Putrescine Supplementation Limits the Expansion of pks+ Escherichia coli and Tumor Development in the Colon.

Escherichia coli that harbor the polyketide synthase (pks) genomic island produce colibactin and are associated with sporadic colorectal cancer development. Given the considerable prevalence of pks+ bacteria in healthy individuals, we sought to identify strategies to limit the growth and expansion of pks+ E. coli. We found that culture supernatants of the probiotic strain E. coli Nissle 1917 were able to inhibit the growth of the murine pathogenic strain pks+ E. coli NC101 (EcNC101). We performed a nontargeted analysis of the metabolome in supernatants from several E. coli strains and identified putrescine as a potential postbiotic capable of suppressing EcNC101 growth in vitro. The effect of putrescine supplementation was then evaluated in the azoxymethane/dextran sulfate sodium mouse model of colorectal cancer in mice colonized with EcNC101. Putrescine supplementation inhibited the growth of pks+ E. coli, reduced the number and size of colonic tumors, and downmodulated the release of inflammatory cytokines in the colonic lumen. Additionally, putrescine supplementation led to shifts in the composition and function of gut microbiota, characterized by an increase in the Firmicutes/Bacteroidetes ratio and enhanced acetate production. The effect of putrescine was further confirmed in vitro using a pks+ E. coli strain isolated from a patient with colorectal cancer. These results suggest that probiotic-derived metabolites can be used as an alternative to live bacteria in individuals at risk of developing colorectal cancer due to the presence of pks+ bacteria in their colon. SIGNIFICANCE: Putrescine supplementation inhibits the growth of cancer-promoting bacteria in the gut, lowers inflammation, and reduces colon cancer development. The consumption of healthy foods rich in putrescine may be a potential prophylactic approach for individuals at risk of developing colorectal cancer due to the presence of pks+ bacteria in their colon.

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.

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.

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.

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.

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.

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.

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.

Putrescine accelerates the differentiation of bone marrow derived dendritic cells via inhibiting phosphorylation of STAT3 at Tyr705.

Dendritic cells (DCs) play pivotal roles in immune responses. The differentiation and function of DCs are regulated by environmental metabolites. Putrescine is ubiquitous in various metabolic microenvironments and its immunoregulation has been of increasing interest. However, the mechanisms associated with its DC-induced immunoregulation remain unclear. In this study, we found putrescine promoted induction of immature bone marrow derived DCs (BMDCs), along with the increased phagocytosis and migration, and altered cytokine secretion in immature BMDCs. Transcriptomic profiles indicated significantly impaired inflammatory-related pathways, elevated oxidative phosphorylation, and decreased p-STAT3 (Tyr705) expression. Additionally, putrescine performed minor influence on the lipopolysaccharide (LPS)-induced maturation of BMDCs but significantly impaired LPS-induced DC-elicited allogeneic T-cell proliferation as well as the cytokine secretion. Furthermore, molecular docking and dynamics on the conjugation between putrescine and STAT3 revealed that putrescine could be stably bound to the hydrophilic cavity in STAT3 and performed significant influence on the Tyr705 phosphorylation. CUT&Tag analysis uncovered altered motifs, downregulated IFN-γ response, and upregulated p53 pathway in Putrescine group compared with Control group. In summary, our results demonstrated for the first time that putrescine might accelerate the differentiation of BMDCs by inhibiting the phosphorylation of STAT3 at Tyr705. Given that both DCs and putrescine have ubiquitous and distinct roles in various immune responses and pathogeneses, our findings may provide more insights into polyamine immunoregulation on DCs, as well as distinct strategies in the clinical utilization of DCs by targeting polyamines.

Effects of Spermidine on Cell Proliferation, Migration, and Inflammatory Response in Porcine Enterocytes.

