Pholiotic acid promotes apoptosis in human metastatic melanoma cells.

Mushrooms produce a great variety of secondary metabolites that can be successful in both prevention and treatment of various cancers. In particular, higher Basidiomycete mushrooms contain various types of biologically active low-molecular compounds in fruiting bodies with suggested anticarcinogenic effects. The polyamine analogue {(2R)-2-[(S)-3-hydroxy-3-methylglutaryloxy] putrescine dicinnamamide} indicated with the name pholiotic acid, isolated for the first time by us from the fruiting bodies of the Basidiomycete Pholiota spumosa (Fr.) Sing. (Strophariaceae), inhibited the viability of human prostate cancer cells, such as other polyamine synthetic analogues that have shown antitumor activity in several types of cancer, including melanoma. Melanoma is an aggressive skin cancer that can metastasize to other organs and presents a high resistance to conventional therapies. In light of these considerations, the present study was therefore designed to assess whether this putrescine derivative could inhibit the growth of human metastatic melanoma cell lines, M14 and A2058. The results obtained demonstrate that this natural compound, at 12.5-50 µM concentration, was able to reduce cell viability of both cancer cells inducing cell death by intrinsic apoptotic pathway that probably involves PTEN activity, inhibition of Hsp70 expression and reactive oxygen species production. On the other hand, the increased expression of enzymes involved in polyamine catabolism trigger apoptotic cell death leading to polyamine depletion and generation of reactive oxygen species as by-products. In conclusion, these findings, starting point for further investigation, implement available our data to support pholiotic acid as an attractive potential chemopreventive agent, and provide a basis for further research into the use of this polyamine derivative as potential anticancer agent for melanoma in combination with existing therapies to improve treatment efficacy and overcome the obstacle of drug resistance.

Molecular Insights into the Synergistic Effects of Putrescine and Ammonium on Dinoflagellates.

Ammonium and polyamines are essential nitrogen metabolites in all living organisms. Crosstalk between ammonium and polyamines through their metabolic pathways has been demonstrated in plants and animals, while no research has been directed to explore this relationship in algae or to investigate the underlying molecular mechanisms. Previous research demonstrated that high concentrations of ammonium and putrescine were among the active substances in bacteria-derived algicide targeting dinoflagellates, suggesting that the biochemical inter-connection and/or interaction of these nitrogen compounds play an essential role in controlling these ecologically important algal species. In this research, putrescine, ammonium, or a combination of putrescine and ammonium was added to cultures of three dinoflagellate species to explore their effects. The results demonstrated the dose-dependent and species-specific synergistic effects of putrescine and ammonium on these species. To further explore the molecular mechanisms behind the synergistic effects, transcriptome analysis was conducted on dinoflagellate Karlodinium veneficum treated with putrescine or ammonium vs. a combination of putrescine and ammonium. The results suggested that the synergistic effects of putrescine and ammonium disrupted polyamine homeostasis and reduced ammonium tolerance, which may have contributed to the cell death of K. veneficum. There was also transcriptomic evidence of damage to chloroplasts and impaired photosynthesis of K. veneficum. This research illustrates the molecular mechanisms underlying the synergistic effects of the major nitrogen metabolites, ammonium and putrescine, in dinoflagellates and provides direction for future studies on polyamine biology in algal species.

Bachmann-Bupp syndrome and treatment.

Bachmann-Bupp syndrome (BABS) is a neurodevelopmental disorder characterized by developmental delay, hypotonia, and varying forms of non-congenital alopecia. The condition is caused by 3'-end mutations of the ornithine decarboxylase 1 (ODC1) gene, which produce carboxy (C)-terminally truncated variants of ODC, a pyridoxal 5'-phosphate-dependent enzyme. C-terminal truncation of ODC prevents its ubiquitin-independent proteasomal degradation and leads to cellular accumulation of ODC enzyme that remains catalytically active. ODC is the first rate-limiting enzyme that converts ornithine to putrescine in the polyamine pathway. Polyamines (putrescine, spermidine, spermine) are aliphatic molecules found in all forms of life and are important during embryogenesis, organogenesis, and tumorigenesis. BABS is an ultra-rare condition with few reported cases, but it serves as a convincing example for drug repurposing therapy. α-Difluoromethylornithine (DFMO, also known as eflornithine) is an ODC inhibitor with a strong safety profile in pediatric use for neuroblastoma and other cancers as well as West African sleeping sickness (trypanosomiasis). Patients with BABS have been treated with DFMO and have shown improvement in hair growth, muscle tone, and development.

