Nutraceutical Properties of the Hydroalcoholic Extract and Phenolic Compounds from Yucca aloifolia Edible Flowers.

The flowers of Yucca aloifolia ("flor de izote") are considered a millenary food in the Northeastern Highlands of Puebla, Mexico. The present investigation reports on the chemical and biological activities of the hydroalcoholic extract (YAHF) obtained from this edible source. HPLC-MS profiling revealed twenty bioactive phenolic compounds with chlorogenic acid (16.5 mg g-1 DW), quercetin (9.5 mg g-1 FW), and their glycosides (rutin and quercitrin), as well as caffeic acid (8.4 mg g-1 DW) and ferulic acid (7.9 mg g-1 DW) as major compounds dissolved in YAHF. Six metabolites had potent anti-lipase (IC50<100 µg mL-1) and anti-ornithine decarboxylase activity (IC50<100 µg mL-1), whereas thirteen exerted strong anti-alpha-glucosidase properties (IC50<100 µg mL-1). The evaluation of YAHF in mice subjected to standard oral glucose tolerance tests and prolonged administration of hypercaloric/atherogenic diet (30 days), unraveled their ability to improve glucose and lipid profiles. YAHF and six phenolic compounds significantly reduced DLD-1 cell viability (IC50, 117.9 µg mL-1) and avoided polyamine accumulation linked to anti-ornithine decarboxylase activity. YAHF and its twenty constituents exerted low toxicity in probiotics (>1000 µg mL-1) and 3T3 fibroblasts (>2.5 mg-mL-1), sustaining their safeness for human consumption.

Spider and Wasp Acylpolyamines: Venom Components and Versatile Pharmacological Leads, Probes, and Insecticidal Agents.

Polyamines (PAs) are polycationic biogenic amines ubiquitously present in all life forms and are involved in molecular signaling and interaction, determining cell fate (e.g., cell proliferation, dif-ferentiation, and apoptosis). The intricate balance in the PAs' levels in the tissues will determine whether beneficial or detrimental effects will affect homeostasis. It's crucial to note that endoge-nous polyamines, like spermine and spermidine, play a pivotal role in our understanding of neu-rological disorders as they interact with membrane receptors and ion channels, modulating neuro-transmission. In spiders and wasps, monoamines (histamine, dopamine, serotonin, tryptamine) and polyamines (spermine, spermidine, acyl polyamines) comprise, with peptides and other sub-stances, the low molecular weight fraction of the venom. Acylpolyamines are venom components exclusively from spiders and a species of solitary wasp, which cause inhibition chiefly of iono-tropic glutamate receptors (AMPA, NMDA, and KA iGluRs) and nicotinic acetylcholine receptors (nAChRs). The first venom acylpolyamines ever discovered (argiopines, Joro and Nephila toxins, and philanthotoxins) have provided templates for the design and synthesis of numerous analogs. Thus far, analogs with high potency exert their effect at nanomolar concentrations, with high se-lectivity toward their ionotropic and ligand receptors. These potent and selective acylpolyamine analogs can serve biomedical purposes and pest control management. The structural modification of acylpolyamine with photolabile and fluorescent groups converted these venom toxins into use-ful molecular probes to discriminate iGluRs and nAchRs in cell populations. In various cases, the linear polyamines, like spermine and spermidine, constituting venom acyl polyamine backbones, have served as cargoes to deliver active molecules via a polyamine uptake system on diseased cells for targeted therapy. In this review, we examined examples of biogenic amines that play an essential role in neural homeostasis and cell signaling, contributing to human health and disease outcomes, which can be present in the venom of arachnids and hymenopterans. With an empha-sis on the spider and wasp venom acylpolyamines, we focused on the origin, structure, derivatiza-tion, and biomedical and biotechnological application of these pharmacologically attractive, chemically modular venom components.

Triclabendazole and clofazimine reduce replication and spermine uptake in vitro in Toxoplasma gondii.

