Putrescine Supplementation Research Articles

Putrescine supplementation improves the developmental competence of in vitro produced bovine embryos

The aim of this study was to investigate the effect of putrescine, anti-apoptotic, antioxidant, and a cell proliferation stimulant, on embryo development and quality by supplementing it to in vitro culture medium. In this study, oocytes were obtained from the ovaries of Holstein cattle. Following maturation and fertilization, the presumptive zygotes were randomly assigned to two groups. The first group (Putrescine, n = 435) was supplemented with putrescine at a concentration of 0.5 mM to in vitro culture. The second group (n = 407) was maintained under standard culture conditions without any supplementations to the medium. Following the determination of the developmental stages of the embryos, only those in the blastocyst stage were subjected to differential staining and the cell numbers of the embryos were determined. Moreover, the TUNEL assay was employed to ascertain the extent of cell death and the apoptotic index in the embryos. Additionally, the levels of ROS were determined in the embryos. Furthermore, gene expression analyses were conducted on blastocyst-stage embryos to ascertain the potential of putrescine supplementation in embryo development along specific pathways. Following in vitro culture, the blastocyst formation rate was 44.37 % in the putrescine group and 32.97 % in the control group (P < 0.05). The counts of ICM (60.60 ± 15.79 vs 50.73 ± 16.74), TE (117.70 ± 23.67 vs 94.0 ± 22.46), and TCC (178.30 ± 26.15 vs 144.73 ± 26.86) were found to be statistically higher in blastocysts developing after putrescine supplementation compared to the control group. Furthermore, the number of apoptotic cells (7.69 ± 2.17 vs 9.96 ± 3.99) and the apoptotic index (5.07 % vs 8.01 %) were found to be lower in the putrescine group in comparison to the control group. Nevertheless, it was established that the ROS level in the control group was approximately two-fold higher than in the putrescine group (P < 0.05). The findings also revealed that putrescine up-regulated the gene expression of SOD, GPX4, CAT, BCL2, NANOG and GATA3 while simultaneously down-regulating the BAX expression level. In conclusion, the supplementation of putrescine to the culture medium during in vitro bovine embryo production was found to contribute to the improvement of embryo quality and early embryonic development.

P-178 Putrescine supplementation enhances embryonic development in patients with Recurrent Implantation Failure (RIF)

Abstract Study question Does putrescine, a type of polyamine, affect embryonic development in patients with recurrent implantation failure (RIF)? Summary answer Putrescine supplementation on cumulus-oocyte complexes (COCs) was associated with better embryologic development from fertilization to blastulation in patients with RIF. What is known already Putrescine, a type of polyamine, plays a role in cell growth and proliferation. Several animal studies suggest the potential impacts of putrescine on embryo development, but its effects on human IVF have yet to be extensively studied. Study design, size, duration This retrospective study analyzed 442 patients who have undergone IVF cycles three or more times from January to December 2023. The average IVF cycle number of RIF patients was 7.4 times. A total of 1356 oocytes from the putrescine group and 1391 oocytes from the control group underwent aspiration and ICSI. Further analysis was conducted by classifying RIF patients into two groups: patients under 40 years of age and patients over 40 years of age. Participants/materials, setting, methods COCs were cultured for 4 hours in a culture medium supplemented with 0.5mM putrescine. 2 hours after oocyte collection, cumulus cells were denuded, and ICSI was performed. Embryonic development was evaluated based on fertilized oocytes (2PN), good quality embryos (GQE) on day 3, and blastulation rates on day 5/6. Group differences were analyzed using t-tests and chi-square tests (p &amp;lt; 0.05). Main results and the role of chance When maternal age was ≥40, the fertilization rate in the putrescine group was significantly higher than that in the control group (86.6% vs. 80.5%, p=0.002). The putrescine group had a numerically higher proportion of embryos with top-grade morphology on day 3 (47.8%, compared with 53.3% for control; p=0.057), without statistical significance. Additionally, the rate of blastulation on day 5 indicated a significant difference between the two groups (14.5% vs. 6.1%, p=0.0001). Further analysis was conducted in younger patients (&amp;lt;40 years old), and it also showed a significantly higher fertilization rate (89.7% vs. 71.1 p=0.0001) and blastocyst development rates on the day 5 in the putrescine group (22.3% vs. 11.1%, p=0.0001), respectively. This study suggests that putrescine supplementation can be utilized to improve embryo quality and facilitate blastocyst formation in IVF procedures for patients with RIF, irrespective of age. Limitations, reasons for caution Putrescine supplementation in IVF showed improved fertilization and blastulation rates, but caution is needed due to the small sample size used. Therefore, a larger sample size will help improve the robustness of the conclusions drawn with respect to the embryologic development in patients with RIF. Wider implications of the findings The study’s findings suggest that putrescine supplementation in IVF may enhance outcomes for patients with RIF, potentially leading to personalized treatments. Trial registration number not applicable

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

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.