Putrescine promotes MMP9-induced angiogenesis in skeletal muscle through hydrogen peroxide/METTL3 pathway.

Blood vessels play a crucial role in the development of skeletal muscle, ensuring the supply of nutrients and oxygen. Putrescine, an essential polyamine for eukaryotic cells, has an unclear impact on skeletal muscle angiogenesis. In this study, we observed lower vessel density and reduced putrescine level in the muscle of low-birth-weight piglet models, and identified a positive correlation between putrescine content and muscle vessel density. Furthermore, putrescine was found to promote angiogenesis in skeletal muscle both in vitro and in vivo by targeting matrix metalloproteinase 9 (MMP9). On a mechanistic level, putrescine augmented the expression of methyltransferase like 3 (METTL3) by attenuating hydrogen peroxide production, thereby increasing the level of N6-methyladenosine (m6A)-modified MMP9 mRNA. This m6A-modified MMP9 mRNA was subsequently recognized and bound by the YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), enhancing the stability of MMP9 mRNA and its protein expression, consequently accelerating angiogenesis in skeletal muscle. In summary, our findings suggest that putrescine enhances MMP9-mediated angiogenesis in skeletal muscle via the hydrogen peroxide/METTL3 pathway.

Influence of exogenous polyamines on the secondary somatic embryogenesis of cork oak (Quercus suber L.).

Quercus suber L. is the main woody tree species in the Mediterranean basin. The in vitro regeneration from adult material, through primary somatic embryogenesis, is a well-known process, but the use of secondary somatic embryos for plant regeneration remains a very sparsely studied process. The main objective of this work is to explore the cork oak regeneration potential by using the secondary somatic embryogenesis process. Mainly, in this work, we report the polyamine effect. Explants used consisted on primary mature embryos, derived from leaves rejuvenated by epicormic shoot of the Moroccan Quercus suber. Three different polyamines were added to the basal medium, which was composed by macronutrients of N30K, 30 g/l glucose, and 7 g/l agar. Three polyamines, Putrescine, Spermine, and Spermidine, were added to the basal medium at 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg/l. Explants were tested after 8 weeks. Morphological analysis showed that the medium with 0.4 mg/l Spermidine provided the best result for secondary embryos, which corresponds to a very significant (p < 0.05) increase of 375%. The number of secondary embryos directly formed was 2.70 ± 0.51. Similarly, the optimum concentrations for high number of clusters (0.50 ± 0.11) and embryo clusters (1.43 ± 0.35) were increased by 145% and 158%. The addition of the polyamine also acted on the quality of embryos formed. A very significant (p < 0.05) increase in the size of secondary embryos was observed compared to the medium without polyamines. Spermidine showed the greatest increase (about 38%).

Specific Gene Expression in Pseudomonas Putida U Shows New Alternatives for Cadaverine and Putrescine Catabolism.

Pseudomonas putida strain U can be grown using, as sole carbon sources, the biogenic amines putrescine or cadaverine, as well as their catabolic intermediates, ɣ-aminobutyrate or δ-aminovalerate, respectively. Several paralogs for the genes that encode some of the activities involved in the catabolism of these compounds, such as a putrescine-pyruvate aminotransferase (spuC1 and spuC2 genes) and a ɣ-aminobutyrate aminotransferase (gabT1 and gabT2 genes) have been identified in this bacterium. When the expression pattern of these genes is analyzed by qPCR, it is drastically conditioned by supplying the carbon sources. Thus, spuC1 is upregulated by putrescine, whereas spuC2 seems to be exclusively induced by cadaverine. However, gabT1 increases its expression in response to different polyamines or aminated catabolic derivatives from them (i.e., ɣ-aminobutyrate or δ-aminovalerate), although gabT2 does not change its expression level concerning no-amine unrelated carbon sources (citrate). These results reveal differences between the mechanisms proposed for polyamine catabolism in P. aeruginosa and Escherichia coli concerning P. putida strain U, as well as allow a deeper understanding of the enzymatic systems used by this last strain during polyamine metabolism.

