Polyamines Spermidine Research Articles

Maize kernel metabolome involved in resistance to fusarium ear rot and fumonisin contamination.

Fusarium verticillioides poses a threat to worldwide maize production due to its ability to infect maize kernel and synthesize fumonisins that can be accumulated above safety levels for humans and animals. Maize breeding has been proposed as key tool to decrease kernel contamination with fumonisins, but metabolic studies complementary to genomic approaches are necessary to disclose the complexity of maize resistance. An untargeted metabolomic study was proposed using inbreds genetically related but with contrasting levels of resistance in order to uncover pathways implicated in resistance to Fusarium ear rot (FER) and fumonisin contamination in the maize kernel and to look for possible biomarkers. Metabolite determinations were performed in kernels collected at 3 and 10 days after inoculation with F. verticillioides (dat). Discriminant metabolites between resistant and susceptible RILs were rather found at 10 than 3 dat, although metabolite differences at later stages of colonization could be driven by subtle variations at earlier stages of infection. Within this context, differences for membrane lipid homeostasis, methionine metabolism, and indolacetic acid conjugation seemed highly relevant to distinguish between resistant and susceptible inbreds, confirming the polygenic nature of resistance to FER and fumonisin contamination in the maize kernels. Nevertheless, some specific metabolites such as the polyamine spermidine and/or the alkaloid isoquinoline seemed to be promising indirect selection traits to improve resistance to FER and reduce fumonisin accumulation. Therefore, in vitro and in vivo experiments will be necessary to validate the inhibitory effects of these compounds on fumonisins biosynthesis.

Determination of selected biogenic amines in fermented vegetables juices

Fermented vegetables and fruits are a good source of selected vitamins, antioxidants, microelements and revealed immune-boosting properties. The appropriate content of health-promoting compounds and probiotics allows you to qualify juices from fermented vegetables or fruits as better-for-you food. Notwithstanding, biogenic amines in this type of food are also detected due to the fermentation process, which may negatively affect their health-promoting properties. In this study, various biogenic amines were determined in juices obtained during the pressing of fermented vegetables (white and red cabbage, cucumbers, tomatoes, cauliflowers, celery, celery leaves, broccoli, and beetroots). The level of monamines (histamine, tyramine, tryptamine, and phenylethylamine), diamines (putrescine, cadaverine), and polyamines (spermidine and spermine) was discussed. The significant discrepancies between all the tested determined compounds as well as the sums of the BAs in tested samples, were observed. The calculated values of BAs sum varied from 6.36 mg L−1 (fermented tomato juice) to 116.52 mg L−1 (fermented cucumber juice). Among the determined amines, the majority were histamine, tyramine, putrescine, and cadaverine (from 66% to 97–98% of total biogenic amines level). Moreover, depending on the type of tested juice, the amount of histamine and tyramine in the full content of BAs was between 0.44% (for fermented red cabbage juice) and 68% (for fermented broccoli juice). According to our knowledge, this is the first time that the determination of biogenic amines in fermented vegetable juices has been studied.

Extensive diversity in RNA termination and regulation revealed by transcriptome mapping for the Lyme pathogen Borrelia burgdorferi

Transcription termination is an essential and dynamic process that can tune gene expression in response to diverse molecular signals. Yet, the genomic positions, molecular mechanisms, and regulatory consequences of termination have only been studied thoroughly in model bacteria. Here, we use several RNA-seq approaches to map RNA ends for the transcriptome of the spirochete Borrelia burgdorferi – the etiological agent of Lyme disease. We identify complex gene arrangements and operons, untranslated regions and small RNAs. We predict intrinsic terminators and experimentally test examples of Rho-dependent transcription termination. Remarkably, 63% of RNA 3′ ends map upstream of or internal to open reading frames (ORFs), including genes involved in the unique infectious cycle of B. burgdorferi. We suggest these RNAs result from premature termination, processing and regulatory events such as cis-acting regulation. Furthermore, the polyamine spermidine globally influences the generation of truncated mRNAs. Collectively, our findings provide insights into transcription termination and uncover an abundance of potential RNA regulators in B. burgdorferi.