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.

Dietary Supplementation with Putrescine Improves Growth Performance and Meat Quality of Wenchang Chickens.

This study was to investigate the effects of dietary supplementation with putrescine on the growth performance and meat quality of chickens. A total of 480 eighty-day-old female Wenchang chickens were randomly assigned into four groups, with 8 replications per group and 15 animals per replicate. The chickens in the control group (Con) were fed a basal diet; the 3 experimental groups were fed a basal diet with 0.01%, 0.03%, and 0.05% putrescine, respectively. The experiment lasted for 40 days. The results showed that dietary supplementation with 0.05% putrescine increased (p < 0.05) the final body weight and average daily weight gain, and decreased the ratio of feed intake to the body weight gain of Wenchang chickens. Dietary supplementation with putrescine decreased the concentrations of putrescine, spermidine, and spermine in serum (p < 0.05). The contents of methionine, phenylalanine, lysine, aspartic acid, tyrosine, total essential amino acids, and total amino acids in breast muscle were higher (p < 0.05) in 0.03% and 0.05% groups than those in Con group. However, the contents of undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, arachidic acid, docosanoic acid, tricosanic acid, lignoceric acid, erucic acid, cis-11,14,17-eicosatrienoate, linoleic acid, and total n-6 monounsaturated fatty acids in breast muscle were lower (p < 0.05) in 0.03% and 0.05% groups than those in Con group. In addition, putrescine supplementation decreased (p < 0.05) the ratio of n-6/n-3 polyunsaturated fatty acids in breast meat. Overall, dietary supplementation with 0.05% putrescine enhanced the growth performance and meat quality of Wenchang chickens.

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.

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.

Role of exogenous putrescine in the status of energy, DNA damage, inflammation, and spermidine/spermine-n(1)- acetyltransferase in brain ischemia-reperfusion in rats.

Objective(s): This study aims to investigate the role of putrescine against brain ischemia-reperfusion (IR) injured rats administered with 250 µmol/kg exogenous putrescine and highlight the IR-associated mechanisms in energy metabolism and inflammatory pathway. Materials and Methods:The rats were divided into six groups: 1-Sham group; 2-IR group, 30 min of ischemia and 30 min of reperfusion was performed with bilateral carotid occlusion (BCAO); 3-IPR group, a single oral dose of putrescine was administered at the start of the 30-minute reperfusion; while in the other treatment groups, 4 doses of putrescine were given within 12-hour intervals. After 30 min of reperfusion, the first dose was administered immediately in the IR-PI (group 4), after 3 hr in IR-PII (group 5), and after 6 hr in IR-PIII (group 6). Interleukin-6 (IL-6), Nuclear factor NF-kappa-B (NF-kB), Adenosine triphosphate (ATP), total Nitric oxide (NO), 8-hydroxyguanosine (8-OHdG), Spermidine/Spermin N-acetyltransferase (SSAT) levels were analyzed in brain tissues.Results:IR reduced brain ATP levels; however, putrescine treatment reversed this state. Brain NO and 8-OHdG levels, and NF-kB and IL-6 levels increased significantly in the IR group and these elevations were decreased in putrescine administered groups. SSAT levels were higher in the IR-PII group. The lowest levels were observed in the IR-PIII group. Conclusion:The exogenous putrescine supplementation after cerebral IR creates neuroprotective effects independent of the time of administration; according to conditions such as formation of radicals in the brain, the spread of the inflammation and the need for consumption of energy are considered as a whole.

Epigenetic effect of putrescine supplementation during in vitro maturation of oocytes on offspring in mice.

