanced mineralization, along with increased caspase activity, indicating increased chondrocyte terminal differentiation. In conclusion these results indicate that the polyamine pathway can represent a potential target to control and correct chondrocyte inappropriate maturation in osteoarthritis.

Keywords: Polyamines; Chondrocytes; Hypertrophy; Terminal differentiation; Osteoarthritis

Translational control of eIF5A in various diseases by Annette Kaiser (pp. 679-684).

Translational control is a crucial component in the development and progression of different diseases. Translational control may involve selective translation of specific mRNAs, which promote cell proliferation or lead to alterations in translation factor levels and activities. Eukaryotic initiation factor 5A (eIF5A) is the only known protein to contain the unusual amino acid hypusine [N ^ε- (4-amino-2-hydroxybutyl)-lysine], which is formed from the polyamine spermidine by two catalytic steps. eIF5A is involved in translation, elongation and stimulating peptide bond formation. Hypusination of eIF5A is essential for its activity in promoting cell proliferation. Meanwhile, there is evidence that eIF5A is a key protein in the pathogenicity of different diseases, such as diabetes, several human cancers, malaria and HIV-1 infections. Hitherto, the available data suggest that eIF5A has a role of a cell context-dependent function being more proliferative in the case of several human cancers and being involved under stress conditions in diabetes. Secondly, in HIV-1 infections and in diabetes, eIF5A also has a nuclear function by its sequence-specific binding of mRNAs as an mRNA-shuttle in conjunction with nuclear membrane export proteins. This binding may also influence the half-lives of mRNAs or their sequestration. Based on these data, there is a considerable therapeutic interest in eIF5A as a selective target for drug development through inhibition of hypusination.

Keywords: eIF5A; Cancer; Diabetes; Malaria; HIV; Hypusine; Translation

Effects of novel C-methylated spermidine analogs on cell growth via hypusination of eukaryotic translation initiation factor 5A by Mervi T. Hyvönen; Tuomo A. Keinänen; Maxim Khomutov; Alina Simonian; Jouko Vepsäläinen; Jong Hwan Park; Alex R. Khomutov; Leena Alhonen; Myung Hee Park (pp. 685-695).

The polyamines, putrescine, spermidine, and spermine, are ubiquitous multifunctional cations essential for cellular proliferation. One specific function of spermidine in cell growth is its role as a butylamine donor for hypusine synthesis in the eukaryotic initiation factor 5A (eIF5A). Here, we report the ability of novel mono-methylated spermidine analogs (α-MeSpd, β-MeSpd, γ-MeSpd, and ω-MeSpd) to function in the hypusination of eIF5A and in supporting the growth of DFMO-treated DU145 cells. We also tested them as substrates and inhibitors for deoxyhypusine synthase (DHS) in vitro. Of these compounds, α-MeSpd, β-MeSpd, and γ-MeSpd (but not ω-MeSpd) were substrates for DHS in vitro, while they all inhibited the enzyme reaction. As racemic mixtures, only α-MeSpd and β-MeSpd supported long-term growth (9–18 days) of spermidine-depleted DU145 cells, whereas γ-MeSpd and ω-MeSpd did not. The S-enantiomer of α-MeSpd, which supported long-term growth, was a good substrate for DHS in vitro, whereas the R-isomer was not. The long-term growth of DFMO-treated cells correlated with the hypusine modification of eIF5A by intracellular methylated spermidine analogs. These results underscore the critical requirement for hypusine modification in mammalian cell proliferation and provide new insights into the specificity of the deoxyhypusine synthase reaction.

Keywords: Polyamine; Methylated spermidine; Cell growth; Deoxyhypusine synthase; Hypusine; eIF5A

eIF5A interacts functionally with eEF2 by Camila A. O. Dias; Ana Paula Borges Gregio; Danuza Rossi; Fábio Carrilho Galvão; Tatiana F. Watanabe; Myung Hee Park; Sandro R. Valentini; Cleslei F. Zanelli (pp. 697-702).

eIF5A is highly conserved from archaea to mammals, essential for cell viability and the only protein known to contain the essential amino acid residue hypusine, generated by a unique posttranslational modification. eIF5A was originally identified as a translation initiation factor due to its ability to stimulate the formation of the first peptide bond. However, recent studies have shown that depletion of eIF5A causes a significant decrease in polysome run-off and an increase in the ribosome transit time, suggesting that eIF5A is actually involved in the elongation step of protein synthesis. We have previously shown that the depletion mutant tif51A-3 (eIF5A^C39Y/G118D) shows a sicker phenotype when combined with the dominant negative mutant eft2 H699K of the elongation factor eEF2. In this study, we used the eIF5A^K56A mutant to further investigate the relationship between eIF5A and eEF2. The eIF5A^K56A mutant is temperature sensitive and has a defect in protein synthesis, but instead of causing depletion of the eIF5A protein, this mutant has a defect in hypusine modification. Like the mutant tif51A-3, the eIF5A^K56A mutant is synthetic sick with the mutant eft2 H699K of eEF2. High-copy eEF2 not only improves cell growth of the eIF5A^K56A mutant, but also corrects its increased cell size defect. Moreover, eEF2 suppression of the eIF5A^K56A mutant is correlated with the improvement of total protein synthesis and with the increased resistance to the protein synthesis inhibitor hygromycin B. Finally, the polysome profile defect of the eIF5A^K56A mutant is largely corrected by high-copy eEF2. Therefore, these results demonstrate that eIF5A is closely related to eEF2 function during translation elongation.

Keywords: eIF5A; Hypusine; eEF2; Translation elongation

Essential role of eIF5A-1 and deoxyhypusine synthase in mouse embryonic development by Kazuhiro Nishimura; Seung Bum Lee; Jong Hwan Park; Myung Hee Park (pp. 703-710).

The eukaryotic initiation factor 5A (eIF5A) contains a polyamine-derived amino acid, hypusine [N^ε-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed post-translationally by the addition of the 4-aminobutyl moiety from the polyamine spermidine to a specific lysine residue, catalyzed by deoxyhypusine synthase (DHPS), and subsequent hydroxylation by deoxyhypusine hydroxylase (DOHH). The eIF5A precursor protein and both of its modifying enzymes are highly conserved, suggesting a vital cellular function for eIF5A and its hypusine modification. To address the functions of eIF5A and the first modification enzyme, DHPS, in mammalian development, we knocked out the Eif5a or the Dhps gene in mice. Eif5a heterozygous knockout mice and Dhps heterozygous knockout mice were viable and fertile. However, homozygous Eif5a1 gt/gt embryos and Dhps gt/gt embryos died early in embryonic development, between E3.5 and E7.5. Upon transfer to in vitro culture, homozygous Eif5a gt/gt or Dhps gt/gt blastocysts at E3.5 showed growth defects when compared to heterozygous or wild type blastocysts. Thus, the knockout of either the eIF5A-1 gene (Eif5a) or of the deoxyhypusine synthase gene (Dhps) caused early embryonic lethality in mice, indicating the essential nature of both eIF5A-1 and deoxyhypusine synthase in mammalian development.

Keywords: eIF5A; Hypusine; Polyamine; Spermidine; Deoxyhypusine synthase; Gene knockout

Recent advances in the molecular biology of metazoan polyamine transport by R. Poulin; R. A. Casero; D. Soulet (pp. 711-723).

Very limited molecular knowledge exists about the identity and protein components of the ubiquitous polyamine transporters found in animal cells. However, a number of reports have been published over the last 5 years on potential candidates for metazoan polyamine permeases. We review the available evidence on these putative polyamine permeases, as well as establish a useful «identikit picture» of the general polyamine transport system, based on its properties as found in a wide spectrum of mammalian cells. Any molecular candidate encoding a putative «general» polyamine permease should fit that provided portrait. The current models proposed for the mechanism of polyamine internalization in mammalian cells are also briefly reviewed.