A back-door insights into the modulation of Src kinase activity by the polyamine spermidine.

Src is a protein tyrosine kinase commonly activated downstream of transmembrane receptors and plays key roles in cell growth, migration and survival signaling pathways. In conventional dendritic cells (cDCs), Src is involved in the activation of the non-enzymatic functions of indoleamine 2,3-dioxygenase 1 (IDO1), an immunoregulatory molecule endowed with both catalytic activity and signal transducing properties. Prompted by the discovery that the metabolite spermidine confers a tolerogenic phenotype on cDCs that is dependent on both the expression of IDO1 and the activity of Src kinase, we here investigated the spermidine mode of action. We found that spermidine directly binds Src in a previously unknown allosteric site located on the backside of the SH2 domain and thus acts as a positive allosteric modulator of the enzyme. Besides confirming that Src phosphorylates IDO1, here we showed that spermidine promotes the protein-protein interaction of Src with IDO1. Overall, this study may pave the way toward the design of allosteric modulators able to switch on/off the Src-mediated pathways, including those involving the immunoregulatory protein IDO1.

Polyamine catabolism is concentrated in tumor-associated histiocytes in diffuse large B-cell lymphoma and classic Hodgkin lymphoma

Polyamines are cationic molecules necessary for cell survival, growth, and replication [1–5]. Polyamines come in a variety of structural forms and are principally regulated by two enzymes, spermine/spermidine acetyltransferase-1 (SAT1) and ornithine decarboxylase-1 (ODC1). SAT1 targets the polyamines spermidine and spermine for degradation via acetylation, while ODC1 is involved in converting the polyamine precursor molecule to more complex polyamines [6–8]. Polyamines and their regulatory enzymes have been implicated in tumor metastasis [9,10] and in crosstalk between oncogenes [11–13] in numerous types of cancer, but their role has never been evaluated in B-cell malignancies. In this study, we examine the expression of SAT1 in diffuse large B-cell lymphoma (DLBCL) and classic Hodgkin lymphoma (HL). We found that SAT1 is expressed in all examined cases of DLBCL (n = 15) and HL (n = 5), though the levels of expression across cases vary. We also note that SAT1 expression appears to be concentrated in tumor-associated histiocytes, rather than tumor cells in both DLBCL and HL. We propose that these findings indicate that the polyamine catabolic enzyme, SAT1, plays an unappreciated role in the pathogenesis of B-cell neoplasms.

The effects of microbial fertilizers application on growth, yield and some biochemical changes in the leaves and seeds of guar (Cyamopsis tetragonoloba L.)

Guar (Cyamopsis tetragonoloba L.) is a summer legume that is becoming a crucial industrial crop because of its high gum and protein content. Thus far, the combined effects of arbuscular mycorrhizal fungi (AMF) and Bradyrhizobium on the yield and chemical composition of guar plants are not well studied. Therefore, the current investigation was designed to estimate the individual as well as the combined effects of AMF and Bradyrhizobium on plant growth, yield and nutritional quality of seeds and leaves of guar. AMF and/or Bradyrhizobium inoculation improved chemical composition of guar seeds and its morpho-physiological (plant height, fresh weight, dry weight, and yield production) traits. In addition to increased guar growth and yield production, the inoculation of AMF and/or Bradyrhizobium increased guar leaf and seed minerals, fiber, lipids, crude protein and ash contents. At primary metabolites, there were increases in sugar levels including raffinose stachyose, verbascose and galactomannan. These increases in sugar provided a route for organic acids, amino acids and fatty acids production. Interestingly, there was an increase in essential amino acids and unsaturated fatty acids. At the bioactive secondary metabolite levels, biofertilizers improved phenols and flavonoids levels and anthocyanin and polyamines biosynthesis. In line with these increases, precursors of anthocyanin (phenylalanine, p-coumaric acid, and cinnamic acid) and the levels of polyamines (diaminopropane, putrescine, cadaverine, spermidine, spermine, and agmatine) were increased. Overall, for the first time, our study shed the light on how AMF and Bradyrhizobium improved guar yield and metabolism. Our findings suggested that the combined inoculation of AMF and Bradyrhizobium is an innovative approach to improve guar growth, yield production and yield quality.