Toxoplasmosis is a worldwide zoonosis caused by the protozoan parasite Toxoplasma gondii. Although this infection is generally asymptomatic in immunocompetent individuals, it can cause serious clinical manifestations in newborns with congenital infection or in immunocompromised patients. As current treatments are not always well tolerated, there is an urgent need to find new drugs against human toxoplasmosis. Drug repurposing has gained considerable momentum in the last decade and is a particularly attractive approach for the search of therapeutic alternatives to treat rare and neglected diseases. Thus, in this study, we investigated the antiproliferative effect of several repurposed drugs. Of these, clofazimine and triclabendazole displayed a higher selectivity against T. gondii, affecting its replication. Furthermore, both compounds inhibited spermine incorporation into the parasite, which is necessary for the formation of other polyamines. The data reported here indicate that clofazimine and triclabendazole could be used for the treatment of human toxoplasmosis and confirms that drug repurposing is an excellent strategy to find new therapeutic targets of intervention.

The Sinorhizobium meliloti NspS-MbaA system affects biofilm formation, exopolysaccharide production and motility in response to specific polyamines.

We previously showed that specific polyamines (PAs) present in the extracellular environment markedly affect extracellular polysaccharide (EPS) production, biofilm formation and motility in Sinorhizobium meliloti Rm8530. We hypothesized that extracellular PA signals were sensed and transduced by the NspS and MbaA proteins, respectively, which are homologs of the PA-sensing, c-di-GMP modulating NspS-MbaA proteins described in Vibrio cholerae. Here we show that the decrease in biofilm formation and EPS production in the quorum-sensing (QS)-deficient S. meliloti wild-type strain 1021 in cultures containing putrescine or spermine did not occur in a 1021 nspS mutant (1021 nspS). The transcriptional expression of nspS in strain 1021 was significantly increased in cultures containing either of these polyamines, but not by exogenous cadaverine, 1,3-diaminopropane (DAP), spermidine (Spd) or norspermidine (NSpd). Cell aggregation in liquid cultures did not differ markedly between strain 1021 and 1021 nspS in the presence or absence of PAs. The S. meliloti QS-proficient Rm8530 wild-type and nspS mutant (Rm8530 nspS) produced similar levels of biofilm under control conditions and 3.2- and 2.2-fold more biofilm, respectively, in cultures with NSpd, but these changes did not correlate with EPS production. Cells of Rm8530 nspS aggregated from two- to several-fold more than the wild-type in cultures without PAs or in those containing Spm. NSpd, Spd and DAP differently affected swimming and swarming motility in strains 1021 and Rm8530 and their respective nspS mutants. nspS transcription in strain Rm8530 was greatly reduced by exogenous Spm. Bioinformatic analysis revealed similar secondary structures and functional domains in the MbaA proteins of S. meliloti and V. cholerae, while their NspS proteins differed in some residues implicated in polyamine recognition in the latter species. NspS-MbaA homologs occur in a small subset of soil and aquatic bacterial species that commonly interact with eukaryotes. We speculate that the S. meliloti NspS-MbaA system modulates biofilm formation, EPS production and motility in response to environmental or host plant-produced PAs.

Spermine reduces the harmful effects of salt stress in Tropaeolum majus.

Flowers, leaves, fruits and buds of Tropaeolum majus are used for ornamental, medicinal and food purposes. However, salt stress limits the development and productivity of T. majus due to biochemical, physiological and anatomical disturbances. Polyamine application is an alternative for mitigating the harmful effects of salt stress. Thus, the objective of this work was to evaluate the effects of spermine application in T. majus grown under salt stress. The experiment was carried out in a completely randomized design, in a 3 × 2 factorial scheme, with 0, 40 mM (moderate salt stress) and 80 mM (severe salt stress) NaCl, and 0 and 1 mM spermine, and with five replicates. Growth (plant height, stem diameter, number of leaves, number of flowers, number of buds, leaf dry mass, stem dry mass and flower dry mass), gas exchange (gs, A, E, Ci and WUE), relative water content, contents of free amino acids, phenolic compounds, reducing and non-reducing sugars, lipid peroxidation and enzymatic activities (CAT, POD and APX) were evaluated. Spermine application decreased the harmful effects of salt stress on the growth and gas exchange and increased flowering in T. majus. Furthermore, the relative water content of T. majus increased under severe salt stress conditions. Spermine application reduced the contents of total phenolic compounds, free amino acids, reducing sugars and non-reducing sugars on leaves of T. majus. Spermine application increased CAT and POD activities in plants under severe salt stress and POD and APX in plants under moderate salt stress.