Keywords: Polyamines; Membrane transport; Permeases; DNA cloning; Vesicular transport

Do mammalian amine oxidases and the mitochondrial polyamine transporter have similar protein structures? by Enzo Agostinelli; Antonio Toninello; Fabio Vianello; Roberto Stevanato (pp. 725-731).

Polyamine transport across the mitochondria membrane occurs by a specific, common uniporter system and appears controlled by electrostatic interactions as for polyamine oxidative deamination by bovine serum and mitochondrial matrix amine oxidases was found. In fact in all the cases, while the catalytic constants or the maximum uptake rate values show little changes with the number of the positive charges of the substrates, Michaelis–Menten constant values demonstrate exponential dependence, confirming that electrostatic forces control the docking of the substrate into the enzyme active site or polyamine channel. By the treatment of the kinetic data in terms of Gibbs equation or Eyring theory, the contribution of each positive charge of the polyamine to the Gibbs energy values for the oxidative deamination of polyamines by two mammalian amine oxidase and for polyamine transport, are obtained. These values were comparable and in good accordance with those reported in literature. Previous studies demonstrated that two negative functional groups in the active site of bovine serum and mitochondrial matrix amine oxidases interact electrostatically with three positive charges of the polyamines in the formation of the enzyme–substrate complex. Remembering the structure–function relationship of proteins, our results suggest analogous interactions in the polyamine transporter and, as a consequence, a partial structural similitude between two proteins. It follows that the primary sequences of the amino oxidases and the mitochondrial transport may, in part, be conserved.

Keywords: Amine oxidases; Mitochondrial polyamines uniporter; Polyamines

Structure and function of polyamine-amino acid antiporters CadB and PotE in Escherichia coli by Hideyuki Tomitori; Keiko Kashiwagi; Kazuei Igarashi (pp. 733-740).

The structure and function of a cadaverine–lysine antiporter CadB and a putrescine–ornithine antiporter PotE in Escherichia coli were evaluated using model structures based on the crystal structure of AdiC, an agmatine–arginine antiporter, and the activities of various CadB and PotE mutants. The central cavity of CadB, containing the substrate binding site, was wider than that of PotE, mirroring the different sizes of cadaverine and putrescine. The size of the central cavity of CadB and PotE was dependent on the angle of transmembrane helix 6 (TM6) against the periplasm. Tyr⁷³, Tyr⁸⁹, Tyr⁹⁰, Glu²⁰⁴, Tyr²³⁵, Asp³⁰³, and Tyr⁴²³ of CadB, and Cys⁶², Trp²⁰¹, Glu²⁰⁷, Trp²⁹², and Tyr⁴²⁵ of PotE were strongly involved in the antiport activities. In addition, Trp⁴³, Tyr⁵⁷, Tyr¹⁰⁷, Tyr³⁶⁶, and Tyr³⁶⁸ of CadB were involved preferentially in cadaverine uptake at neutral pH, while only Tyr⁹⁰ of PotE was involved preferentially in putrescine uptake. The results indicate that the central cavity of CadB consists of TMs 2, 3, 6, 7, 8, and 10, and that of PotE consists of TMs 2, 3, 6, and 8. These results also suggest that several amino acid residues are necessary for recognition of cadaverine in the periplasm because the level of cadaverine is much lower than that of putrescine in the periplasm at neutral pH. All the amino acid residues identified as being strongly involved in both the antiport and uptake activities were located on the surface of the transport path consisting of the central cavity and TM12.

Keywords: Cadaverine; Putrescine; CadB; PotE; Antiporter

Effect of peroxides on spermine transport in rat brain and liver mitochondria by Valentina Battaglia; Elena Tibaldi; Silvia Grancara; Francesca Zonta; Anna Maria Brunati; Pamela Martinis; Marcantonio Bragadin; Maria Angelica Grillo; Giampiero Tempera; Enzo Agostinelli; Antonio Toninello (pp. 741-749).

The polyamine spermine is transported into the matrix of various types of mitochondria by a specific uniporter system identified as a protein channel. This mechanism is regulated by the membrane potential; other regulatory effectors are unknown. This study analyzes the transport of spermine in the presence of peroxides in both isolated rat liver and brain mitochondria, in order to evaluate the involvement of the redox state in this mechanism, and to compare its effect in both types of mitochondria. In liver mitochondria peroxides are able to inhibit spermine transport. This effect is indicative of redox regulation by the transporter, probably due to the presence of critical thiol groups along the transport pathway, or in close association with it, with different accessibility for the peroxides and performing different functions. In brain mitochondria, peroxides have several effects, supporting the hypothesis of a different regulation of spermine transport. The fact that peroxovanadate can inhibit tyrosine phosphatases in brain mitochondria suggests that mitochondrial spermine transport is regulated by tyrosine phosphorylation in this organ. In this regard, the evaluation of spermine transport in the presence of Src inhibitors suggests the involvement of Src family kinases in this process. It is possible that phosphorylation sites for Src kinases are present in the channel pathway and have an inhibitory effect on spermine transport under regulation by Src kinases. The results of this study suggest that the activity of the spermine transporter probably depends on the redox and/or tyrosine phosphorylation state of mitochondria, and that its regulation may be different in distinct organs.

Keywords: Spermine transport; Mitochondria; Peroxides; Tyrosine phosphorylation; Src kinases family

Mitochondrial oxidative stress induced by Ca²⁺ and monoamines: different behaviour of liver and brain mitochondria in undergoing permeability transition by Silvia Grancara; Valentina Battaglia; Pamela Martinis; Nikenza Viceconte; Enzo Agostinelli; Antonio Toninello; Renzo Deana (pp. 751-759).

Mitochondrial permeability transition (MPT) is correlated with the opening of a nonspecific pore, the so-called transition pore, that triggers bidirectional traffic of inorganic solutes and metabolites across the mitochondrial membrane. This phenomenon is caused by supraphysiological Ca²⁺ concentrations and by other compounds leading to oxidative stress, while cyclosporin A, ADP, bongkrekic acid, antioxidant agents and naturally occurring polyamines strongly inhibit it. The effects of polyamines, including the diamine agmatine, have been widely studied in several types of mitochondria. The effects of monoamines on MPT have to date, been less well-studied, even if they are involved in a variety of neurological and neuroendocrine processes. This study shows that in rat liver mitochondria (RLM), monoamines such as tyramine, serotonin and dopamine amplify the swelling induced by calcium, and increase the oxidation of thiol groups and the production of hydrogen peroxide, effects that are counteracted by the above-mentioned inhibitors. In rat brain mitochondria (RBM), the monoamines do not amplify calcium-induced swelling, even if they demonstrate increases in the extent of oxidation of thiol groups and hydrogen peroxide production. In these mitochondria, the antioxidants are not at all or scarcely effective in suppressing mitochondrial swelling. In conclusion, we hypothesize that different mechanisms induce the MPT in the two different types of mitochondria evaluated. Calcium and monoamines induce oxidative stress in RLM, which in turn appears to induce and amplify MPT. This process is not apparent in RBM, where MPT seems resistant to oxidative stress.

Keywords: Mitochondria; Ca²⁺ ; Monoamines; Oxidative stress; Mitochondrial permeability transition pore

Further characterization of agmatine binding to mitochondrial membranes: involvement of imidazoline I₂ receptor by Pamela Martinis; Valentina Battaglia; Silvia Grancara; Lisa Dalla Via; Vito Di Noto; Stefania Saccoccio; Enzo Agostinelli; Marcantonio Bragadin; Maria Angelica Grillo; Antonio Toninello (pp. 761-768).