Hypusination Maintains Intestinal Homeostasis and Prevents Colitis and Carcinogenesis by Enhancing Aldehyde Detoxification

The amino acid hypusine, synthesized from the polyamine spermidine by the enzyme deoxyhypusine synthase (DHPS), is essential for the activity of eukaryotic translation initiation factor 5A (EIF5A). The role of hypusinated EIF5A (EIF5AHyp) remains unknown in intestinal homeostasis. Our aim was to investigate EIF5AHyp in the gut epithelium in inflammation and carcinogenesis. We used human colon tissue messenger RNA samples and publicly available transcriptomic datasets, tissue microarrays, and patient-derived colon organoids. Mice with intestinal epithelial-specific deletion of Dhps were investigated at baseline and in models of colitis and colon carcinogenesis. We found that patients with ulcerative colitis and Crohn's disease exhibit reduced colon levels of DHPS messenger RNA and DHPS protein and reduced levels of EIF5AHyp. Similarly, colonic organoids from colitis patients also show down-regulated DHPS expression. Mice with intestinal epithelial-specific deletion of Dhps develop spontaneous colon hyperplasia, epithelial proliferation, crypt distortion, and inflammation. Furthermore, these mice are highly susceptible to experimental colitis and show exacerbated colon tumorigenesis when treated with a carcinogen. Transcriptomic and proteomic analysis on colonic epithelial cells demonstrated that loss of hypusination induces multiple pathways related to cancer and immune response. Moreover, we found that hypusination enhances translation of numerous enzymes involved in aldehyde detoxification, including glutathione S-transferases and aldehyde dehydrogenases. Accordingly, hypusination-deficient mice exhibit increased levels of aldehyde adducts in the colon, and their treatment with a scavenger of electrophiles reduces colitis. Hypusination in intestinal epithelial cells has a key role in the prevention of colitis and colorectal cancer, and enhancement of this pathway via supplementation of spermidine could have a therapeutic impact.

Heterologous expression of genes from a cyanobacterial endosymbiont highlights substrate exchanges with its diatom host.

A few genera of diatoms are widespread and thrive in low-nutrient waters of the open ocean due to their close association with N2-fixing, filamentous heterocyst-forming cyanobacteria. In one of these symbioses, the symbiont, Richelia euintracellularis, has penetrated the cell envelope of the host, Hemiaulus hauckii, and lives inside the host cytoplasm. How the partners interact, including how the symbiont sustains high rates of N2 fixation, is unstudied. Since R. euintracellularis has evaded isolation, heterologous expression of genes in model laboratory organisms was performed to identify the function of proteins from the endosymbiont. Gene complementation of a cyanobacterial invertase mutant and expression of the protein in Escherichia coli showed that R. euintracellularis HH01 possesses a neutral invertase that splits sucrose producing glucose and fructose. Several solute-binding proteins (SBPs) of ABC transporters encoded in the genome of R. euintracellularis HH01 were expressed in E. coli, and their substrates were characterized. The selected SBPs directly linked the host as the source of several substrates, e.g. sugars (sucrose and galactose), amino acids (glutamate and phenylalanine), and a polyamine (spermidine), to support the cyanobacterial symbiont. Finally, transcripts of genes encoding the invertase and SBPs were consistently detected in wild populations of H. hauckii collected from multiple stations and depths in the western tropical North Atlantic. Our results support the idea that the diatom host provides the endosymbiotic cyanobacterium with organic carbon to fuel N2 fixation. This knowledge is key to understanding the physiology of the globally significant H. hauckii-R. euintracellularis symbiosis.

Fecal Microbial Structure and Metabolic Profile in Post-Weaning Diarrheic Piglets.