The citrus plant pathogen Xanthomonas citri has a dual polyamine-binding protein.

ATP-Binding Cassette transporters (ABC transporters) are protein complexes involved in the import and export of different molecules, including ions, sugars, peptides, drugs, and others. Due to the diversity of substrates, they have large relevance in physiological processes such as virulence, pathogenesis, and antimicrobial resistance. In Xanthomonas citri subsp. citri, the phytopathogen responsible for the citrus canker disease, 20% of ABC transporters components are expressed under infection conditions, including the putative putrescine/polyamine ABC transporter, PotFGHI. Polyamines are ubiquitous molecules that mediate cell growth and proliferation and play important role in bacterial infections. In this work, we characterized the X. citri periplasmic-binding protein PotF (XAC2476) using bioinformatics, biophysical and structural methods. PotF is highly conserved in Xanthomonas sp. genus, and we showed it is part of a set of proteins related to the import and assimilation of polyamines in X. citri. The interaction of PotF with putrescine and spermidine was direct and indirectly shown through fluorescence spectroscopy analyses, and experiments of circular dichroism (CD) and small-angle X-ray scattering (SAXS), respectively. The protein showed higher affinity for spermidine than putrescine, but both ligands induced structural changes that coincided with the closing of the domains and increasing of thermal stability.

Polyamine homeostasis-based strategies for cancer: The role of combination regimens.

Spermine, spermidine and putrescine polyamines are naturally occurring ubiquitous positively charged amines and are essential metabolites for biological functions in our life. These compounds play a crucial role in many cell processes, including cellular proliferation, growth, and differentiation. Intracellular levels of polyamines depend on their biosynthesis, transport and degradation. Polyamine levels are high in cancer cells, which leads to the promotion of tumor growth, invasion and metastasis. Targeting polyamine metabolism as an anticancer strategy is considerably rational. Due to compensatory mechanisms, a single strategy does not achieve satisfactory clinical effects when using a single agent. Combination regimens are more clinically promising for cancer chemoprevention because they work synergistically with causing little or no adverse effects due to each individual agent being used at lower doses. Moreover, bioactive substances have advantages over single chemical agents because they can affect multiple targets. In this review, we discuss anticancer strategies targeting polyamine metabolism and describe how combination treatments and effective natural active ingredients are promising therapies. The existing research suggests that polyamine metabolic enzymes are important therapeutic targets and that combination therapies can be more effective than monotherapies based on polyamine depletion.

Metabolic changes associated with differential salt tolerance in sorghum genotypes.

MAIN CONCLUSION: Accumulation of specific metabolites, mainly γ-aminobutyric acid, polyamines, and proline, was essential to homeostasis regulation and differential salt tolerance in sorghum genotypes. Salinity is severe abiotic stress that limits plant growth and development in arid and semi-arid regions. Survival to abiotic stresses depends on metabolic and sometimes even morphological adjustments. We measured the growth parameters, water relations, the content of ions (Na+, K+, Cl-), compatible solutes [some free amino acids (FAAs) including γ-aminobutyric acid (GABA) and proline and soluble carbohydrates) and polyamines (PAs), the activity of PAs metabolism enzymes, and metabolomic profile in plants after 14 days of salt stress treatment. These analyses were to evaluate the influence of metabolomic responses of sorghum genotypes exhibiting sensitivity (CSF18) or tolerance (CSF20) to salinity on plant growth. The salinity promoted growth reductions and induced increases in Na+ and Cl- content and decreases in K+ content. The water status and osmotic potential (Ψo) were reduced by salt stress, but to minimize damage, especially in the CSF20, the osmolytes and PAs contributed to the osmotic adjustment. The results showed that salinity induced an increase in putrescine (Put) in the sensitive genotype. However, it raised spermidine (Spd), spermine (Spm), and cadaverine (Cad) in the tolerant genotype. In addition, the regulation of polyamine oxidase can be related to Spm and GABA biosynthesis. Differential metabolic changes to salt tolerance include metabolites associated with tricarboxylic acid (TCA) cycle intermediates and the metabolisms of sugars, FAAs, and PAs.