To investigate the epigenetic safety of putrescine supplementation during in vitro maturation (IVM) to offspring. Germinal vesicle oocytes retrieved from 12-week-old mice were randomly divided into two groups and cultured in IVM medium with or without 1mmol/L putrescine for 16h. Then, in vitro fertilization and embryo transplantation were conducted to produce the F1 offspring. The F1 mated with ordinary mice and bred the F2 offspring. The DNA methylation patterns in the brain and heart of F1 were investigated by reduced representation bisulfite sequencing. Imprinted gene expression levels of F1 oocytes were tested. The global methylation of F2 was examined by dot blot. The weight, organ coefficient, and histology were normal in the F1 and F2 offspring from the putrescine-treated oocytes. An overall methylation level of 31.23 to 32.53% was observed for all CpG sites in the brain and heart of the two groups. The DNA methylation patterns of the brain and heart in F1 were not altered in general, with subtle differences. The expression levels of imprinted genes including H19, Snrpn, Peg3, Igf2, and Igf2r did not statistically change. The global 5mC level of F2 was consistent with the control group. Putrescine supplementation during IVM did not directly affect the development, health, and reproduction, and did not affect the genome and global epigenetics of mouse offspring derived from those oocytes. The transient putrescine treatment for improving oocyte maturation shows its long-term safety of genome and epigenetics in the offspring of mice.

Effects of putrescine on the quality and epigenetic modification of mouse oocytes during in vitro maturation.

Low ovarian putrescine levels and decreased peak values following luteinising hormone peaks are related to poor oocyte quantity and quality in ageing women. To investigate the effects of putrescine supplementation in in vitro maturation (IVM) medium on oocyte quality and epigenetic modification. Germinal vesicle oocytes retrieved from the ovaries of 8-week-old and 9-month-old mice were divided into four groups (the young, young+difluoromethylornithine (DFMO), ageing and ageing+putrescine groups) and cultured in IVM medium with or without 1mM putrescine or DFMO for 16h. The first polar body extrusion (PBE), cleavage and embryonic development were evaluated. Spindles, chromosomes, mitochondria and reactive oxygen species (ROS) were measured. The expression levels of SIRT1, H3K9ac, H3K9me2, H3K9me3, and 5mC levels were evaluated. Sirt1 and imprinted genes were detected. The PBE was higher in the ageing+putrescine group than in the ageing group. Putrescine increased the total and inner cell mass cell numbers of blastocysts in ageing oocytes. Putrescine decreased aberrant spindles and chromosome aneuploidy, increased the mitochondrial membrane potential and decreased ROS levels. Putrescine increased SIRT1 expression and attenuated the upregulation of H3K9ac levels in ageing oocytes. Putrescine did not affect 5mC, H3K9me2 or H3K9me3 levels or imprinted gene expression. Putrescine supplementation during IVM improved the maturation and quality of ageing oocytes and promoted embryonic development by decreasing ROS generation, maintaining mitochondrial and spindle function and correcting aberrant epigenetic modification. Putrescine shows application potential for human-assisted reproduction, especially for IVM of oocytes from ageing women.

Systemic L-ornithine supplementation specifically increases ovarian putrescine levels during ovulation in mice†.

In all mammalian species examined thus far, the ovaries produce a burst of ornithine decarboxylase (ODC) and putrescine during ovulation or after application of human chorionic gonadotropin (hCG). Aged mice have significantly reduced levels of this periovulatory ODC and putrescine rise. Putrescine supplementation, in vitro during oocyte maturation or in mouse drinking water during the periovulatory period, reduces egg aneuploidies and embryo resorption, improving fertility of aged mice. These studies suggest that periovulatory putrescine supplementation may be a simple and effective therapy for reproductive aging for women. However, putrescine supplementation is expected to increase widespread tissue putrescine levels, raising concerns of nonspecific and unwanted side effects. Given that ODC is highly expressed in the ovaries during ovulation but otherwise exhibits low activity in most tissues, we hypothesized that periovulatory supplementation of L-ornithine, the substrate of ODC, might be suitable for delivering putrescine specifically to the ovaries. In this study, we have demonstrated that systemic application of L-ornithine via oral gavage or subcutaneous injection increased ovarian putrescine levels; the increase was restricted to animals that had been injected with hCG. Furthermore, L-ornithine specifically increased ovarian putrescine levels without affecting putrescine levels in any other tissues. However, our attempts to improve fertility of aged mice through L-ornithine supplementation in mouse drinking water produced either no effects (1% L-ornithine) or negative impact on fertility (4% ornithine). Our results suggest that it might not be feasible to achieve fertility-enhancing ovarian putrescine levels via L-ornithine supplementation in drinking water without encountering undesired consequences of high dose of exogenous L-ornithine.

Exogenous kinetin and putrescine synergistically mitigate salt stress in Luffa acutangula by modulating physiology and antioxidant defense.