(1) Background: Piglet diarrhea is one of the most serious diseases in pigs and has brought great economic losses to the pig industry. Alteration of the gut microbiota is an important factor in the etiology of piglet diarrhea. Therefore, this study aimed to analyze the differences in the gut microbial structures and fecal metabolic profile between post-weaning diarrhea and healthy Chinese Wannan Black pigs. (2) Methods: An integrated approach of 16S rRNA gene sequencing combined with LC/MS-based metabolomics was employed in this study. (3) Results: We found an increase in the relative abundance of the bacterial genus Campylobacter and a decrease in phylum Bacteroidetes and the species Streptococcus gallolyticus subsp. macedonicus. (S. macedonicus) in piglet diarrhea. Meanwhile, obvious changes in the fecal metabolic profile of diarrheic piglets were also detected, particularly higher levels of polyamines (spermine and spermidine). Moreover, there were substantial associations between the disturbed gut microbiota and the altered fecal metabolites, especially a strong positive relationship between spermidine and Campylobacter. (4) Conclusions: These observations may provide novel insights into potential etiologies related to post-weaning diarrhea and further enhance our understanding of the role of gut microbiota in host homeostasis and in modulating gut microbial structure.

Strikingly conserved gene expression changes of polyamine regulating enzymes among various forms of acute and chronic kidney injury

The polyamines spermidine and spermine and their common precursor molecule putrescine are involved in tissue injury and repair. Here, we test the hypothesis that impaired polyamine homeostasis contributes to various kidney pathologies in mice during experimental models of ischemia-reperfusion, transplantation, rhabdomyolysis, cyclosporine treatment, arterial hypertension, diabetes, unilateral ureteral obstruction, high oxalate feeding, and adenine-induced injuries. We found a remarkably similar pattern in most kidney pathologies with reduced expression of enzymes involved in polyamine synthesis together with increased expression of polyamine degrading enzymes. Transcript levels of amine oxidase copper-containing 1 (Aoc1), an enzyme which catalyzes the breakdown of putrescine, were barely detectable by in situ mRNA hybridization in healthy kidneys. Aoc1 was highly expressed upon various experimental kidney injuries resulting in a significant reduction of kidney putrescine content. Kidney levels of spermine were also significantly reduced, whereas spermidine was increased in response to ischemia-reperfusion injury. Increased Aoc1 expression in injured kidneys was mainly accounted for by an Aoc1 isoform that harbors 22 additional amino acids at its N-terminus and shows increased secretion. Mice with germline deletion of Aoc1 and injured kidneys showed no decrease of kidney putrescine content; although they displayed no overt phenotype, they had fewer tubular casts upon ischemia-reperfusion injury. Hyperosmotic stress stimulated AOC1 expression at the transcriptional and post-transcription levels in metanephric explants and kidney cell lines. AOC1 expression was also significantly enhanced after kidney transplantation in humans. These data demonstrate that the kidneys respond to various forms of injury with down-regulation of polyamine synthesis and activation of the polyamine breakdown pathway. Thus, an imbalance in kidney polyamines may contribute to various etiologies of kidney injury.

A Fine-Tuning of the Plant Hormones, Polyamines and Osmolytes by Ectomycorrhizal Fungi Enhances Drought Tolerance in Pedunculate Oak.