Agmatine, a divalent diamine with two positive charges at physiological pH, is transported into the matrix of liver mitochondria by an energy-dependent mechanism, the driving force of which is the electrical membrane potential. Its binding to mitochondrial membranes is studied by applying a thermodynamic treatment of ligand–receptor interactions on the analyses of Scatchard and Hill. The presence of two mono-coordinated binding sites S₁ and S₂, with a negative influence of S₂ on S₁, has been demonstrated. The calculated binding energy is characteristic for weak interactions. S₁ exhibits a lower binding capacity and higher binding affinity both of about two orders of magnitude than S₂. Experiments with idazoxan, a ligand of the mitochondrial imidazoline receptor I₂, demonstrate that S₁ site is localized on this receptor while S₂ is localized on the transport system. S₁ would act as a sensor of exogenous agmatine concentration, thus modulating the transport of the amine by its binding to S₂.

Keywords: Agmatine; Binding; Mitochondria; Imidazoline

Chronic exposure to agmatine results in the selection of agmatine-resistant hepatoma cells by Bandino Andrea; Battaglia Valentina; Bravoco Vittoria; Busletta Chiara; Compagnone Alessandra; Cravanzola Carlo; Meli Floriana; Agostinelli Enzo; Parola Maurizio; Colombatto Sebastiano (pp. 769-774).

During our study of the cytostatic effect of agmatine, we were able to isolate an agmatine resistant clone from a parental hepatoma cell line, HTC. These cells, called Agres, had slower growth rate than the parental cells when cultured in normal medium. The modification in polyamine content induced by agmatine was much lower in these cells and ornithine decarboxylase, S-adenosylmethionine decarboxylase and spermidine/spermine acetyltransferase activities were much less affected. By investigating the mechanism responsible for these modifications, it was shown that agmatine and polyamines were not taken up by Agres cells. Their resistance to the antiproliferative effects of agmatine may thus arise from a lack of the polyamine transport system. Moreover, Agres cells were able to take up both glutamic acid and arginine at a rate significantly higher than that detected for HTC cells, most likely to provide components for compensatory increase of PA synthesis. These results emphasize the importance of polyamine transport for cell growth.

Keywords: Agmatine; Polyamines; Uptake; Glutamic acid; Arginine; HTC cells

Protective effects of agmatine in rotenone-induced damage of human SH-SY5Y neuroblastoma cells: Fourier transform infrared spectroscopy analysis in a model of Parkinson’s disease by Salvatore Condello; Emanuele Calabrò; Daniela Caccamo; Monica Currò; Nadia Ferlazzo; Joseph Satriano; Salvatore Magazù; Riccardo Ientile (pp. 775-781).

Agmatine is a novel neuromodulator that plays a protective role in the CNS in several models of cellular damage. However, the mechanisms involved in these protective effects in neurodegenerative diseases are poorly understood. Fourier transform infrared (FTIR) spectroscopy analysis detects biomolecular changes in disordered cells and tissues. In this report, we utilize FTIR spectroscopy to characterize the changes in rotenone-induced damage in neuronal-like differentiated SH-SY5Y neuroblastoma cells in the presence or absence of agmatine. The analysis of the FTIR spectra demonstrates significant alterations in rotenone-treated cells, whereas the FTIR spectra obtained after pre-incubation with agmatine (250 nM) significantly reduces these redox alterations and more closely resembles those of the control cells. In particular, rotenone-damaged cells demonstrate spectral alterations related to amide I, which correspond to an increase in β-sheet components, and decreases in the amide II absorption intensity, suggesting a loss of N–H bending and C–N stretching. These alterations were also evident by Fourier self-deconvolution analysis. Thus, rotenone-induced increases in the levels of stretching vibration band related to the protein carboxyl group would account for a significant amount of misfolded proteins in the cell. Agmatine effectively reduces these effects of rotenone on protein structure. In conclusion, antioxidant and scavenging properties of agmatine reduce rotenone-produced cellular damage at the level of protein structure. These, together with other previous observations, demonstrate the therapeutic potential of agmatine in the treatment of Parkinson’s disease.

Keywords: Agmatine; Fourier transform infrared spectroscopy; SH-SY5Y neuroblastoma cells; Reactive oxygen species; Membrane mitochondrial potential; Neuroprotection

Polyamines modulate epithelial-to-mesenchymal transition by Compagnone Alessandra; Bandino Andrea; Meli Floriana; Bravoco Vittoria; Cravanzola Carlo; Parola Maurizio; C. Sebastiano (pp. 783-789).

Epithelial-to-mesenchymal transition and mesenchymal-to-epithelial transition are biologic processes responsible for conversion of epithelial cells into a mesenchymal phenotype or viceversa, respectively. They occur during embryo- and foetal-development and, in adult organisms, are involved in wound healing, in the genesis and progression of organ fibrosis as well as in the invasiveness of epithelial cancer cells. The key event of epithelial-to-mesenchymal transition is the loss of E-cadherin expression due to repressor activity of the transcriptional factor Snai1. Intracellular Snai1 levels are controlled through translational and post-translational events such as phosphorylation and de-phosphorylation, potentially modulated by polyamine content. Epithelial MDCK cells exposed to TGF-β₁ acquired a fibroblastoid phenotype and expressed mesenchymal markers. These changes were emphasized in cells that were also exposed to DFMO in order to decrease the intracellular levels of polyamines. Addition of exogenous polyamines almost completely abolished the combined action of DFMO and TGF-β₁ and rapidly reverted to epithelial phenotype MDCK cells previously undergone to mesenchymal phenotype. Nuclear extracts of cells treated with DFMO + TGF-β₁ revealed the presence of Snai1 immunopositive bands in a range of molecular weight between 55 and 72 kDa, with additional positive bands detected at MW greater than 170 kDa. Same bands resulted positive to anti-Sumo 2/3 antibody, suggesting that an intracellular low level of polyamines favours Snai1 nuclear accumulation under the form of polysumoylated proteins.

Keywords: MDCK; Polyamines; EMT; MET; Snai1; Sumo

Polyamines modulate epithelial-to-mesenchymal transition by Compagnone Alessandra; Bandino Andrea; Meli Floriana; Bravoco Vittoria; Cravanzola Carlo; Parola Maurizio; C. Sebastiano (pp. 783-789).

Keywords: MDCK; Polyamines; EMT; MET; Snai1; Sumo

Modification of secondary head-forming activity of microinjected ∆β-catenin mRNA by co-injected spermine and spermidine in Xenopus early embryos by Takamichi Mishina; Kota Fuchimukai; Kazuei Igarashi; Kosuke Tashiro; Koichiro Shiokawa (pp. 791-801).

Polyamines are essential for cell growth and differentiation. In Xenopus early embryos, per embryo level of spermine is extremely low as compared with that of spermidine. To disclose the possible function of polyamines in Xenopus early embryos, we tested the effect of co-injection of spermine and spermidine on the functioning of exogenously microinjected in vitro-synthesized, ∆β-catenin mRNA which is known to induce a secondary head after being microinjected into a ventral vegetal blastomere in 8-celled Xenopus embryos. Microinjection of ∆β-catenin mRNA in fact induced a secondary axis with a secondary head, and co-injection of spermine or spermidine suppresses induction of the secondary head and secondary axis without drastic effects like induction of immediate cell death or execution of apoptosis at blastula stage. The inhibitory effects were dosage dependent, and at lower doses the inhibition was mainly on secondary head formation rather than on secondary axis formation. We performed similar experiments using GFP mRNA and confirmed that expression of GFP mRNA was also suppressed by co-injection of spermine. We mixed ∆β-catenin mRNA with different amounts of spermine and performed electrophoresis on agarose gels, with a finding that the prior mixing greatly suppressed mRNA migration. These results suggest that an excess amount of spermine as well as spermidine exerts inhibitory effects on mRNA translation, and that the inhibition may be due to direct binding of polyamines to mRNA and a reduction of negative charges on mRNA molecules that might also induce the formation of cross link-like networks among mRNAs.