Self-assembled peptide dendrigraft supraparticles with potential application in pH/enzyme-triggered multistage drug release.

Multistage delivery systems with size reduction capacity have been proposed as a powerful strategy for improving tissue drug penetration. Here we developed a simple and fast supramolecular approach to construct size-shrinkable polyamine-salt aggregates by ionic cross-linking of biodegradable poly-L-lysine dendrigraft with tripolyphosphate anion. The use of a peptide dendrimer as a nanobuilding block (∼7 nm in diameter) allows the formation of supraparticles (SPs) with well-defined dimensions (∼200 nm in diameter), narrow size distribution and great capacity to encapsulate different molecules, including chemotherapeutic agents as Curcumin and Doxorubicin. When exposed to slightly acidic environments, the crosslinked matrix is instantaneously disassembled to free dendrimer units. Subsequently, model cargo molecules entrapped in the dendrimer architecture can be released by the action of trypsin enzyme through peptide biodegradation. Therefore, these SPs with proved sequential pH and enzyme-responsiveness could be exploited as nanocarriers in multistage drug delivery systems.

Synthesis and Application of a Cationic Polyamine as Yankee Dryer Coating Agent for the Tissue Paper-Making Process.

Tissue paper is of high importance worldwide and, continuously, research is focused on improvements of the softening and durability properties of the paper which depend specifically on the production process. Polyamide-amine-epichlorohydrin (PAE) resins along with release agents are widely used to adhere the paper to the yankee dryer (creping cylinder) in paper manufacture. Nevertheless, these resins are highly cationic and they normally adhere in excess to the paper which negatively affects the creping process and the quality of the paper. For this reason, a low cationic polyamine-epichlorohydrin coating (Polycoat 38®) was synthesized from a diamine supplied by Disproquin S.A.S. and epichlorohydrin. The analysis of the synthesized polymer was carried out by Fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (1H-NMR). The molecular weight of the polymer was obtained by gel permeation chromatography (GPC), physical-chemical properties such as kinematic viscosity, percentage of solids, density, charge density were measured and compared with a commercial PAE resin (Dispro620®) Thermal stability of the Polycoat 38® and glass transition temperature in presence of a release agent (Disprosol 17®) were also evaluated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), respectively. Finally, a peel adhesion test and an absorption durability assessment were carried out together with the evaluation of the creeping efficiency of the paper by caliber and tensile measurements in a tissue (towel paper) production plant, demonstrating a superior performance in the paper creping process as compared to some commercially available products.

Antibacterial and antitumoral activities of the spider acylpolyamine mygalin silver nanoparticles

Mygalin is a synthetic analog of polyamine spermidine isolated from spider hemocytes. Polyamines show potential therapeutic activity against a wide range of human diseases such as cancer and microbial infections. In this work, we analyzed the antibacterial and antitumoral activities of Mygalin silver nanoparticles synthesized by the photoreduction method. The formation and distribution of MygAgNPs were confirmed by UV-visible spectroscopy, zeta potential, and transmission electron microscopy. The obtained nanoparticles were mostly spherical with a particle size distribution in the range of ~ 10–60 nm. We have demonstrated that MygAgNPs increased the effectiveness of the native Mygalin by approximately 6400-fold. Cytotoxicity tests were performed, and it was possible to reach a concentration that was not toxic to healthy cells (NHI-3T3) and at the same time toxic to the tumor cell line (MCF-7). The obtained results suggest that this system shows potential enhanced antibacterial activity against Escherichia coli, DH5a and anticancer activity against MCF-7 cell line

Identification of Trypanosoma cruzi Polyamine Transport Inhibitors by Computational Drug Repurposing.