Salinity is one ofthe most vicious environmental constraints that hamper agricultural production. Experiments were done to explore the significant role of sole and synergistic supplementation of kinetin (100µM KN) and putrescine (100µM PUT) on Luffa acutangula in NaCl (100mM) treatment. The harmful effects of salinity on growth were manifested by decreased seedling length, biomass, and pigment contents. We studied the effect of KN, and PUT in preventing salt (NaCl) induced physiological disorders and oxidative damages in 20-day-old Luffa acutangula seedlings. The individual application of KN and PUT increased growth and biochemical parameters, whereas combined KN + PUT treatment showed significant enhancement in growth, photosynthetic pigment content, and osmolyte accumulationin salt-affected plants. Application of KN and PUT also prevented hydrogen peroxide and superoxide production as confirmed by inhibition in electrolyte leakage and lipid peroxidation. Kinetin and PUT application upregulated the antioxidant defense system by enhancing antioxidant enzymes and non-enzymatic contents. Luffa seedlings treated with NaCl + KN + PUT showed 79, 26, 74, and 73% rise in superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase enzymes, respectively, in comparison to NaCl-stressed Luffa acutangula. Findings revealed that synergistic utilization of KN and PUT modulate growth and biochemical processes in seedlings efficaciously in comparison to the individual application under salt stress, and it may be due to a regulatory crosstalk mechanism.

The Polyamine Putrescine Promotes Human Epidermal Melanogenesis

Hyperpigmentary conditions can arise when melanogenesis in the epidermis is misregulated. Understanding the pathways underlying melanogenesis is essential for the development of effective treatments. Here, we report that a group of metabolites called polyamines are important in the control of melanogenesis in human skin. Polyamines are cationic molecules present in all cells and are essential for cellular function. We report that polyamine regulator ODC1 is upregulated in melanocytes from melasma lesional skin. We report that the polyamine putrescine can promote pigmentation in human skin explants and primary normal human epidermal melanocytes through induction of tyrosinase which is rate-limiting for the synthesis of melanin. Putrescine supplementation on normal human epidermal melanocytes results in the activation of polyamine catabolism, which results in increased intracellular H2O2. Polyamine catabolism is also increased in human skin explants that have been treated with putrescine. We further report that inhibition of polyamine catabolism prevents putrescine-induced promotion of tyrosinase levels and pigmentation in normal human epidermal melanocytes, showing that polyamine catabolism is responsible for the putrescine induction of melanogenesis. Our data showing that putrescine promotes pigmentation has important consequences for hyperpigmented and hypopigmented conditions. Further understanding of how polyamines control epidermal pigmentation could open the door for the development of new therapeutics.

Altered gene expression in CHO cells following polyamine starvation.

To investigate the impact of polyamine deprivation on the transcriptome of CHO cells RESULTS: Polyamines play a central but poorly-understood role in cell proliferation. Most studies to date have utilised chemical inhibitors to probe polyamine function. Here we exploit the fact that CHO cells grown in serum-free medium have an absolute requirement for putrescine supplementation due to their deficiency in activity of the enzyme arginase. A gene expression microarray (Affymetrix) analysis of CHO-K1 cells starved of polyamines for 3days showed that cessation of growth, associated with increased G1/S transition and inhibition ofM/G1 transitionwas accompanied by increased mRNA levels of mitotic complex checkpoint genes (Mad2l1, Tkk, Bub1b) and in the transition of G1- to S-phase (such as Skp2 and Tfdp1). mRNAs associated with DNA homologous recombination and repair (including Fanconi's anaemia-related genes) and with RNA splicing were consistently increased. Alterations in mRNA levels for genes related to protein processing in the ER, to ER stress, and to p53-related and apoptosis pathways were also observed. mRNAs showing highest levels of fold-change included several which code for membrane-localised proteins and receptors (Thbs1, Tfrc1, Ackr3, Extl1). Growth-arrest induced by polyamine deprivation was associated with significant alterations in levels of mRNAs associated with cell cycle progression, DNA repair, RNA splicing, ER trafficking and membrane signalling as well as p53 and apoptosis-related pathways.

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.