Putrescine alleviates the oxidative damage of cumulus-oocyte complex via improving fatty acid oxidation.

BACKGROUND: Fatty acid oxidation of cumulus-oocyte complex (COC) provides sufficient energy for oocyte maturation. But, the relationship between fatty acid oxidation and oxidative stress in aging follicles, as well as the effect of putrescine, is still unclear. METHODS: The porcine COCs were randomly divided into four groups and cultured in in vitro maturation (IVM) medium with or without 1 mmol/L putrescine, with 50 µmol/L hydrogen peroxide (H2O2) or with 50 µmol/L H2O2 plus 1 mmol/L putrescine. Oocyte maturation was assessed by the first polar body extrusion. The expressions of genes involved in fatty acid oxidation were detected, and the mitochondrial function was analyzed by themembrane potential. RESULTS: The maturation rate of oocyte was significantly lower in the H2O2 group when compared with the control group (P＜0.001), and putrescine significantly increased this rate in the H2O2 plus putrescine group when compared with the H2O2 group (P＜0.001). The expressions of LKB1, STRAD, ACC2, AMPKα1 and AMPKα2 mRNAs in cumulus cells (CCs) were significantly downregulated by H2O2 treatment, and partly rescued by putrescine addition (P＜0.05-0.001). However, the changes of LKB1, STRAD, ACC2, AMPKα1 and AMPKα2 mRNAs in oocytes were inapparent. The mitochondrial membrane potential of CCs in the H2O2 group was significantly lower than that in the control group, while putrescine addition significantly increased the mitochondrial membrane potential (P＜0.001). CONCLUSION: The decrease of oocyte maturation due to oxidative stress is related with the decreased fatty acid oxidation, and putrescine may alleviate the COCs damage via improving fatty acid oxidation.

Polyamines modulate mouse sperm motility.

Polyamines are polycationic molecules which contains two or more amino groups (-NH3+) highly charged at physiological pH, and among them we found spermine, spermidine, putrescine, and cadaverine. They interact with proteins, nucleic acids, modulate Ca2+, K+, and Na+ channels, and protect sperm from oxidative stress. In this work, we evaluate the effect of spermine, spermidine, and putrescine on the total, progressive and kinematic parameters of motility, capacitation, acrosome reaction, also in presence and absence of the dbcAMP, an analogue of the cAMP, and the IBMX, a phosphodiesterase inhibitor. In addition, we evaluated the intracellular concentrations of cAMP [cAMP]i, and performed an in silico analysis between polyamines and the sAC from mouse to predict the possible interaction among them. Our results showed that all polyamines decrease drastically the total, progressive and the kinetic parameters of sperm motility, decrease the capacitation, and only spermidine and putrescine impeded the acquisition of acrosome reaction. Moreover, the effect of polyamines was attenuated but not countered by the addition of db-cAMP and IBMX, suggesting a possible inhibition of the sAC. Also, the presence of polyamines induced a decrease of the [cAMP]i, and the in silico analysis predicted a strong interaction among polyamines and the sAC. Overall, the evidence suggests that probably the polyamines interact and inhibit the activity of the sAC.

Exogenous Putrescine Changes Biochemical (Antioxidant Activity, Polyphenol, Flavonoid, and Total Phenol Compounds) and Essential Oil Constituents of Salvia officinalis L.