BACKGROUND: Polyamines have been demonstrated to be beneficial to porcine intestinal development. Our previous study showed that putrescine mitigates intestinal atrophy in weanling piglets and suppresses inflammatory response in porcine intestinal epithelial cells, it is still unknown the role of spermidine in mediating putrescine function. OBJECTIVE: The current study aimed to investigate the effect of spermidine on the proliferation, migration, and inflammatory response in porcine intestinal epithelial cells (IPEC-J2 cell line). METHODS: The effects of spermidine on proliferation and migration of IPEC-J2 cells were measured. Difluoromethyl ornithine (DFMO) and diethylglyoxal bis (guanylhydrazone) (DEGBG) were used to block the production of putrescine and spermidine, respectively. A cell inflammation model was established with lipopolysaccharides (LPS) stimulation. Gene expression and protein abundance were determined by real-time quantitative PCR and western blotting, respectively. RESULT: Spermidine significantly enhanced cell proliferation in DFMO (or/and) DEGBG treated IPEC-J2 cells (p < 0.05). Pretreatment with putrescine restored cell growth inhibited by DFMO but did not prevent the decrease in cell proliferation caused by DEGBG (p > 0.05). Similarly, spermidine but not putrescine significantly elevated the rate of migration in DEGBG treated IPEC-J2 cells (p < 0.05). Spermidine deprivation by DEGBG dramatically enhanced mRNA abundance of pro-inflammatory cytokines IL-8, IL-6, and TNF-α (p < 0.05), and the addition of spermidine attenuated excessive expression of those inflammatory pro-inflammatory cytokines, moreover, spermidine but not putrescine suppressed the phosphorylation of NF-κB induced by DEGBG. Spermidine supplementation also significantly suppressed LPS-induced the expression of TNF-α. CONCLUSIONS: The present study highlights a novel insight that putrescine may be converted into spermidine to modulate cell proliferation, migration, and inflammatory response on porcine enterocytes.

Urea transporter B downregulates polyamines levels in melanoma B16 cells via p53 activation.

Urea transporter B (UT-B, encoded by the SLC14A1 gene) is a membrane channel protein involved in urea transmembrane transport. Compared with normal tissues, UT-B expression is significantly decreased in most tumours, especially melanoma. However, the UT-B role in tumorigenesis and development is still unclear. Herein, we investigated the effects of UT-B overexpression on polyamine metabolism and the urea cycle in murine melanoma B16 cells, to explore the roles of mitochondrial dysfunction and p53 activation in cell growth and polyamines metabolism. UT-B overexpression in B16 cells decreased cell growth, increased apoptosis, and significantly altered metabolic pathways related to the urea cycle, which were characterized by reduced production of urea and polyamines and increased production of nitric oxide. Subsequently, we observed that activation of the p53 pathway may be the main cause of the above phenomena. The p53 inhibitor pifithrin-α partially restored the production of polyamines, but the mitochondrial morphology and function were still impaired. Further treatment of UT-B-overexpressing B16 cells with reactive oxygen species scavenging agent N-acetyl-l-cysteine and coenzyme Q10 restored cell viability and mitochondrial function and increased polyamine production. In conclusion, UT-B overexpression caused mitochondrial dysfunction and increased oxidative stress in B16 cells, and then activated p53 expression, which may be one of the mechanisms leading to the decrease in intracellular polyamines.

Short divalent ethacrynic amides as pro-inhibitors of glutathione S-transferase isozyme Mu and potent sensitisers of cisplatin-resistant ovarian cancers.

The linking of ethacrynic acid with ethylenediamine and 1,4-butanediamine gave EDEA and BDEA, respectively, as membrane-permeable divalent pro-inhibitors of glutathione S-transferase (GST). Their divalent glutathione conjugates showed subnanomolar inhibition and divalence-binding to GSTmu (GSTM) (PDB: 5HWL) at ∼0.35 min-1. In cisplatin-resistant SK-OV-3, COC1, SGC7901 and A549 cells, GSTM activities probed by 15 nM BDEA or EDEA revealed 5-fold and 1.0-fold increases in cisplatin-resistant SK-OV-3 and COC1 cells, respectively, in comparison with the susceptible parental cells. Being tolerable by HEK293 and LO2 cells, BDEA at 0.2 µM sensitised resistant SK-OV-3 and COC1 cells by ∼3- and ∼5-folds, respectively, released cytochrome c and increased apoptosis; EDEA at 1.0 µM sensitised resistant SK-OV-3 and A549 cells by ∼5- and ∼7-fold, respectively. EDEA at 1.7 µg/g sensitised resistant SK-OV-3 cells to cisplatin at 3.3 µg/g in nude mouse xenograft model. BDEA and EDEA are promising leads for probing cellular GSTM and sensitising cisplatin-resistant ovarian cancers.

miR27a, a fine-tuning molecule, interacts with growth hormone (GH) signaling and ornithine decarboxylase (ODC) via targeting STAT5.