Trypanosoma cruzi is the causative agent of Chagas disease, a parasitic infection endemic in Latin America. In T. cruzi the transport of polyamines is essential because this organism is unable to synthesize these compounds de novo. Therefore, the uptake of polyamines from the extracellular medium is critical for survival of the parasite. The anthracene-putrescine conjugate Ant4 was first designed as a polyamine transport probe in cancer cells. Ant4 was also found to inhibit the polyamine transport system and produced a strong trypanocidal effect in T. cruzi. Considering that Ant4 is not currently approved by the FDA, in this work we performed computer simulations to find trypanocidal drugs approved for use in humans that have structures and activities similar to Ant4. Through a similarity ligand-based virtual screening using Ant4 as reference molecule, four possible inhibitors of polyamine transport were found. Three of them, promazine, chlorpromazine, and clomipramine, showed to be effective inhibitors of putrescine uptake, and also revealed a high trypanocidal activity against T. cruzi amastigotes (IC50 values of 3.8, 1.9, and 2.9 µM, respectively) and trypomastigotes (IC50 values of 3.4, 2.7, and 1.3 µM, respectively) while in epimastigotes the IC50 were significantly higher (34.7, 41.4, and 39.7 µM, respectively). Finally, molecular docking simulations suggest that the interactions between the T. cruzi polyamine transporter TcPAT12 and all the identified inhibitors occur in the same region of the protein. However, this location is different from the site occupied by the natural substrates. The value of this effort is that repurposing known drugs in the treatment of other pathologies, especially neglected diseases such as Chagas disease, significantly decreases the time and economic cost of implementation.

Amino Acid and Polyamine Membrane Transporters in Trypanosoma cruzi: Biological Function and Evaluation as Drug Targets.

Amino acids and polyamines are involved in relevant processes for the parasite Trypanosoma cruzi, like protein synthesis, stress resistance, life cycle progression, infection establishment and redox balance, among others. In addition to the biosynthetic routes of amino acids, T. cruzi possesses transport systems that allow the active uptake from the extracellular medium; and in the case of polyamines, the uptake is the unique way to obtain these compounds. The TcAAAP protein family is absent in mammals and its members are responsible for amino acid and derivative uptake, thus the TcAAAP permeases are not only interesting and promising therapeutic targets but could also be used to direct the entry of toxic compounds into the parasite. Although there is a treatment available for Chagas disease, its limited efficacy in the chronic stage of the disease, as well as the side effects reported, highlight the urgent need to develop new therapies. Discovery of new drugs is a slow and cost-consuming process, and even during clinical trials the drugs can fail. In this context, drug repositioning is an interesting and recommended strategy by the World Health Organization since costs and time are significantly reduced. In this article, amino acids and polyamines transport and their potential as therapeutic targets will be revised, including examples of synthetic drugs and drug repurposing.

Melatonin and Leishmania amazonensis Infection Altered miR-294, miR-30e, and miR-302d Impacting on Tnf, Mcp-1, and Nos2 Expression.