Keywords: ∆β-Catenin mRNA; Secondary head formation; Secondary axis formation; Xenopus 8-celled embryos; Microinjection; Polyamines; Spermine; Spermidine; GFP mRNA; Gel electrophoretic mobility

A gold-containing drug against parasitic polyamine metabolism: the X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition by Andrea Ilari; Paola Baiocco; Luigi Messori; Annarita Fiorillo; Alberto Boffi; Marina Gramiccia; Trentina Di Muccio; Gianni Colotti (pp. 803-811).

Auranofin is a gold(I)-containing drug in clinical use as an antiarthritic agent. Recent studies showed that auranofin manifests interesting antiparasitic actions very likely arising from inhibition of parasitic enzymes involved in the control of the redox metabolism. Trypanothione reductase is a key enzyme of Leishmania infantum polyamine-dependent redox metabolism, and a validated target for antileishmanial drugs. As trypanothione reductase contains a dithiol motif at its active site and gold(I) compounds are known to be highly thiophilic, we explored whether auranofin might behave as an effective enzyme inhibitor and as a potential antileishmanial agent. Notably, enzymatic assays revealed that auranofin causes indeed a pronounced enzyme inhibition. To gain a deeper insight into the molecular basis of enzyme inhibition, crystals of the auranofin-bound enzyme, in the presence of NADPH, were prepared, and the X-ray crystal structure of the auranofin–trypanothione reductase–NADPH complex was solved at 3.5 Å resolution. In spite of the rather low resolution, these data were of sufficient quality as to identify the presence of the gold center and of the thiosugar of auranofin, and to locate them within the overall protein structure. Gold binds to the two active site cysteine residues of TR, i.e. Cys52 and Cys57, while the thiosugar moiety of auranofin binds to the trypanothione binding site; thus auranofin appears to inhibit TR through a dual mechanism. Auranofin kills the promastigote stage of L. infantum at micromolar concentration; these findings will contribute to the design of new drugs against leishmaniasis.

Keywords: Gold; Auranofin; Leishmania ; Trypanothione reductase

Profiling the aminopropyltransferases in plants: their structure, expression and manipulation by Lin Shao; Rajtilak Majumdar; Subhash C. Minocha (pp. 813-830).

Polyamines are organic polycations that are involved in a wide range of cellular activities related to growth, development, and stress response in plants. Higher polyamines spermidine and spermine are synthesized in plants and animals by a class of enzymes called aminopropyltransferases that transfer aminopropyl moieties (derived from decarboxylated S-adenosylmethionine) to putrescine and spermidine to produce spermidine and spermine, respectively. The higher polyamines show a much tighter homeostatic regulation of their metabolism than the diamine putrescine in most plants; therefore, the aminopropyltransferases are of high significance. We present here a comprehensive summary of the current literature on plant aminopropyltransferases including their distribution, biochemical properties, genomic organization, pattern of expression during development, and their responses to abiotic stresses, and manipulation of their cellular activity through chemical inhibitors, mutations, and genetic engineering. This minireview complements several recent reviews on the overall biosynthetic pathway of polyamines and their physiological roles in plants and animals. It is concluded that (1) plants often have two copies of the common aminopropyltransferase genes which exhibit redundancy of function, (2) their genomic organization is highly conserved, (3) direct enzyme activity data on biochemical properties of these enzymes are scant, (4) often there is a poor correlation among transcripts, enzyme activity and cellular contents of the respective polyamine, and (5) transgenic work mostly confirms the tight regulation of cellular contents of spermidine and spermine. An understanding of expression and regulation of aminopropyltransferases at the metabolic level will help us in effective use of genetic engineering approaches for the improvement in nutritional value and stress responses of plants.

Keywords: Polyamines; Spermidine synthase; Spermine synthase; Transgenic manipulation; Genetic manipulation; Putrescine

The members of Arabidopsis thaliana PAO gene family exhibit distinct tissue- and organ-specific expression pattern during seedling growth and flower development by Paola Fincato; Panagiotis N. Moschou; Abdellah Ahou; Riccardo Angelini; Kalliopi A. Roubelakis-Angelakis; Rodolfo Federico; Paraskevi Tavladoraki (pp. 831-841).

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine catabolism. In Arabidopsis thaliana, five PAOs (AtPAO1-5) are present with cytosolic or peroxisomal localization. Here, we present a detailed study of the expression pattern of AtPAO1, AtPAO2, AtPAO3 and AtPAO5 during seedling and flower growth and development through analysis of promoter activity in AtPAO::β-glucuronidase (GUS) transgenic Arabidopsis plants. The results reveal distinct expression patterns for each studied member of the AtPAO gene family. AtPAO1 is mostly expressed in the transition region between the meristematic and the elongation zone of roots and anther tapetum, AtPAO2 in the quiescent center, columella initials and pollen, AtPAO3 in columella, guard cells and pollen, and AtPAO5 in the vascular system of roots and hypocotyls. Furthermore, treatment with the plant hormone abscisic acid induced expression of AtPAO1 in root tip and AtPAO2 in guard cells. These data suggest distinct physiological role(s) for each member of the AtPAO gene family.

Keywords: Polyamines; Polyamine oxidase; Arabidopsis thaliana ; GUS; Guard cells; Pollen; Tapetum; Roots

Methyl jasmonate deficiency alters cellular metabolome, including the aminome of tomato (Solanum lycopersicum L.) fruit by Kurt D. Kausch; Anatoly P. Sobolev; Ravinder K. Goyal; Tahira Fatima; Rekha Laila-Beevi; Robert A. Saftner; Avtar K. Handa; Autar K. Mattoo (pp. 843-856).

Exogenous treatment with jasmonates (JA) has been shown to reduce the levels of polyamines in many plants. But the role of endogenous JA on polyamine biosynthesis or other cellular metabolites has thus far remained uninvestigated. We developed transgenic tomato (Solanum lycopersicum L.) having severely reduced methyl JA levels by silencing a fruit ripening-associated lipoxygenase (LOX), SlLoxB, using a truncated LOX gene under the control of the constitutive CaMV35S promoter. The LOX suppressed and MeJA-deficient fruits had lowered polyamine levels. Thus, these transgenic fruits were used as a plant model to evaluate the effects of reduced endogenous MeJA on cellular metabolites in ripening tomato fruits using NMR spectroscopy. During on-shelf ripening, transgenic fruits were significantly reduced in the content of 19 out of 30 metabolites examined, including Ile, Val, Ala, Thr, Asn Tyr, Glu, Gln, His, Phe, Trp, GABA, citrate, succinate, myo-inositol, unidentified compound B, nucleic acid compound Nucl1, choline, and trigonelline as compared to the wild-type azygous counterparts. A significant increase in β-glucose levels in transgenic fruits was observed at the pink stage. The transgenic fruits were equivalent to the wild type in lycopene level and chlorophyll degradation rates. Taken together, these results show that intracellular MeJA significantly regulates overall primary metabolism, especially aminome (amino acids and polyamines) of ripening fruits.

Keywords: Aminome; Fruit metabolism; Lipoxygenase; Oxylipins; Tomato; Transgenics

Polyamines modulate nitrate reductase activity in wheat leaves: involvement of nitric oxide by Eliana Paola Rosales; María Florencia Iannone; María Daniela Groppa; María Patricia Benavides (pp. 857-865).

In the present work, the effect of polyamines (PAs) on nitrate reductase (NR) activity was studied in wheat leaves exposed to exogenously added PAs while assessing the nitric oxide (NO) involvement in the regulation of the enzyme activity. A biphasic response was observed along the time of treatment using 0.1 mM of putrescine (Put), spermidine (Spd) or spermine (Spm). At 3 h, Spd and Spm significantly reduced NR activity by 29 or 35%, respectively, whereas at 6 h, the activity of the enzyme decreased by an average of 25%. At 21 h, Put increased NR activity by 63%, while Spd and Spm elevated the enzyme activity by 114%. NR activity, that was reduced by 0.1 mM Spm at 3 and 6 h, returned almost to control values when c-PTIO (an NO scavenger) was used, confirming that NO was involved in the inhibition of NR activity. Nitric oxide was also mediating the PA-increase of the enzyme activity at longer incubation times, evidenced when the raise in NR activity produced by 0.1 mM Spm at the longest incubation time returned to the value of the control in the presence of cPTIO. Neither the protein expression nor the nitrate content were modified by PAs treatments. The involvement of PAs and NO in the regulation of NR activity is discussed.