Autocrine growth hormone (GH) expression triggers cell proliferation, invasion-metastasis in vitro and in vivo models, but GH gene mutations inhibit postnatal growth. Natural polyamines (PA); putrescine, spermidine, spermine trigger cell growth and differentiation. The importance of miR27a has shown to exert a suppressive effect on ornithine decarboxylase (ODC) expression in dwarf mice models. We aimed to modulate the role of A13S, F166Δ, T24 GH gene mutations' impact on PA metabolism and epithelial-mesencyhmal transition (EMT) pathway through miR27a. Biologically active GH signaling triggered cell viability, growth, and colony formation, but T24A alteration significantly decreases aggressive profiles due to inactive GH signaling through a decline in STAT5 activity and expressions of STAT5, c-myc and ODC. Although statistically significant increase in intracellular PA levels in wt GH signaling HEK293 cells compared to HEK293 cells with a lack of GH signaling, a sharp decline in PA levels measured in each mutant GH expressing HEK293 cells. When we inhibited miR27a, proliferation and colony formation accelerated through a significant increase in putrescine levels and upregulation of ODC, STAT5 expression. In contrast, a substantial decline in GH-mediated colony enlargement observed via ODC, STAT5 downregulation, and PA depletion in both wt and mutant GH expressing HEK293 cell lines by miR27a mimic transfection. In conclusion, T24A mutant GH expression declines the GH signaling through STAT5 activity, and mutant GH signaling decreased cell proliferation, division, and colony formation via EMT inhibition. The autocrine GH-mediated proliferative profiles were under the control of miR27a that depletes intracellular putrescine levels via targeting ODC.

Polyamine biomarkers as indicators of human disease.

The significant increase of periodontitis, chronic kidney disease (CKD), Alzheimer's disease and cancer can be attributed to an ageing population. Each disease produces a range of biomarkers that can be indicative of disease onset and progression. Biomarkers are defined as cellular (intra/extracellular components and whole cells), biochemical (metabolites, ions and toxins) or molecular (nucleic acids, proteins and lipids) alterations which are measurable in biological media such as human tissues, cells or fluids. An interesting group of biomarkers that merit further investigation are the polyamines. Polyamines are a group of molecules consisting of cadaverine, putrescine, spermine and spermidine and have been implicated in the development of a range of systemic diseases, in part due to their production in periodontitis. Cadaverine and putrescine within the periodontal environment have demonstrated cell signalling interfering abilities, by way of leukocyte migration disruption. The polyamines spermine and spermidine in tumour cells have been shown to inhibit cellular apoptosis, effectively prolonging tumorigenesis and continuation of cancer within the host. Polyamine degradation products such as acrolein have been shown to exacerbate renal damage in CKD patients. Thus, the use of such molecules has merit to be utilized in the early indication of such diseases in patients.

High-Throughput Screen Identifies Host and Microbiota Regulators of Intestinal Barrier Function.

BACKGROUND & AIMS: The intestinal barrier protects intestinal cells from microbes and antigens in the lumen-breaches can alter the composition of the intestinal microbiota, the enteric immune system, and metabolism. We performed a screen to identify molecules that disrupt and support the intestinal epithelial barrier and tested their effects in mice. METHODS: We performed an imaging-based, quantitative, high-throughput screen (using CaCo-2 and T84 cells incubated with lipopolysaccharide; tumor necrosis factor; histamine; receptor antagonists; and libraries of secreted proteins, microbial metabolites, and drugs) to identify molecules that altered epithelial tight junction (TJ) and focal adhesion morphology. We then tested the effects of TJ stabilizers on these changes. Molecules we found to disrupt or stabilize TJs were administered mice with dextran sodium sulfate-induced colitis or Citrobacter rodentium-induced intestinal inflammation. Colon tissues were collected and analyzed by histology, fluorescence microscopy, and RNA sequencing. RESULTS: The screen identified numerous compounds that disrupted or stabilized (after disruption) TJs and monolayers of epithelial cells. We associated distinct morphologic alterations with changes in barrier function, and identified a variety of cytokines, metabolites, and drugs (including inhibitors of actomyosin contractility) that prevent disruption of TJs and restore TJ integrity. One of these disruptors (putrescine) disrupted TJ integrity in ex vivo mouse colon tissues; administration to mice exacerbated colon inflammation, increased gut permeability, reduced colon transepithelial electrical resistance, increased pattern recognition receptor ligands in mesenteric lymph nodes, and decreased colon length and survival times. Putrescine also increased intestine levels and fecal shedding of viable C rodentium, increased bacterial attachment to the colonic epithelium, and increased levels of inflammatory cytokines in colon tissues. Colonic epithelial cells from mice given putrescine increased expression of genes that regulate metal binding, oxidative stress, and cytoskeletal organization and contractility. Co-administration of taurine with putrescine blocked disruption of TJs and the exacerbated inflammation. CONCLUSIONS: We identified molecules that disrupt and stabilize intestinal epithelial TJs and barrier function and affect development of colon inflammation in mice. These agents might be developed for treatment of barrier intestinal impairment-associated and inflammatory disorders in patients, or avoided to prevent inflammation.