Leishmaniases are neglected diseases that cause a large spectrum of clinical manifestations, from cutaneous to visceral lesions. The initial steps of the inflammatory response involve the phagocytosis of Leishmania and the parasite replication inside the macrophage phagolysosome. Melatonin, the darkness-signaling hormone, is involved in modulation of macrophage activation during infectious diseases, controlling the inflammatory response against parasites. In this work, we showed that exogenous melatonin treatment of BALB/c macrophages reduced Leishmania amazonensis infection and modulated host microRNA (miRNA) expression profile, as well as cytokine production such as IL-6, MCP-1/CCL2, and, RANTES/CCL9. The role of one of the regulated miRNA (miR-294-3p) in L. amazonensis BALB/c infection was confirmed with miRNA inhibition assays, which led to increased expression levels of Tnf and Mcp-1/Ccl2 and diminished infectivity. Additionally, melatonin treatment or miR-30e-5p and miR-302d-3p inhibition increased nitric oxide synthase 2 (Nos2) mRNA expression levels and nitric oxide (NO) production, altering the macrophage activation state and reducing infection. Altogether, these data demonstrated the impact of melatonin treatment on the miRNA profile of BALB/c macrophage infected with L. amazonensis defining the infection outcome.

Defining novel plant polyamine oxidase subfamilies through molecular modeling and sequence analysis.

BACKGROUND: The polyamine oxidases (PAOs) catabolize the oxidative deamination of the polyamines (PAs) spermine (Spm) and spermidine (Spd). Most of the phylogenetic studies performed to analyze the plant PAO family took into account only a limited number and/or taxonomic representation of plant PAOs sequences. RESULTS: Here, we constructed a plant PAO protein sequence database and identified four subfamilies. Subfamily PAO back conversion 1 (PAObc1) was present on every lineage included in these analyses, suggesting that BC-type PAOs might play an important role in plants, despite its precise function is unknown. Subfamily PAObc2 was exclusively present in vascular plants, suggesting that t-Spm oxidase activity might play an important role in the development of the vascular system. The only terminal catabolism (TC) PAO subfamily (subfamily PAOtc) was lost in Superasterids but it was present in all other land plants. This indicated that the TC-type reactions are fundamental for land plants and that their function could being taken over by other enzymes in Superasterids. Subfamily PAObc3 was the result of a gene duplication event preceding Angiosperm diversification, followed by a gene extinction in Monocots. Differential conserved protein motifs were found for each subfamily of plant PAOs. The automatic assignment using these motifs was found to be comparable to the assignment by rough clustering performed on this work. CONCLUSIONS: The results presented in this work revealed that plant PAO family is bigger than previously conceived. Also, they delineate important background information for future specific structure-function and evolutionary investigations and lay a foundation for the deeper characterization of each plant PAO subfamily.

Searching for Drug Synergy Against Cancer Through Polyamine Metabolism Impairment: Insight Into the Metabolic Effect of Indomethacin on Lung Cancer Cells.

Non-small cell lung cancer (NSCLC) is the most lethal and prevalent type of lung cancer. In almost all types of cancer, the levels of polyamines (putrescine, spermidine, and spermine) are increased, playing a pivotal role in tumor proliferation. Indomethacin, a non-steroidal anti-inflammatory drug, increases the abundance of an enzyme termed spermidine/spermine-N1-acetyltransferase (SSAT) encoded by the SAT1 gene. This enzyme is a key player in the export of polyamines from the cell. The aim of this study was to compare the effect of indomethacin on two NSCLC cell lines, and their combinatory potential with polyamine-inhibitor drugs in NSCLC cell lines. A549 and H1299 NSCLC cells were exposed to indomethacin and evaluations included SAT1 expression, SSAT levels, and the metabolic status of cells. Moreover, the difference in polyamine synthesis enzymes among these cell lines as well as the synergistic effect of indomethacin and chemical inhibitors of the polyamine pathway enzymes on cell viability were investigated. Indomethacin increased the expression of SAT1 and levels of SSAT in both cell lines. In A549 cells, it significantly reduced the levels of putrescine and spermidine. However, in H1299 cells, the impact of treatment on the polyamine pathway was insignificant. Also, the metabolic features upstream of the polyamine pathway (i.e., ornithine and methionine) were increased. In A549 cells, the increase of ornithine correlated with the increase of several metabolites involved in the urea cycle. Evaluation of the levels of the polyamine synthesis enzymes showed that ornithine decarboxylase is increased in A549 cells, whereas S-adenosylmethionine-decarboxylase and polyamine oxidase are increased in H1299 cells. This observation correlated with relative resistance to polyamine synthesis inhibitors eflornithine and SAM486 (inhibitors of ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, respectively), and MDL72527 (inhibitor of polyamine oxidase and spermine oxidase). Finally, indomethacin demonstrated a synergistic effect with MDL72527 in A549 cells and SAM486 in H1299 cells. Collectively, these results indicate that indomethacin alters polyamine metabolism in NSCLC cells and enhances the effect of polyamine synthesis inhibitors, such as MDL72527 or SAM486. However, this effect varies depending on the basal metabolic fingerprint of each type of cancer cell.