Keywords: Nitrate reductase; Nitric oxide; Polyamines; Wheat

Constitutively and highly expressed Oryza sativa polyamine oxidases localize in peroxisomes and catalyze polyamine back conversion by Yusuke Ono; Dong Wook Kim; Kanako Watanabe; Ayano Sasaki; Masaru Niitsu; Thomas Berberich; Tomonobu Kusano; Yoshihiro Takahashi (pp. 867-876).

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine (PA) catabolism. Recent studies have revealed that plant PAOs are not only active in the terminal catabolism of PAs as demonstrated for maize apoplastic PAO but also in a polyamine back-conversion pathway as shown for most Arabidopsis PAOs. We have characterized Oryza sativa PAOs at molecular and biochemical levels. The rice genome contains 7 PAO isoforms that are termed OsPAO1 to OsPAO7. Of the seven PAOs, OsPAO3, OsPAO4, and OsPAO5 transcripts were most abundant in 2-week-old seedlings and mature plants, while OsPAO1, OsPAO2, OsPAO6, and OsPAO7 were expressed at very low levels with different tissue specificities. The more abundantly expressed PAOs—OsPAO3, OsPAO4, and OsPAO5—were cloned, and their gene products were produced in Escherichia coli. The enzymatic activities of the purified OsPAO3 to OsPAO5 proteins were examined. OsPAO3 favored spermidine (Spd) as substrate followed by thermospermine (T-Spm) and spermine (Spm) and showed a full PA back-conversion activity. OsPAO4 substrate specificity was similar to that of OsPAO5 preferring Spm and T-Spm but not Spd. Those enzymes also converted Spm and T-Spm to Spd, again indicative of PA back-conversion activities. Lastly, we show that OsPAO3, OsPAO4, and OsPAO5 are localized in peroxisomes. Together, these data revealed that constitutively and highly expressed O. sativa PAOs are localized in peroxisomes and catalyze PA back-conversion processes.

Keywords: Oryza sativa ; Peroxisome; Polyamine; Polyamine oxidase; Substrate specificity; Tissue specificity

Determination of biogenic amines in infusions of tea (Camellia sinensis) by HPLC after derivatization with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) by H. Brückner; S. Flassig; J. Kirschbaum (pp. 877-885).

The reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was used for the pre-column derivatization of the biogenic amines (BAs) cadaverine (Cad), histamine (Him), octopamine (Ocp), phenylethylamine (Pea), putrescine (Put), spermidine (Spd), spermine (Spm), tyramine (Tym) and the internal standard 1,6-diaminohexane (Dhx). The resulting Fmoc-derivatives were resolved by high-performance liquid chromatography on a Superspher^© C₁₈ column using a binary gradient generated from sodium acetate and acetonitrile. For quantification, the fluorescence of derivatives was used at 263 nm excitation and 313 nm emission wavelength. This approach was applied to free BAs extractable with boiling water from 14 black, 5 green, 1 Oolong, and 1 instant tea. Infusions were prepared by adding 35 ml boiling water to one gram of tea and extracted for 20 min. In the Oolong tea and two black teas, no BAs could be detected. Limits of detection were 0.07–1.0 pmol for BAs at signal-to-noise ratio 3:1. Besides most abundant Tym and Spm lower quantities of Pea, Put, and Spd were detected, albeit not in all teas. Quantities of Tym ranged from 16 to 431 μg Tym/L infusion (1.1–25.3 μgTym/g tea) and 31 to 319 μg Spm/L infusion (1.5–16.9 μg Spm/g tea). In none of the teas, Him was detected. Owing to the low amounts of free BAs in tea infusions, no health risks are to be expected even on consumption of large quantities of tea as beverage.

Keywords: Tea infusions; High-performance liquid chromatography; Polyamines; 9-fluorenylmethyl chloroformate; Risk assessment

Polyamine analogs modulate gene expression by inhibiting lysine-specific demethylase 1 (LSD1) and altering chromatin structure in human breast cancer cells by Qingsong Zhu; Yi Huang; Laurence J. Marton; Patrick M. Woster; Nancy E. Davidson; Robert A. Casero Jr. (pp. 887-898).

Aberrant epigenetic repression of gene expression has been implicated in most cancers, including breast cancer. The nuclear amine oxidase, lysine-specific demethylase 1 (LSD1) has the ability to broadly repress gene expression by removing the activating mono- and di-methylation marks at the lysine 4 residue of histone 3 (H3K4me1 and me2). Additionally, LSD1 is highly expressed in estrogen receptor α negative (ER−) breast cancer cells. Since epigenetic marks are reversible, they make attractive therapeutic targets. Here we examine the effects of polyamine analog inhibitors of LSD1 on gene expression, with the goal of targeting LSD1 as a therapeutic modality in the treatment of breast cancer. Exposure of the ER-negative human breast cancer cells, MDA-MB-231 to the LSD1 inhibitors, 2d or PG11144, significantly increases global H3K4me1 and H3K4me2, and alters gene expression. Array analysis indicated that 98 (75 up and 23 down) and 477 (237 up and 240 down) genes changed expression by at least 1.5-fold or greater after treatment with 2d and PG11144, respectively. The expression of 12 up-regulated genes by 2d and 14 up-regulated genes by PG11144 was validated by quantitative RT-PCR. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated that up-regulated gene expression by polyamine analogs is associated with increase of the active histone marks H3K4me1, H3K4me2 and H3K9act, and decrease of the repressive histone marks H3K9me2 and H3K27me3, in the promoter regions of the relevant target genes. These data indicate that the pharmacologic inhibition of LSD1 can effectively alter gene expression and that this therapeutic strategy has potential.

Keywords: Epigenetics; Chromatin; Histone methylation; Acetylation; Gene silencing

A potential estrogen mimetic effect of a bis(ethyl)polyamine analogue on estrogen receptor positive MCF-7 breast cancer cells by Irina Nayvelt; Shali John; Hui-Chen Hsu; PingAr Yang; Wensheng Liu; Gokul Das; Mervi T. Hyvönen; Leena Alhonen; Tuomo A. Keinänen; Akira Shirahata; Rajesh Patel; Thresia Thomas; T. J. Thomas (pp. 899-911).

BE-3-3-3-3 (1,15-(ethylamino)4,8,12-triazapentadecane) is a bis(ethyl)polyamine analogue under investigation as a therapeutic agent for breast cancer. Since estradiol (E₂) is a critical regulatory molecule in the growth of breast cancer, we examined the effect of BE-3-3-3-3 on estrogen receptor α (ERα) positive MCF-7 cells in the presence and absence of E₂. In the presence of E₂, a concentration-dependent decrease in DNA synthesis was observed using [³H]-thymidine incorporation assay. In the absence of E₂, low concentrations (2.5–10 μM) of BE-3-3-3-3 increased [³H]-thymidine incorporation at 24 and 48 h. BE-3-3-3-3 induced the expression of early response genes, c-myc and c-fos, in the absence of E₂, but not in its presence, as determined by real-time quantitative polymerase chain reaction (qPCR). BE-3-3-3-3 had no significant effect on these genes in an ERα-negative cell line, MDA-MB-231. Chromatin immunoprecipitation assay demonstrated enhanced promoter occupation by either E₂ or BE-3-3-3-3 of an estrogen-responsive gene pS2/Tff1 by ERα and its co-activator, steroid receptor co-activator 3 (SRC-3). Confocal microscopy of BE-3-3-3-3-treated cells revealed membrane localization of ERα, similar to that induced by E₂. The failure of BE-3-3-3-3 to inhibit cell proliferation was associated with autophagic vacuole formation, and the induction of Beclin 1 and MAP LC3 II. These results indicate a differential effect of BE-3-3-3-3 on MCF-7 cells in the absence and presence of E₂, and suggest that pre-clinical and clinical development of polyamine analogues might require special precautions and selection of sensitive subpopulation of patients.