The drought sensitivity of the pedunculate oak (Quercus robur L.) poses a threat to its survival in light of climate change. Mycorrhizal fungi, which orchestrate biogeochemical cycles and particularly have an impact on the plant's defense mechanisms and metabolism of carbon, nitrogen, and phosphorus, are among the microbes that play a significant role in the mitigation of the effects of climate change on trees. The study's main objectives were to determine whether ectomycorrhizal (ECM) fungi alleviate the effects of drought stress in pedunculate oak and to investigate their priming properties. The effects of two levels of drought (mild and severe, corresponding to 60% and 30% of field capacity, respectively) on the biochemical response of pedunculate oak were examined in the presence and absence of ectomycorrhizal fungi. To examine whether the ectomycorrhizal fungi modulate the drought tolerance of pedunculate oak, levels of plant hormones and polyamines were quantified using UPLC-TQS and HPLC-FD techniques in addition to gas exchange measurements and the main osmolyte amounts (glycine betaine-GB and proline-PRO) which were determined spectrophotometrically. Droughts increased the accumulation of osmolytes, such as proline and glycine betaine, as well as higher polyamines (spermidine and spermine) levels and decreased putrescine levels in both, mycorrhized and non-mycorrhized oak seedlings. In addition to amplifying the response of oak to severe drought in terms of inducible proline and abscisic acid (ABA) levels, inoculation with ECM fungi significantly increased the constitutive levels of glycine betaine, spermine, and spermidine regardless of drought stress. This study found that compared to non-mycorrhized oak seedlings, unstressed ECM-inoculated oak seedlings had higher levels of salicylic (SA) and abscisic acid (ABA) but not jasmonic acid (JA), indicating a priming mechanism of ECM is conveyed via these plant hormones. According to a PCA analysis, the effect of drought was linked to the variability of parameters along the PC1 axe, such as osmolytes PRO, GB, polyamines, and plant hormones such as JA, JA-Ile, SAG, and SGE, whereas mycorrhization was more closely associated with the parameters gathered around the PC2 axe (SA, ODPA, ABA, and E). These findings highlight the beneficial function of the ectomycorrhizal fungi, in particular Scleroderma citrinum, in reducing the effects of drought stress in pedunculate oak.

Genomic and structural basis for evolution of tropane alkaloid biosynthesis

The tropane alkaloids (TAs) cocaine and hyoscyamine have been used medicinally for thousands of years. To understand the evolutionary origins and trajectories of serial biosynthetic enzymes of TAs and especially the characteristic tropane skeletons, we generated the chromosome-level genome assemblies of cocaine-producing Erythroxylum novogranatense (Erythroxylaceae, rosids clade) and hyoscyamine-producing Anisodus acutangulus (Solanaceae, asterids clade). Comparative genomic and phylogenetic analysis suggested that the lack of spermidine synthase/N-methyltransferase (EnSPMT1) in ancestral asterids species contributed to the divergence of polyamine (spermidine or putrescine) methylation in cocaine and hyoscyamine biosynthesis. Molecular docking analysis and key site mutation experiments suggested that ecgonone synthases CYP81AN15 and CYP82M3 adopt different active-site architectures to biosynthesize the same product ecgonone from the same substrate in Erythroxylaceae and Solanaceae. Further synteny analysis showed different evolutionary origins and trajectories of CYP81AN15 and CYP82M3, particularly the emergence of CYP81AN15 through the neofunctionalization of ancient tandem duplication genes. The combination of structural biology and comparative genomic analysis revealed that ecgonone methyltransferase, which is responsible for the biosynthesis of characteristic 2-substituted carboxymethyl group in cocaine, evolved from the tandem copies of salicylic acid methyltransferase by the mutations of critical E216 and S153 residues. Overall, we provided strong evidence for the independent origins of serial TA biosynthetic enzymes on the genomic and structural level, underlying the chemotypic convergence of TAs in phylogenetically distant species.

Polyamines in Parkinson's Disease: Balancing Between Neurotoxicity and Neuroprotection.

The polyamines putrescine, spermidine, and spermine are abundant polycations of vital importance in mammalian cells. Their cellular levels are tightly regulated by degradation and synthesis, as well as by uptake and export. Here, we discuss the delicate balance between the neuroprotective and neurotoxic effects of polyamines in the context of Parkinson's disease (PD). Polyamine levels decline with aging and are altered in patients with PD, whereas recent mechanistic studies on ATP13A2 (PARK9) demonstrated a driving role of a disturbed polyamine homeostasis in PD. Polyamines affect pathways in PD pathogenesis, such as α-synuclein aggregation, and influence PD-related processes like autophagy, heavy metal toxicity, oxidative stress, neuroinflammation, and lysosomal/mitochondrial dysfunction. We formulate outstanding research questions regarding the role of polyamines in PD, their potential as PD biomarkers, and possible therapeutic strategies for PD targeting polyamine homeostasis.