Spermine modulates fungal morphogenesis and activates plasma membrane H+-ATPase during yeast to hyphae transition.

Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA) induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

Techniques Used for Functional Characterization of Polyamine Transporters.

Transport systems are key processes in every living organism: they allow the entry of all essential nutrients into the cell and its compartments and regulate the intracellular concentrations of metabolites. The transport of cell nutrients represents the first step of many metabolic routes and may also regulate such processes. They are also responsible for reaching the effective intracellular concentration of therapeutic drugs and some mechanisms of resistance and tolerance also depend on them. However, the common techniques used to evaluate the metabolites transport in different cells types are not easy to carry out and require extensive training. In this chapter, we report detailed protocols and tips about the expression of transporters, different activity assays and transporter kinetics determination.

Diamine derivative anti-Trichomonas vaginalis and anti-Tritrichomonas foetus activities by effect on polyamine metabolism.

Human and bovine trichomoniasis are sexually transmitted diseases (STD) caused by Trichomonas vaginalis and Tritrichomonas foetus, respectively. Human trichomoniasis is the most common non-viral STD in the world and bovine trichomoniasis causes significant economic losses to breeders. Considering the significant impact of the infections caused by these protozoa and the treatment failures, the search for new therapeutic alternatives becomes crucial. In this study the effect of diamines and amino alcohols in the in vitro viability of trichomonads was evaluated. Screening demonstrated the high activity of diamine 4 against these protozoa. Although cytotoxicity against HMVII cell line and slight hemolysis were observed in vitro, the compound showed no toxic effect on the Galleria mellonella in vivo model. Importantly, diamine 4 was active against both trichomonads species at 6h and 24h of incubation, and these effects was reverted by putrescine, a polyamine, suggesting competition for the same metabolic pathway. These findings indicate that the mechanism of action of diamine 4 is through the polyamine metabolism, a pathway distinct from that presented by metronidazole, the drug usually used to treat trichomoniasis and to which resistance is widely reported. These data demonstrate the importance of diamines as potential novel candidates as anti-T. vaginalis and anti-T. foetus agents.

A protein chimera including PspA in fusion with PotD is protective against invasive pneumococcal infection and reduces nasopharyngeal colonization in mice.

Despite the success of the available polysaccharide-based vaccines against Streptococcus pneumoniae in preventing invasive diseases, this bacterium remains a major cause of death in many parts of the world. New vaccine strategies are needed in order to increase protection. Thus, the utilization of fusion proteins is being investigated as an alternative to the current formulations. In the present work, we demonstrate that a chimeric protein, composed of PspA and PotD in fusion is able to maintain the protective characteristics of both parental proteins, providing protection against systemic infection while reducing nasal colonization. The hybrid was not able to improve the response against invasive disease elicited by PspA alone, but the inclusion of PotD was able to reduce colonization, an effect never observed using subcutaneous immunization with PspA. The mechanisms underlying the protective efficacy of the rPspA-PotD hybrid protein were investigated, revealing the production of antibodies with an increased binding capacity to pneumococcal strains of diverse serotypes and genetic backgrounds, enhanced opsonophagocytosis, and secretion of IL-17 by splenocytes. These findings reinforce the use of chimeric proteins based on surface antigens as an effective strategy against pneumococcal infections.