Keywords: Polyamines; Polyamine analogue; Breast cancer; Estrogen receptor; Autophagy

Synthetic polyamines as potential amine oxidase inhibitors: a preliminary study by Bonaiuto Emanuela; Anna Minarini; Vincenzo Tumiatti; Andrea Milelli; Michele Lunelli; Maurizio Pegoraro; Valeria Rizzoli; Maria Luisa Di Paolo (pp. 913-928).

In the last few decades, medicinal chemists have carried out extensive research on synthetic polyamines for use as anticancer drugs and multitarget-directed ligands in neurodegenerative diseases. The aim of this study was to evaluate the effect of some synthetic polyamines as inhibitors of two new potential targets, human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1 (SSAO/VAP-1) and monoamine oxidases B (MAO B), enzymes involved in various multi-factorial diseases such as Alzheimer’s disease. N,N′-Dibenzyl-dodecane-1,12-diamine (Bis-Bza-Diado), a newly synthesised compound, and ELP 12, a muscarinic cholinergic M₂ receptor antagonist, were found to behave as reversible and mixed non-competitive inhibitors of both amine oxidases (dissociation constants of about 100 μM). ELP 12 was found to be more selective for SSAO/VAP-1. Combining kinetic and structural approaches, the binding mode of ELP 12 to SSAO/VAP-1 was investigated. ELP 12 may bind at the entrance of the active site channel by ionic interactions with ASP446 and/or ASP180; one end of the polyamine may be accommodated inside the channel, reaching the TPQ cofactor area. The binding of ELP 12 induces rearrangement of the secondary structure of the enzyme and impedes substrate entry and/or product release and catalysis. These structural data reveal that the entrance and the first part of the SSAO/VAP-1 channel may be considered as a new target area, or a “secondary binding site”, for modulators of human SSAO/VAP-1 activity. These results indicate ELP 12 and Bis-Bza-Diado as new “skeletons” for the development of novel SSAO/VAP-1 and MAO B inhibitors.

Keywords: Synthetic polyamines; Semicarbazide-sensitive amine oxidases; Monoamine oxidase B; Amine oxidase inhibitors; Structure–function relationships

Novel anti-apoptotic effect of the retinoblastoma protein: implications for polyamine analogue toxicity by Veronica M. Johansson; Iréne Thuvesson; Kersti Alm; Stina M. Oredsson (pp. 929-937).

The retinoblastoma protein (pRb) pathway is frequently altered in breast cancer cells. pRb is involved in the regulation of cell proliferation and cell death. The breast cancer cell line L56Br-C1 does not express pRb and is extremely sensitive to treatment with the polyamine analogue N ¹,N ¹¹-diethylnorspermine (DENSPM) which causes apoptosis. Polyamines are essential for the regulation of cell proliferation, cell differentiation and cell death. DENSPM depletes cells of polyamines, e.g., by inducing the activity of the polyamine catabolic enzyme spermidine/spermine N ¹-acetyltransferase (SSAT). In this study, L56Br-C1 cells were transfected with human pRb–cDNA. Overexpression of pRb inhibited DENSPM-induced cell death and DENSPM-induced SSAT activity. This suggests that the pRb protein level is a promising marker for polyamine depletion sensitivity and that there is a connection between pRb and the regulation of SSAT activity. We also show that SSAT protein levels and SSAT activity do not always correlate, suggesting that there is an unknown regulation of SSAT.

Keywords: pRb; Polyamine deficiency; Breast cancer; SSAT; DENSPM

TG2, a novel extracellular protein with multiple functions by Zhuo Wang; Martin Griffin (pp. 939-949).

TG2 is multifunctional enzyme which can be secreted to the cell surface by an unknown mechanism where its Ca²⁺-dependent transamidase activity is implicated in a number of events important to cell behaviour. However, this activity may only be transient due to the oxidation of the enzyme in the extracellular environment including its reaction with NO probably accounting for its many other roles, which are transamidation independent. In this review, we discuss the novel roles of TG2 at the cell surface and in the ECM acting either as a transamidating enzyme or as an extracellular scaffold protein involved in cell adhesion. Such roles include its ability to act as an FN co-receptor for β integrins or in a heterocomplex with FN interacting with the cell surface heparan sulphate proteoglycan syndecan-4 leading to activation of PKCα. These different properties of TG2 involve this protein in various physiological processes, which if not regulated appropriately can also lead to its involvement in a number of diseases. These include metastatic cancer, tissue fibrosis and coeliac disease, thus increasing its attractiveness as both a therapeutic target and diagnostic marker.

Keywords: Tissue transglutaminase; Extracellular matrix; Cell adhesion and crosslinking

Mutual adaptation between mouse transglutaminase 4 and its native substrates in the formation of copulatory plug by Huan-Chin Tseng; Jyh-Bing Tang; P. S. Sudhakar Gandhi; Ching-Wei Luo; Chung-Mao Ou; Chia-Jen Tseng; Han-Jia Lin; Yee-Hsiung Chen (pp. 951-960).

Formation of copulatory plugs by male animals is a common means of reducing competition with rival males. In mice, copulatory plugs are formed by the coagulation of seminal vesicle secretion (SVS), which is a very viscous and self-clotting fluid containing high concentration of proteins. In its native state, mouse SVS contains a variety of disulfide-linked high-molecular-weight complexes (HMWCs) composed of mouse SVS I–III, which are the major components of mouse SVS. Further, mouse SVS I–III are the substrates for transglutaminase 4 (TGM4), a cross-linking enzyme secreted from the anterior prostate. According to activity assays, mouse TGM4 prefers a mild reducing and alkaline environment. However, under these conditions, the activity of mouse TGM4 toward SVS I–III was much lower than that of a common tissue-type TGM, TGM2. On the other hand, mouse TGM4 exhibited much higher cross-linking activity than TGM2 when native HMWCs containing SVS I–III were used as substrates under non-reducing condition. By the action of TGM4, the clot of SVS became more resistant to proteolysis. This indicates that the activity of TGM4 can further rigidify the copulatory plug and extend its presence in the female reproductive tract. Together with the properties of TGM4 and the nature of its disulfide-linked SVS protein substrates, male mice can easily transform the semen into a rigid and durable copulatory plug, which is an important advantage in sperm competition.

Keywords: Copulatory plug; Mouse; Prostate; Seminal vesicle secretion; Transglutaminase

Transglutaminase 2 is secreted from smooth muscle cells by transamidation-dependent microparticle formation by Jeroen van den Akker; Angela van Weert; Gijs Afink; Erik N. T. P. Bakker; Edwin van der Pol; Anita N. Böing; Rienk Nieuwland; Ed VanBavel (pp. 961-973).

Transglutaminase 2 (TG2) is a pleiotropic enzyme involved in both intra- and extracellular processes. In the extracellular matrix, TG2 stabilizes the matrix by both covalent cross-linking and disulfide isomerase activity. These functions become especially apparent during matrix remodeling as seen in wound healing, tumor development and vascular remodeling. However, TG2 lacks the signal sequence for a classical secretory mechanism, and the cellular mechanism of TG2 secretion is currently unknown. We developed a green fluorescent TG2 fusion protein to study the hypothesis that TG2 is secreted via microparticles. Characterization of TG2/eGFP, using HEK/293T cells with a low endogenous TG2 expression, showed that cross-linking activity and fibronectin binding were unaffected. Transfection of TG2/eGFP into smooth muscle cells resulted in the formation of microparticles (MPs) enriched in TG2, as detected both by immunofluorescent microscopy and flow cytometry. The fraction of TG2-positive MPs was significantly lower for cross-linking-deficient mutants of TG2, implicating a functional role for TG2 in the formation of MPs. In conclusion, the current data suggest that TG2 is secreted from the cell via microparticles through a process regulated by TG2 cross-linking.