Abstract 3093: Synthesis and bioevaluation of LAT-1 efflux agonists for the treatment of pancreatic cancers

Abstract The purpose of this report is to describe the discovery and development of the world’s first L-type amino acid transporter 1 (LAT-1) efflux agonist. LAT-1 is used for the import of amino acid substrates (leucine, phenylalanine, and methionine) and LAT-1 expression has been showed to be increased in pancreatic ductal adenocarcinoma (PDAC) cells. LAT-1 is a promising target for the treatment of rapidly growing cancers, such as PDAC, in part, due to its key role in methionine import. Methionine is required for various cellular processes, including the generation of S-adenosylmethionine (SAM) and the maintenance of intracellular polyamine pools, which are essential for PDAC growth. Indeed, the biosynthesis of the higher polyamines (spermidine and spermine) requires the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dc-SAM), illustrating the molecular connection between methionine and polyamine pools. LAT-1 inhibition, such as with the known LAT-1 inhibitor JPH203, has shown efficacy in suppressing tumor growth in pancreatic cancer cell lines. While the early pre-clinical data is promising, competitive inhibitors of LAT-1 may have limited potency in the presence of high extracellular levels of LAT-1 substrates. In contrast, LAT-1 efflux agonists are insensitive to extracellular LAT-1 substrates. These novel compounds reverse the flow of substrates through LAT-1, effectively depleting cells of key intracellular amino acids, rather than blocking the import of extracellular substrates like methionine. The chiral piperazine (1), our lead efflux agonist, was discovered by a high throughput screen and was shown to deplete intracellular methionine, leucine and phenylalanine pools by LC-MS as well as decrease the synthesis of the higher polyamines (spermidine and spermine), a process which is reliant upon methionine as a precursor. A structure activity relationship (SAR) analysis of the four possible diastereomers of 1 was performed. The performance of these diastereomers and other derivatives were evaluated by LC-MS, HPLC, and Western blot. These molecules were shown to increase 3H-leucine efflux, decrease intracellular polyamine, SAM, and S-adenosylhomocysteine (SAH) levels, decrease phosphatidylethanolamine levels and induce the autophagic marker LC3-II. In summary, this novel method of methionine restriction allows one to alter intracellular methionine levels in a dose-dependent manner and provides a novel way to control pancreatic cancer cell growth. Citation Format: Holly Moots, Houssine Ikhlef, Vandana Sekhar, Otto Phanstiel. Synthesis and bioevaluation of LAT-1 efflux agonists for the treatment of pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3093.

Abstract NG07: Targeting polyamines metabolism in acute myeloid leukemia stem cells

Abstract NG07: Targeting polyamines metabolism in acute myeloid leukemia stem cells

Abstract 3693: Single-cell flux estimation analysis reveals the metabolic states of tumor-specific CD4+ T cells