Polyamines are small polycationic molecules containing amines that are present in almost all cells of living organisms and act in a wide range of physiological processes, growth, and development, biological and protection of cells against free radicals. This research is based on principal component analysis (PCA) and calculation of selection criteria (SC) to investigate the effect of foliar spraying of polyamine putrescine on essential oil yield, essential oil compounds, antioxidant activity, and biochemical compounds (polyphenol, flavonoid, and total phenol compounds) of Salvia officinalis. The treatments used included four levels of putrescine, Put (Control: 0, Put1: 500, Put2: 1000, and Put3: 1500 mg L-1 ) with five replications. Based on our results, four factors had eigenvalues≥1 and showed a cumulative variance percentage of 92.57 % by applying different concentrations of putrescine. According to the results of this research, putrescine had significant effects on the amount of total phenolic compounds, flavonoids, and antioxidant activity. The best attention to improving the essential oil yield of sage was 1000 mg L-1 . The crucial essential oil compounds of different Put treated sage were: cis-thujone (35.34 %), camphor (15.60 %), trans-thujone (9.90 %), 1,8-cineole (9.46 %), α-humulene (3.85 %), viridiflorol (3.62 %), camphene (3.58 %), α-pinene (3.50 %), ß-pinene (2.78 %), and limonene (1.23 %). The results showed that the amount of total phenol, the phenolic composition of catechin, and the antioxidant activity of sage plant extract increased significantly when putrescine was used at 1000 mg/liter. Results can use the current research to optimize the production management of medicinal plants and improve the quality of their products. In addition, the advantage of using putrescine is that it increases antioxidants and reduces oxidative damage, and can replace medicinal plants as suitable natural preservatives, thus improving food quality and safety.

Putrescine (1,4-Diaminobutane) enhances antifungal activity in postharvest mango fruit against Colletotrichum gloeosporioides through direct fungicidal and induced resistance mechanisms.

Anthracnose decay caused by Colletotrichum gloeosporioides greatly shortens the shelf life and commercial quality of mango fruit. Putrescine (1,4-Diaminobutane) is involved in modulating plant defense to various environmental stresses. In this research, in vivo and in vitro tests were used to explore the antifungal activity and the underlying mechanism of putrescine against C. gloeosporioides in mango fruit after harvested. In vivo tests suggested that putrescine markedly delayed the occurrence of disease and limited the spots expansion on inoculated mango fruit. Further analysis exhibited that putrescine treatment enhanced disease resistance, along with enhanced activities of chitinase (CHI), ß-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL), polyphenol oxidase (PPO) and the accumulation of lignin, flavonoid, phenolics, and anthocyanin in infected mango fruit. In addition, in vitro tests showed that putrescine exerted strongly antifungal activity against C. gloeosporioides. Putrescine induced the production of reactive oxygen species (ROS) and severe lipid peroxidation damage in C. gloeosporioides mycelia, resulting in the leakage of soluble protein, soluble sugar, nucleic acids, K+ and Ca2+ of C. gloeosporioides mycelia. The mycelium treated with putrescine showed severe deformity and shrinkage, and even cracking. Taken together, putrescine could effectively reduce the incidence rate and severity of anthracnose disease possibly through direct fungicidal effect and indirect induced resistance mechanism, thus showing great potential to be applied to disease control.

Foliar application of chitosan-putrescine nanoparticles (CTS-Put NPs) alleviates cadmium toxicity in grapevine (Vitis vinifera L.) cv. Sultana: modulation of antioxidant and photosynthetic status.

BACKGROUND: Cadmium (Cd) stress displays critical damage to the plant growth and health. Uptake and accumulation of Cd in plant tissues cause detrimental effects on crop productivity and ultimately impose threats to human beings. For this reason, a quite number of attempts have been made to buffer the adverse effects or to reduce the uptake of Cd. Of those strategies, the application of functionalized nanoparticles has lately attracted increasing attention. Former reports clearly noted that putrescine (Put) displayed promising effects on alleviating different stress conditions like Cd and similarly chitosan (CTS), as well as its nano form, demonstrated parallel properties in this regard besides acting as a carrier for many loads with different applications in the agriculture industry. Herein, we, for the first time, assayed the potential effects of nano-conjugate form of Put and CTS (CTS-Put NP) on grapevine (Vitis vinifera L.) cv. Sultana suffering from Cd stress. We hypothesized that their nano conjugate combination (CTS-Put NPs) could potentially enhance Put proficiency, above all at lower doses under stress conditions via CTS as a carrier for Put. In this regard, Put (50 mg L- 1), CTS (0.5%), Put 50 mg L- 1 + CTS 0.5%" and CTS-Put NPs (0.1 and 0.5%) were applied on grapevines under Cd-stress conditions (0 and 10 mg kg- 1). The interactive effects of CTS-Put NP were investigated through a series of physiological and biochemical assays. RESULTS: The findings of present study clearly revealed that CTS-Put NPs as optimal treatments alleviated adverse effects of Cd-stress condition by enhancing chlorophyll (chl) a, b, carotenoids, Fv/Fm, Y(II), proline, total phenolic compounds, anthocyanins, antioxidant enzymatic activities and decreasing Y (NO), leaf and root Cd content, EL, MDA and H2O2. CONCLUSIONS: In conclusion, CTS-Put NPs could be applied as a stress protection treatment on plants under diverse heavy metal toxicity conditions to promote plant health, potentially highlighting new avenues for sustainable crop production in the agricultural sector under the threat of climate change.