Keywords: Transglutaminase; Microparticle; Smooth muscle cell; GFP; Flow cytometry

Evidences for a role of protein cross-links in transglutaminase-related disease by Claudio Tabolacci; Alessandro Lentini; Bruno Provenzano; Simone Beninati (pp. 975-986).

Transglutaminases (TGs) are a large family of related and ubiquitous enzymes that catalyze the cross-linking of a glutaminyl residue of a protein/peptide substrate to a lysyl residue of a protein/peptide co-substrate. Considerable and intense progress has been made in the understanding of the chemistry, molecular biology and cell biology of TGs. The knowledge that very different physiological and pathological processes are dependent on the presence of adequate levels of these cross-linking enzymes and on the amount of both free and protein-conjugated polyamines by TG, has generated an incredible amount of original research and review articles. It is clear that TG-mediated reactions are essential for some biological processes, such as blood coagulation, skin barrier formation and extracellular matrix assembly, but may also be involved in pathogenetic mechanisms responsible for several human diseases, such as cancer, AIDS, neurodegenerative disorders, celiac disease, and eye lens opacification. We present here a comprehensive review of recent insights into the pathophysiology of TGs related to their protein cross-linking activity.

Keywords: Transglutaminases; Polyamines; Protein cross-link; Post-translational modifications

Increased thermostability of microbial transglutaminase by combination of several hot spots evolved by random and saturation mutagenesis by Karin Buettner; Thomas C. Hertel; Markus Pietzsch (pp. 987-996).

The thermostability of microbial transglutaminase (MTG) of Streptomyces mobaraensis was further improved by saturation mutagenesis and DNA-shuffling. High-throughput screening was used to identify clones with increased thermostability at 55°C. Saturation mutagenesis was performed at seven “hot spots”, previously evolved by random mutagenesis. Mutations at four positions (2, 23, 269, and 294) led to higher thermostability. The variants with single amino acid exchanges comprising the highest thermostabilities were combined by DNA-shuffling. A library of 1,500 clones was screened and variants showing the highest ratio of activities after incubation for 30 min at 55°C relative to a control at 37°C were selected. 116 mutants of this library showed an increased thermostability and 2 clones per deep well plate were sequenced (35 clones). 13 clones showed only the desired sites without additional point mutations and eight variants were purified and characterized. The most thermostable mutant (triple mutant S23V-Y24N-K294L) exhibited a 12-fold higher half-life at 60°C and a 10-fold higher half-life at 50°C compared to the unmodified recombinant wild-type enzyme. From the characterization of different triple mutants differing only in one amino acid residue, it can be concluded that position 294 is especially important for thermostabilization. The simultaneous exchange of amino acids at sites 23, 24, 269 and 289 resulted in a MTG-variant with nearly twofold higher specific activity and a temperature optimum of 55°C. A triple mutant with amino acid substitutions at sites 2, 289 and 294 exhibits a temperature optimum of 60°C, which is 10°C higher than that of the wild-type enzyme.

Keywords: Microbial transglutaminase; Thermostability; Saturation mutagenesis; DNA-shuffling

Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag by Christian Sommer; Thomas C. Hertel; Christian E. H. Schmelzer; Markus Pietzsch (pp. 997-1006).

In order to produce recombinant microbial transglutaminase (rMTG) which is free of the activating protease, dispase was used to activate the pro-rMTG followed by immobilized metal affinity chromatography (IMAC). As shown by MALDI-MS, the dispase does not only cleave the pro-sequence, but unfortunately also cleaves within the C-terminal histidine-tag. Hence, the active rMTG cannot properly bind to the IMAC material. As an alternative, proteinase K was investigated. This protease was successfully applied for the activation of purified pro-rMTG either as free or immobilized enzyme and the free enzyme was also applicable directly in the crude cell extract of E. coli. Thus, it enables a simple two-step activation/purification procedure resulting in protease-free and almost pure transglutaminase preparations. The protocol has been successfully applied to both, wild-type transglutaminase of Streptomyces mobaraensis as well as to the highly active variant S2P. Proteinase K activates the pro-rMTG without unwanted degradation of the histidine-tag. It turned out to be very important to inhibit proteinase K activity, e.g., by PMSF, prior to protein separation by SDS–PAGE.

Keywords: Dispase; E. coli ; Proteinase K; Proteolytic activation; Recombinant microbial transglutaminase; Transglutaminase variants; IMAC purification

Simulated environmental criticalities affect transglutaminase of Malus and Corylus pollens having different allergenic potential by Rosa Anna Iorio; Alessia Di Sandro; Roberta Paris; Giulia Pagliarani; Stefano Tartarini; Giampaolo Ricci; Donatella Serafini-Fracassini; Elisabetta Verderio; Stefano Del Duca (pp. 1007-1024).

Increases in temperature and air pollution influence pollen allergenicity, which is responsible for the dramatic raise in respiratory allergies. To clarify possible underlying mechanisms, an anemophilous pollen (hazel, Corylus avellana), known to be allergenic, and an entomophilous one (apple, Malus domestica), the allergenicity of which was not known, were analysed. The presence also in apple pollen of known fruit allergens and their immunorecognition by serum of an allergic patient were preliminary ascertained, resulting also apple pollen potentially allergenic. Pollens were subjected to simulated stressful conditions, provided by changes in temperature, humidity, and copper and acid rain pollution. In the two pollens exposed to environmental criticalities, viability and germination were negatively affected and different transglutaminase (TGase) gel bands were differently immunodetected with the polyclonal antibody AtPng1p. The enzyme activity increased under stressful treatments and, along with its products, was found to be released outside the pollen with externalisation of TGase being predominant in C. avellana, whose grain presents a different cell wall composition with respect to that of M. domestica. A recombinant plant TGase (AtPng1p) stimulated the secreted phospholipase A₂ (sPLA₂) activity, that in vivo is present in human mucosa and is involved in inflammation. Similarly, stressed pollen, hazel pollen being the most efficient, stimulated to very different extent sPLA₂ activity and putrescine conjugation to sPLA₂. We propose that externalised pollen TGase could be one of the mediators of pollen allergenicity, especially under environmental stress induced by climate changes.

Keywords: Climate changes; Environmental pollution; Phospholipase A₂ ; Pollen allergenicity; Respiratory allergies; Transglutaminase

Polyamines and transglutaminase activity are involved in compatible and self-incompatible pollination of Citrus grandis by Alessandra Gentile; Fabiana Antognoni; Rosa Anna Iorio; Gaetano Distefano; Giuseppina Las Casas; Stefano La Malfa; Donatella Serafini-Fracassini; Stefano Del Duca (pp. 1025-1035).

Pollination of pummelo (Citrus grandis L. Osbeck) pistils has been studied in planta by adding compatible and self-incompatible (SI) pollen to the stigma surface. The pollen germination has been monitored inside the pistil by fluorescent microscopy showing SI altered morphologies with irregular depositions of callose in the tube walls, and heavy callose depositions in enlarged tips. The polyamine (PA) content as free, perchloric acid (PCA)-soluble and -insoluble fractions and transglutaminase (TGase) activity have been analyzed in order to deepen their possible involvement in the progamic phase of plant reproduction. The conjugated PAs in PCA-soluble fraction were definitely higher than the free and the PCA-insoluble forms, in both compatible and SI pollinated pistils. In pistils, pollination caused an early decrease of free PAs and increase of the bound forms. The SI pollination, showed highest values of PCA-soluble and -insoluble PAs with a maximum in concomitance with the pollen tube arrest. As TGase mediates some of the effects of PAs by covalently binding them to proteins, its activity, never checked before in Citrus, was examined with two different assays. In addition, the presence of glutamyl-PAs confirmed the enzyme assay data and excluded the possibility of a misinterpretation. The SI pollination caused an increase in TGase activity, whereas the compatible pollination caused its decrease. Similarly to bound PAs, the glutamyl-PAs and the enzyme activity peaked in the SI pollinated pistils in concomitance with the observed block of the pollen tube growth, suggesting an involvement of TGase in SI response.