Abstract Adoptive Cell Therapy (ACT) holds great promise to transform cancer treatments. However, applying ACT to a broad patient population still faces major challenges. The significant barriers to the success of ACT have been attributed to multiple factors, including functional exhaustion, poor persistence, and the inability of T cells to efficiently compete against tumor cells for metabolic resources in the tumor microenvironment (TME). These functional deficiencies highlight the importance of targeting T cell metabolism to improve ACT treatment outcomes. In the TME, tumor cells preferentially utilize glycolysis to rapidly generate ATP, converting pyruvate into lactate and creating a hypoxic environment that is inconducive for effector T cells due to a lack of access to the metabolites needed for sustenance, leading to T cell exhaustion, and declined proliferation. Consequently, T cells lose their function and are unable to persist against tumor cells. The expression of Constitutively Active Signal Transducer and Activator of Transcription 5A (CASTAT5) on tumor-specific CD4+ T cells has been shown to improve their persistence, polyfunctionality, and anti-tumor effects. Our hypothesis is that the improved polyfunctionality and persistence of CASTAT5-CD4+ T cells are partly due to the reprogramming of their metabolic landscape by CASTAT5, resulting in improved metabolic fitness. Single-cell metabolic pathway enrichment analysis of the identified polyfunctional CASTAT5-CD4+ T cells revealed increased capacity for glycolysis, electron transport chain, oxidative phosphorylation, and amino acid metabolism. Single-cell Flux Estimation Analysis (scFEA) further revealed increased metabolic fluxes in amino acid uptake and synthesis, glycolysis, tricarboxylic acid (TCA) cycle, and polyamine synthesis. Interestingly, CASTAT5-CD4+ T cells showed increased polyamine (spermine and spermidine) synthesis, with the upregulation of the metabolic fluxes from putrescine to spermine, catalyzed by spermidine synthase (Srm) and spermine synthase (Srs), both of which are also upregulated in the polyfunctional CD4+ T cell populations. Spermidine and spermine have been shown to enhance mitochondrial metabolism in T cells, increasing their proliferation and survival. Here, we show that there is increased synthesis of spermine in CASTAT5-CD4+ T cells, which may contribute to their increased capacity for oxidative phosphorylation and TCA cycle, leading to the observed improvements in their persistence and anti-tumor function. These findings suggest a role for the polyamine synthesis pathway in the improvement of T cell function. Further studies modulating targets within the polyamine synthesis pathway will help provide an improved understanding of the underlying mechanisms affecting T cell function, thus, unveiling potential metabolic targets to improve the efficacy of tumor-specific T cells. Citation Format: Mercy Kehinde-Ige, Ogacheko Okoko, Zhi-Chun Ding, Gang Zhou, Huidong Shi. Single-cell flux estimation analysis reveals the metabolic states of tumor-specific CD4+ T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3693.

Putrescine supplementation shifts macrophage L-arginine metabolism related-genes reducing Leishmania amazonensis infection.

Leishmania is a protozoan that causes leishmaniasis, a neglected tropical disease with clinical manifestations classified as cutaneous, mucocutaneous, and visceral leishmaniasis. In the infection context, the parasite can modulate macrophage gene expression affecting the microbicidal activity and immune response. The metabolism of L-arginine into polyamines putrescine, spermidine, and spermine reduces nitric oxide (NO) production, favoring Leishmania survival. Here, we investigate the effect of supplementation with L-arginine and polyamines in infection of murine BALB/c macrophages by L. amazonensis and in the transcriptional regulation of genes involved in arginine metabolism and proinflammatory response. We showed a reduction in the percentage of infected macrophages upon putrescine supplementation compared to L-arginine, spermidine, and spermine supplementation. Unexpectedly, deprivation of L-arginine increased nitric oxide synthase (Nos2) gene expression without changes in NO production. Putrescine supplementation increased transcript levels of polyamine metabolism-related genes Arg2, ornithine decarboxylase (Odc1), Spermidine synthase (SpdS), and Spermine synthase (SpmS), but reduced Arg1 in L. amazonensis infected macrophages, while spermidine and spermine promoted opposite effects. Putrescine increased Nos2 expression without leading to NO production, while L-arginine plus spermine led to NO production in uninfected macrophages, suggesting that polyamines can induce NO production. Besides, L-arginine supplementation reduced Il-1b during infection, and L-arginine or L-arginine plus putrescine increased Mcp1 at 24h of infection, suggesting that polyamines availability can interfere with cytokine/chemokine production. Our data showed that putrescine shifts L-arginine-metabolism related-genes on BALB/c macrophages and affects infection by L. amazonensis.

Spermidine Ameliorates Colitis via Induction of Anti-Inflammatory Macrophages and Prevention of Intestinal Dysbiosis.