Effect of putrescine supplementation to in vitro maturation medium on embryo development and quality in cattle.

This study aimed to investigate the effect of putrescine supplementation to maturation medium during in vitro embryo production in cattle on maturation and embryo development/quality. Oocytes obtained from the ovaries of Holstein cattle were used in the study. Obtained cumulus-oocyte complexes were evaluated according to morphological structure, cytoplasmic features, and cumulus cell number, and only Category-I ones were used in the study. Before the in vitro maturation step, oocytes were randomly divided into two groups. In the first group (Putrescine group, n = 159), 0.5 mM putrescine was added to the maturation medium before in vitro maturation. No addition was applied to the maturation medium of the second group (Control group, n = 149). Cumulus expansion degrees of oocytes following maturation (Grade I: poor, Grade II: partial, and Grade III: complete) were determined. In addition, the meiosis of oocytes after maturation was evaluated by differential staining. Then the oocytes were left for fertilization with sperm and finally, possible zygotes were transferred to the culture medium. After determining the developmental stages and quality of the embryos after in vitro culture, only the embryos at the blastocyst stage were stained with the differential staining method to determine the cell numbers. When the cumulus expansion degrees of the groups were evaluated, the Grade III cumulus expansion rate in the putrescine group was higher than the control group (74.21% and 60.4%; respectively) and the Grade I expansion rate (11.95% and 26.17%; respectively) was found lower (p < .05). When the resumption of meiosis was evaluated according to the cumulus expansion degrees, it was determined that the rate of resumption of meiosis increased as the cumulus expansion increased. In addition, the cleavage rates of oocytes and reaching the blastocyst in the putrescine group were found to be higher than in the control group (p < .05). Moreover, inner cell mass, trophectoderm cells, and total cell counts were found to be higher in blastocysts obtained after the putrescine supplementation to the maturation medium compared to the control group (p < .05). As a result, it was determined that the putrescine supplementation to the maturation medium during in vitro embryo production in cattle increased the degree of cumulus expansion and the rate of resumption of meiosis. In addition, putrescine supplementation was thought to increase the rate of reaching the blastocyst of oocytes due to better cell development in embryos.

Levilactobacillus brevis with High Production of Putrescine Isolated from Blue Cheese and Its Application.

Polyamine intake has been reported to help extend the lifespan of animals. Fermented foods contain high concentrations of polyamines, produced by fermenting bacteria. Therefore, the bacteria, isolated from fermented foods that produce large amounts of polyamines, are potentially used as a source of polyamines for humans. In this study, the strain Levilactobacillus brevis FB215, which has the ability to accumulate approximately 200 µM of putrescine in the culture supernatant, was isolated from fermented foods, specifically the Blue Stilton cheese. Furthermore, L. brevis FB215 synthesized putrescine from agmatine and ornithine, which are known polyamine precursors. When cultured in the extract of Sakekasu, a byproduct obtained during the brewing of Japanese rice wine containing high levels of both agmatine and ornithine, L. brevis FB215 grew to OD600 = 1.7 after 83 h of cultivation and accumulated high concentrations (~1 mM) of putrescine in the culture supernatant. The fermentation product also did not contain histamine or tyramine. The Sakekasu-derived ingredient fermented by the food-derived lactic acid bacteria developed in this study could contribute to increasing polyamine intake in humans.

Foliar application of putrescine, salicylic acid, and ascorbic acid mitigates frost stress damage in Vitis vinifera cv. ̒Giziluzum̕.