Keywords: Citrus ; Pollen; Polyamines; Reproduction; Self-incompatibility; Transglutaminase

Monitoring of transglutaminase2 under different oxidative stress conditions by Daniela Caccamo; Monica Currò; Nadia Ferlazzo; Salvatore Condello; Riccardo Ientile (pp. 1037-1043).

Transglutaminase 2 (TG2) is a multifunctional calcium-dependent enzyme which catalyzes the post-translational protein crosslinking with formation of intra- or inter-molecular epsilon(gamma-glutamyl)lysine bonds or polyamine incorporation. The up-regulation and activation of TG2 have been reported in a variety of physiological events, including cell differentiation, signal transduction, apoptosis, and wound healing, as well as in cell response to stress evoked by different internal and external stimuli. Here we review TG2 role in cell response to redox state imbalance both under physiological and pathological conditions, such as neurodegenerative disorders, inflammation, autoimmune diseases and cataractogenesis, in which oxidative stress plays a pathogenetic role and also accelerates disease progression. The increase in TG activity together with mitochondrial impairment and collapse of antioxidant enzymatic cell defences have been reported to be the prominent biochemical alterations becoming evident prior to neurodegeneration. Moreover, oxidative stress-induced TG2 pathway is involved in autophagy inhibition and aggresome formation, and TG2 has been suggested to function as a link between oxidative stress and inflammation by driving the decision as to whether a protein should undergo SUMO-mediated regulation or proteasomal degradation. Literature data suggest a strong association between oxidative stress and TG2 up-regulation, which in turn may result in cell survival or apoptosis, depending on cell type, kind of stressor, duration of insult, as well as TG2 intracellular localization and activity state. In particular, it may be suggested that TG2 plays a pro-survival role when the alteration of cell redox state homeostasis is not associated with intracellular calcium increase triggering TG2 transamidation activity.

Keywords: Transglutaminase 2; Oxidative stress; Neurodegeneration; Autoimmune diseases

Tissue transglutaminase regulates chondrogenesis in mesenchymal stem cells on collagen type XI matrices by Shobana Shanmugasundaram; Sheila Logan-Mauney; Kaitlin Burgos; Maria Nurminskaya (pp. 1045-1053).

Tissue transglutaminase (tTG) is a multifunctional enzyme with a plethora of potential applications in regenerative medicine and tissue bioengineering. In this study, we examined the role of tTG as a regulator of chondrogenesis in human mesenchymal stem cells (MSC) using nanofibrous scaffolds coated with collagen type XI. Transient treatment of collagen type XI films and 3D scaffolds with tTG results in enhanced attachment of MSC and supports rounded cell morphology compared to the untreated matrices or those incubated in the continuous presence of tTG. Accordingly, enhanced cell aggregation and augmented chondrogenic differentiation have been observed on the collagen type XI-coated poly- (L-lactide) nanofibrous scaffolds treated with tTG prior to cell seeding. These changes implicate that MSC chondrogenesis is enhanced by the tTG-mediated modifications of the collagen matrix. For example, exogenous tTG increases resistance to collagenolysis in collagen type XI matrices by catalyzing intermolecular cross-linking, detected by a shift in the denaturation temperature. In addition, tTG auto-crosslinks to collagen type XI as detected by western blot and immunofluorescent analysis. This study identifies tTG as a novel regulator of MSC chondrogenesis further contributing to the expanding use of these cells in cartilage bioengineering.

Keywords: Transglutaminase; Collagen type XI; Adult mesenchymal stem cells; Tissue engineering; Cartilage

Autoantibodies from patients with celiac disease inhibit transglutaminase 2 binding to heparin/heparan sulfate and interfere with intestinal epithelial cell adhesion by Kaupo Teesalu; Marina Panarina; Oivi Uibo; Raivo Uibo; Meeme Utt (pp. 1055-1064).

Autoantibodies from patients with celiac disease (CD) can influence transglutaminase 2 (TG2) activity and its cellular functions, but the exact mechanisms have remained unknown. Our objective was to study whether autoantibodies could modulate TG2 binding to heparin/heparan sulfate (HS) and intestinal epithelial cell attachment to fibronectin-TG2 matrix. Anti-TG2 antibodies were purified by TG2 affinity chromatography from sera of patients with active CD. Serum and antibody effects on TG2 binding to heparin/HS, on transamidase activity of TG2, as well as on Caco-2 cell attachment to fibronectin-TG2 matrix were assessed using microplate assays. Both sera and purified anti-TG2 antibodies from CD patients with high anti-TG2 IgA levels reduced TG2 binding to heparin/HS as compared with those with low anti-TG2 IgA or controls. There was a negative correlation between anti-TG2 IgA levels and TG2 binding to heparin/HS. Treatment of fibronectin-TG2 coated wells with CD patients’ sera or purified anti-TG2 antibodies reduced attachment of Caco-2 cells onto the plate as compared with the control samples. The effect of CD patients’ antibodies on Caco-2 cell attachment to fibronectin-TG2 matrix occurred independently of the inhibition of cell adhesion by Arg-Gly-Asp sequence containing peptides. Anti-TG2 autoantibodies had no effect on transamidase activity of TG2 in vitro. We suggest that modulation of adhesion function of TG2 by autoantibodies from patients with CD could be related to the inhibition of TG2 binding to HS residues of cell surface proteoglycans and could have possible implications for CD pathogenesis.

Keywords: Transglutaminase 2; Autoantibodies; Heparin/heparan sulfate; Adhesion; Caco-2 cells

Characterization of the transglutaminase gene family in zebrafish and in vivo analysis of transglutaminase-dependent bone mineralization by Stephanie Deasey; Olga Grichenko; Shaojun Du; Maria Nurminskaya (pp. 1065-1075).

We have characterized the protein cross-linking enzyme transglutaminase (TGs) genes in zebrafish, Danio rerio, based on the analysis of their genomic organization and phylogenetics. Thirteen zebrafish TG genes (zTGs) have been identified, of which 11 show high homology to only 3 mammalian enzymes: TG1, TG2 and FXIIIa. No zebrafish homologues were identified for mammalian TGs 3-7. Real-time PCR analysis demonstrated distinct temporal expression profiles for zTGs in larvae and adult fish. Analysis by in situ hybridization revealed restricted expression of zTG2b and zFXIIIa in skeletal elements, resembling expression of their mammalian homologues in osteo-chondrogenic cells. Mammalian TG2 and FXIIIa have been implicated in promoting osteoblast differentiation and bone mineralization in vitro, however, mouse models lacking either gene have no skeletal phenotype likely due to a compensation effect. We show in this study that mineralization of the newly formed vertebrae is significantly reduced in fish grown for 5 days in the presence of TG inhibitor KCC-009 added at 3–5 days post fertilization. This treatment reduces average vertebrae mineralization by 30%, with complete inhibition in some fish, and no effect on the overall growth and vertebrae number. This is the first in vivo demonstration of the crucial requirement for the TG-catalyzed cross-linking activity in bone mineralization.

Keywords: Transglutaminase; Inhibitors; Zebrafish; Bone development

Concluding remarks by Uriel Bachrach (pp. 1077-1082).

Contributors (pp. 1083-1084).

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.42, #2-3)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.42, #2-3)