Exacerbated immune activation, intestinal dysbiosis and a disrupted intestinal barrier are common features among inflammatory bowel disease [IBD] patients. The polyamine spermidine, which is naturally present in all living organisms, is an integral component of the human diet, and exerts beneficial effects in human diseases. Here, we investigated whether spermidine treatment ameliorates intestinal inflammation and offers therapeutic potential for IBD treatment. We assessed the effect of oral spermidine administration on colitis severity in the T cell transfer colitis model in Rag2-/- mice by endoscopy, histology and analysis of markers of molecular inflammation. The effects on the intestinal microbiome were determined by 16S rDNA sequencing of mouse faeces. The impact on intestinal barrier integrity was evaluated in co-cultures of patient-derived macrophages with intestinal epithelial cells. Spermidine administration protected mice from intestinal inflammation in a dose-dependent manner. While T helper cell subsets remained unaffected, spermidine promoted anti-inflammatory macrophages and prevented the microbiome shift from Firmicutes and Bacteroides to Proteobacteria, maintaining a healthy gut microbiome. Consistent with spermidine as a potent activator of the anti-inflammatory molecule protein tyrosine phosphatase non-receptor type 2 [PTPN2], its colitis-protective effect was dependent on PTPN2 in intestinal epithelial cells and in myeloid cells. The loss of PTPN2 in epithelial and myeloid cells, but not in T cells, abrogated the barrier-protective, anti-inflammatory effect of spermidine and prevented the anti-inflammatory polarization of macrophages. Spermidine reduces intestinal inflammation by promoting anti-inflammatory macrophages, maintaining a healthy microbiome and preserving epithelial barrier integrity in a PTPN2-dependent manner.

Changes in the Localization of Polyamine Spermidine in the Rat Retina with Age.

Polyamines (PAs) in the nervous system has a key role in regeneration and aging. Therefore, we investigated age-related changes in the expression of PA spermidine (SPD) in the rat retina. Fluorescent immunocytochemistry was used to evaluate the accumulation of SPD in retinae from rats of postnatal days 3, 21, and 120. Glial cells were identified using glutamine synthetase (GS), whereas DAPI, a marker of cell nuclei, was used to differentiate between retinal layers. SPD localization in the retina was strikingly different between neonates and adults. In the neonatal retina (postnatal day 3-P3), SPD is strongly expressed in practically all cell types, including radial glia and neurons. SPD staining showed strong co-localization with the glial marker GS in Müller Cells (MCs) in the outer neuroblast layer. In the weaning period (postnatal day 21-P21), the SPD label was strongly expressed in all MCs, but not in neurons. In early adulthood (postnatal day 120-P120), SPD was localized in MCs only and was co-localized with the glial marker GS. A decline in the expression of PAs in neurons was observed with age while glial cells accumulated SPD after the differentiation stage (P21) and during aging in MC cellular endfoot compartments.

Understanding the Effects of Trenbolone Acetate, Polyamine Precursors, and Polyamines on Proliferation, Protein Synthesis Rates, and the Abundance of Genes Involved in Myoblast Growth, Polyamine Biosynthesis, and Protein Synthesis in Murine Myoblasts

Research suggests that androgens increase skeletal muscle growth by modulating polyamine biosynthesis. As such, the objective of this study was to investigate effects of anabolic hormones, polyamine precursors, and polyamines relative to proliferation, protein synthesis, and the abundance of mRNA involved in polyamine biosynthesis, proliferation, and protein synthesis in C2C12 and Sol8 cells. Cultures were treated with anabolic hormones (trenbolone acetate and/or estradiol), polyamine precursors (methionine or ornithine), or polyamines (putrescine, spermidine, or spermine). Messenger RNA was isolated 0.5 or 1, 12, or 24 h post-treatment. The cell type had no effect (p > 0.10) on proliferation, protein synthesis, or mRNA abundance at any time point. Each treatment increased (p < 0.01) proliferation, and anabolic hormones increased (p = 0.04) protein synthesis. Polyamines increased (p < 0.05) the abundance of mRNA involved in polyamine biosynthesis, proliferation, and protein synthesis. Treatment with polyamine precursors decreased (p < 0.05) the abundance of mRNA involved in proliferation and protein synthesis. Overall, C2C12 and Sol8 myoblasts do not differ (p > 0.10) in proliferation, protein synthesis, or mRNA abundance at the time points assessed. Furthermore, anabolic hormones, polyamines, and polyamine precursors increase proliferation and protein synthesis, and polyamines and their precursors alter the abundance of mRNA involved in growth.