BACKGROUND: Cold stress is an effective factor in reducing production and injuring fruit trees. Various materials, such as salicylic acid, ascorbic acid, and putrescine, are used to alleviate the damage of abiotic stress. RESULTS: The effect of different treatments of putrescine, salicylic acid, and ascorbic acid on alleviating the damage of frost stress (- 3 °C) to grapes 'Giziluzum' was investigated. Frost stress increased the amount of H2O2, MDA, proline, and MSI. On the other hand, it decreased the concentration of chlorophyll and carotenoids in the leaves. Putrescine, salicylic acid and ascorbic acid significantly increased the activities of catalase, guaiacol peroxidase, ascorbate peroxidase, and superoxide dismutase under frost stress. Following frost stress, the grapes treated with putrescine, salicylic acid, and ascorbic acid showed higher levels of DHA, AsA, and AsA/DHA than the untreated grapes. Our results showed that the treatment with ascorbic acid outperformed the other treatments in adjusting frost stress damages. CONCLUSION: The use of compounds, such as ascorbic ac id, salicylic acid, and putrescine, modulates the effects of frost stress, thereby increasing the antioxidant defense system of cells, reducing its damage, and stabilizing stable cell conditions, so it can be used to reduce frost damage to different grape cultivars.

Putrescine-functionalized carbon quantum dot (put-CQD) nanoparticle: A promising stress-protecting agent against cadmium stress in grapevine (Vitis vinifera cv. Sultana).

Due to their sessile nature, plant cannot escape from stress factors in their growing environment, in either biotic or abiotic nature. Amid the abiotic stress factors; high levels of soil cadmium (Cd) impose heavy metal stress on plants, resulting in critical injuries and reduced agronomic performance. In order to buffer the adverse effects of Cd stress, novel nanoparticles (NP) have been applied and notable improvements have been reported. According to the literature, the protective roles of polyamines (e.g., Putrescine; Put) and carbon quantum dots (CQD) have been reported with respect to the plant productivity under either stress or non-stress conditions. Those reports led us to hypothesize that the conjugation of Put and CQD (Put-CQD NPs) might lead to further augmented performance of plants under stress and non-stress conditions. In this regard, we successfully synthesized a novel nanomaterial Put-CQD NPs. In this respect, Put (50 mg L-1), CQD (50 mg L-1) and Put-CQD NPs (25 and 50 mg L-1) were sprayed in 'Sultana' grapevines under Cd stress (10 mg kg-1). As expected, upon stress, Cd content in leaf and root tissues increased by 103.40% and 65.15%, respectively (p < 0.05). The high uptake and accumulation of Cd in plant tissues were manifested in significant alterations of physiological and biochemical attributes of the plant. Concerning stress markers, Cd stress caused increases in content of induced MDA, H2O2, and proline as well as electrolyte leakage rate. As expected, Cd stress caused critical reductions in fresh and dry leaf weight by 21.31% and 42.34%, respectively (p < 0.05). On the other hand, both Put-CQD NPs increased fresh and dry leaf weigh up to approximately 30%. The Cd-mediated disturbances in photosynthetic pigments and chlorophyll fluorescence were buffered with Put-CQD NPs. Of the defence system, enzymatic (SOD, APX, GP) as well as anthocyanin and phenolics were induced by both Cd stress and Put-CQD NPs (p < 0.05). On the other hand, Cd stress reduced content of polyamines (putrescine (Put), spermine (Spm) and spermidine (Spd) by 39.28%, 53.36%, and 39.26%, respectively (p < 0.05). However, the reduction levels were buffered by the treatments. Considering the effectiveness of both NP concentrations, the lower dose (25 mg L-1) could be considered as an optimal concentration. To our knowledge, this is the first report of its kind as a potential agent to reduce the adverse effects of Cd stress in grapevines.

Analysis of gene expression related to polyamine concentration and dimorphism induced in ornithine decarboxylase (odc) and spermidine synthase (spd) Ustilago maydis mutants.