Regulation of the hypothalamic GnRH-GnIH system by putrescine in adult female rats and GT1-7 neuronal cell line.

The gonadotropin-releasing hormone-gonadotropin inhibitor (GnRH-GnIH) system in the hypothalamus of mammals is the key factor that controls the entire reproductive system. The aim of this study was to immunolocalize GnIH (RFRP-3) in the hypothalamus during the estrous cycle and to study the effect of putrescine on the expression of GnRH-I and GnIH through both in vivo and in vitro (GT1-7 cells) approach and the circulatory levels of GnRH-I, GnIH, and gonadotropins were also investigated. The study also aims in analyzing all the immunofluorescence images by measuring the relative pixel count of an image. This study showed the effect of putrescine on the morphology of ovary, uterus, and the expression of the steroidogenic acute regulatory protein in the ovary. This study showed GnIH expression was intense during the diestrus and moderate during proestrus and estrus, whereas mild staining during the metestrus. The study further showed that putrescine supplementation to adult female rats increased both GnRH-I expression in the hypothalamus as well as the GnRH-I levels in circulation. The study, for the first time, also showed that putrescine supplementation decreased the expression and release of GnIH. These effects of upregulating GnRH-I expression and downregulating GnIH expression were confirmed by in vitro experiments using GT1-7 cells. Putrescine supplementation also increased the gonadotropin levels in the serum. To summarize, putrescine can regulate the hypothalamic-pituitary-gonadal axis by increasing the GnRH-I, luteinizing hormone, and follicle-stimulating hormone levels and suppressing GnIH levels. This is the first report showing the simultaneous effects of putrescine on the regulation of both GnRH-I and GnIH in the hypothalamus.

Polyamine stimulation perturbs intracellular Ca2+ homeostasis and decreases viability of breast cancer BT474 cells.

Intracellular polyamines such as spermine and spermidine are essential to cell growth in normal and especially in cancer cells. However, whether extracellular polyamines affect cancer cell survival is unknown. We therefore examined the actions of extracellular polyamines on breast cancer BT474 cells. Our data showed that spermine, spermidine, and putrescine decreased cell viability by apoptosis. These polyamines also elicited Ca2+ signals, but the latter were unlikely triggered via Ca2+-sensing receptor (CaSR) as BT474 cells have been demonstrated previously to lack CaSR expression. Spermine-elicited Ca2+ response composed of both Ca2+ release and Ca2+ influx. Spermine caused a complete discharge of the cyclopiazonic acid (CPA)-sensitive Ca2+ pool and, expectedly, endoplasmic reticulum (ER) stress. The Ca2+ influx pore opened by spermine was Mn2+-impermeable, distinct from the CPA-triggered store-operated Ca2+ channel, which was Mn2+-permeable. Spermine cytotoxic effects were not due to oxidative stress, as spermine did not trigger reactive oxygen species formation. Our results therefore suggest that spermine acted on a putative polyamine receptor in BT474 cells, causing cytotoxicity by Ca2+ overload, Ca2+ store depletion, and ER stress.

Role of putrescine in ovary and embryo development in fruit bat Cynopterus sphinx during embryonic diapause.