Polyamines are ubiquitous small organic cations, and their roles as regulators of several cellular processes are widely recognized. They are implicated in the key stages of the fungal life cycle. Ustilago maydis is a phytopathogenic fungus, the causal agent of common smut of maize and a model system to understand dimorphism and virulence. U. maydis grows in yeast form at pH 7 and it can develop its mycelial form in vitro at pH 3. Δodc mutants that are unable to synthesize polyamines, grow as yeast at pH 3 with a low putrescine concentration, and to complete its dimorphic transition high putrescine concentration is require. Δspd mutants require spermidine to grow and cannot form mycelium at pH 3. In this work, the increased expression of the mating genes, mfa1 and mfa2, on Δodc mutants, was related to high putrescine concentration. Global gene expression analysis comparisons of Δodc and Δspd U. maydis mutants indicated that 2,959 genes were differentially expressed in the presence of exogenous putrescine at pH 7 and 475 genes at pH 3. While, in Δspd mutant, the expression of 1,426 genes was affected by exogenous spermine concentration at pH 7 and 11 genes at pH 3. Additionally, we identified 28 transcriptional modules with correlated expression during seven tested conditions: mutant genotype, morphology (yeast, and mycelium), pH, and putrescine or spermidine concentration. Furthermore, significant differences in transcript levels were noted for genes in modules relating to pH and genotype genes involved in ribosome biogenesis, mitochondrial oxidative phosphorylation, N-glycan synthesis, and Glycosylphosphatidylinositol (GPI)-anchor. In summary, our results offer a valuable tool for the identification of potential factors involved in phenomena related to polyamines and dimorphism.

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.

Enhanced cell growth, production, and mAb quality produced in Chinese hamster ovary-K1 cells by supplementing polyamine in the media.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. KEY POINTS: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing.

Effects of putrescine on oxidative stress, spermidine/spermine-N(1)-acetyltransferase, inflammation and energy levels in liver and serum in rats with brain ischemia-reperfusion.

We aimed to examine the effects of brain ischemia-reperfusion (IR) especially on serum parameters or liver enzymes, free radicals, cytokines, oxidatively damaged DNA, spermidine/spermine N-1-acetyltransferase (SSAT). The effects of addition of putrescine on IR will be evaluated in terms of inflammation and oxidant-antioxidant balance in liver.The study was conducted on 46 male Albino Wistar rats weighing 200-250 g. The rats were grouped into: 1-Sham group (n = 6). 2-IR group (n = 8): The carotid arteries were ligated for 30-min and reperfusion was achieved for 30-min under general anesthesia. 3-Ischemia + putrescine + reperfusion group (IPR) (n = 8): Unlike the IR group, a single dose of 250 µmol kg-1 putrescine was given by gavage at the beginning of reperfusion. In putrescine treatment groups in addition to the procedures performed in the IR group a total of 4 doses of 250 µmol kg-1 putrescine were given at 12-h intervals, with the first dose immediately after 30-min reperfusion (4-IR+putrescine group (IR+P1) (n = 8)); 3 h after the 30-min reperfusion (5-IR+putrescine group (IR+P2) (n = 8)); 6 h after the 30-min reperfusion (6-IR+putrescine group (IR+P3) (n = 8)). ALT, AST, ATP, NO, SSAT, 8-OHdG levels were analyzed in the serum, and liver samples. NF-κB and IL-6 levels were analyzed in the liver samples.Brain IR causes inflammatory, oxidative and DNA damage in the liver, and putrescine supplementation through gavage reduces liver damage by showing anti-inflammatory and antioxidant effects.

The Polyamine Putrescine Is a Positive Regulator of Group 3 Innate Lymphocyte Activation.

Group 3 innate lymphocytes (ILC3s) rapidly respond to invading pathogens or inflammatory signals, which requires shifting cellular metabolic demands. Metabolic adaptations regulating ILC3 function are not completely understood. Polyamines are polycationic metabolites that have diverse roles in cellular functions and in immunity regulate immune cell biology, including Th17 cells. Whether polyamines play a role in ILC3 activation is unknown. In this article, we report that the polyamine synthesis pathway is important for ILC3 activation. IL-23-activated mouse ILC3s upregulate ornithine decarboxylase, the enzyme catalyzing the rate-limiting step of the conversion of ornithine to putrescine in polyamine synthesis, with a subsequent increase in putrescine levels. Inhibition of ornithine decarboxylase via a specific inhibitor, α-difluoromethylornithine, reduced levels of IL-22 produced by steady-state or IL-23-activated ILC3s in a putrescine-dependent manner. Thus, the polyamine putrescine is a positive regulator of ILC3 activation. Our results suggest that polyamines represent a potential target for therapeutic modulation of ILC3 activation during infection or inflammatory disorders.