The aim of this study was to evaluate the effect of putrescine on ovarian activity and the rate of embryonic development in Cynopterus sphinx during delayed development. The result showed the presence of a rate-limiting enzyme, ornithine decarboxylase-1, in both ovary and utero-embryonic unit of C. sphinx suggests a synthesis of putrescine in these sites. The corpus luteum showed increased, whereas utero-embryonic unit showed decreased production of putrescine during delayed development as compared with the normal development. The bat treated in vivo with putrescine during delayed development showed increase in progesterone and estradiol synthesis, correlated with increased expression of luteinizing hormone receptor, steroidogenic acute receptor protein, and 3ß-hydroxysteroid dehydrogenase through extracellular signal-regulated kinase (ERK1/2)-mediated pathway in the ovary; but showed increase in the weight and expression of progesterone receptor (PR), B-cell lymphoma 2, proliferating cell nucleus antigen, and vascular endothelial growth factor proteins in utero-embryonic unit. The in vitro treatment of putrescine showed stimulatory whereas treatment with an inhibitor of putrescine, 2-difluoromethylornithine caused an inhibitory effect on ovarian progesterone synthesis and cell proliferation, and cell survival in the utero-embryonic unit. In conclusion, the putrescine showed two separate roles during embryonic diapause, high concentration of putrescine in the ovary may support corpus luteum and basal synthesis of progesterone, whereas a low level of putrescine causes retarded embryonic development by inhibiting cell proliferation in the utero-embryonic unit. The bat treated with putrescine either directly promotes cell proliferation, cell survival, and angiogenic activities or acts indirectly increasing PR on utero-embryonic unit thereby activating development in delayed embryo in C. sphinx.

Putrescine enhances intestinal immune function and regulates intestinal bacteria in weaning piglets.

This study aimed to investigate the effect of putrescine on the immune function and intestinal bacteria of weaning piglets. Twenty-four male castrated weaning piglets on their 21st day were randomly assigned into four groups: control (basal diet) and treatment groups given basal diets supplemented with 0.05%, 0.1%, and 0.15% putrescine for 11 days. Results were as follows: (1) Dietary putrescine increased the villus height, width, height/crypt depth and surface area, and decreased the diarrhea index (P < 0.05). (2) Dietary putrescine increased the lysozyme and acid phosphatase activities and the amount of immunoglobulin M, antibacterial peptides, and transforming growth factor ß1, but decreased the mRNA levels of tumor necrosis factor α, interleukin-6, interleukin-8 and inducible nitric oxide synthase (P < 0.05). (3) Dietary putrescine increased the mRNA expression of the mammalian target of rapamycin, signal transducer and activator of transcription, and Janus kinase 2 but decreased the mRNA expression of nuclear factor-kappa B P65 (P < 0.05). (4) Dietary putrescine increased the population of total bacteria, Lactobacillus, and Bifidobacterium and decreased that of Escherichia coli in the colon and cecum (P < 0.05). (5) Finally, dietary putrescine increased the concentrations of butyrate and total volatile fatty acids in the colon and those of acetate, propionate, and total volatile fatty acids in the cecum (P < 0.05). Overall, putrescine can enhance intestinal development, improve immune functions, and regulate the population of intestinal bacteria in weaning piglets.

Exogenous putrescine alleviates photoinhibition caused by salt stress through cooperation with cyclic electron flow in cucumber.

When plants suffer from abiotic stresses, cyclic electron flow (CEF) is induced for photo-protection. Putrescine (Put), a primary polyamine in chloroplasts, plays a critical role in stress tolerance. However, the relationship between CEF and Put in chloroplasts for photo-protection is unknown. In this study, we investigated the role of Put-induced CEF for salt tolerance in cucumber plants (Cucumis sativus L). Treatment with 90 mM NaCl and/or Put did not influence the maximum photochemical efficiency of PSII (Fv/Fm), but the photoactivity of PSI was severely inhibited by NaCl. Salt stress induced a high level of CEF; moreover, plants treated with both NaCl and Put exhibited much higher CEF activity and ATP accumulation than those exhibited by single-salt-treated plants to provide an adequate ATP/NADPH ratio for plant growth. Furthermore, Put decreased the trans-membrane proton gradient (ΔpH), which was accompanied by reduced pH-dependent non-photochemical quenching (NPQ) and an increased the effective quantum yield of PSII (Y(II)). The ratio of NADP+/NADPH increased significantly with Put in salt-stressed leaves compared with the ratio in leaves treated with NaCl, indicating that Put relieved over-reduction pressure at the PSI acceptor side caused by salt stress. Collectively, our results suggest that exogenous Put creates an excellent condition for CEF promotion: a large amount of pmf is predominantly stored as Δψ, resulting in moderate lumen pH and low NPQ, while maintaining high rates of ATP synthesis (high pmf).