An arginase-based system for selection of transfected CHO cells without the use of toxic chemicals

Polyamines have essential roles in cell proliferation, DNA replication, transcription, and translation processes, with intracellular depletion of putrescine, spermidine, and spermine resulting in cellular growth arrest and eventual death. Serum-free media for CHO-K1 cells require putrescine supplementation, because these cells lack the first enzyme of the polyamine production pathway, arginase. On the basis of this phenotype, we developed an arginase-based selection system. We transfected CHO-K1 cells with a bicistronic vector co-expressing GFP and arginase and selected cells in media devoid of l-ornithine and putrescine, resulting in mixed populations stably expressing GFP. Moreover, single clones in these selective media stably expressed GFP for a total of 42 generations. Using this polyamine starvation method, we next generated recombinant CHO-K1 cells co-expressing arginase and human erythropoietin (hEPO), which also displayed stable expression and healthy growth. The hEPO-expressing clones grew in commercial media, such as BalanCD and CHO-S serum-free media (SFM)-II, as well as in a defined serum-free, putrescine-containing medium for at least 9 passages (27 generations), with a minimal decrease in hEPO titer by the end of the culture. We observed a lack of arginase activity also in several CHO cell strains (CHO-DP12, CHO-S, and DUXB11) and other mammalian cell lines, including BHK21, suggesting broader utility of this selection system. In conclusion, we have established an easy-to-apply alternative selection system that effectively generates mammalian cell clones expressing biopharmaceutically relevant or other recombinant proteins without the need for any toxic selective agents. We propose that this system is applicable to mammalian cell lines that lack arginase activity.

Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

BackgroundPolyamines are essential for cell growth and beneficial for intestinal maturation. To evaluate the effects of putrescine on alleviating intestinal atrophy and underlying molecular mechanisms, both in vivo feeding trial and in vitro cell culture were conducted. Weanling pigs were fed a diet supplemented with 0, 0.1%, 0.2% or 0.3% putrescine dihydrochloride, whereas porcine intestinal epithelial cells (IPEC-J2) were challenged with lipopolysaccharide (LPS) in the presence of 200 μmol/L putrescine.ResultsDietary supplementation with 0.2% putrescine dihydrochloride decreased the incidence of diarrhea with an improvement in intestinal integrity. Inhibition of ornithine decarboxylase activity decreased the proliferation and migration of IPEC-J2 cells, and this effect was alleviated by the supplementation with putrescine. The phosphorylation of extracellular signal regulated kinase and focal adhesion kinase was enhanced by putrescine. LPS increased the expression of inflammatory cytokines [tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-8], and inhibited cell proliferation and migration in IPEC-J2 cells. Adding exogenous putrescine suppressed the expression of TNF-α, IL-6 and IL-8, and recovered cell migration and proliferation in LPS-treated IPEC-J2 cells. Dietary putrescine supplementation also reduced the mRNA levels of TNF-α, IL-6 and IL-8 and their upstream regulator nuclear receptor kappa B p65 subunit in the jejunal mucosa of piglets.ConclusionsDietary supplementation with putrescine mitigated mucosal atrophy in weanling piglets through improving anti-inflammatory function and suppressing inflammatory response. Our results have important implications for nutritional management of intestinal integrity and health in weanling piglets and other neonates.

Putrescine supplementation promote the maturation of oocytes and improve the quality of oocytes and the potential of embryos development

Putrescine supplementation promote the maturation of oocytes and improve the quality of oocytes and the potential of embryos development

Citryl Ornithine Is an Intermediate in a Three-Step Biosynthetic Pathway for Rhizoferrin in Francisella.

The Gram-negative bacterium Francisella tularensis secretes the siderophore rhizoferrin to scavenge necessary iron from the environment. Rhizoferrin, also produced by a variety of fungi and bacteria, comprises two citrate molecules linked by amide bonds to a central putrescine (diaminobutane) moiety. Genetic analysis has determined that rhizoferrin production in F. tularensis requires two enzymes: FslA, a siderophore synthetase of the nonribosomal peptide synthetase-independent siderophore synthetase (NIS) family, and FslC, a pyridoxal-phosphate-dependent decarboxylase. To discern the steps in the biosynthetic pathway, we tested F. tularensis strain LVS and its ΔfslA and ΔfslC mutants for the ability to incorporate potential precursors into rhizoferrin. Unlike putrescine supplementation, supplementation with ornithine greatly enhanced siderophore production by LVS. Radioactivity fromL-[U-14C] ornithine, but not from L-[1-14C] ornithine, was efficiently incorporated into rhizoferrin by LVS. Although neither the ΔfslA nor the ΔfslC mutant produced rhizoferrin, a putative siderophore intermediate labeled by both [U-14C] ornithine and [1-14C] ornithine was secreted by the ΔfslC mutant. Rhizoferrin was identified by liquid chromatography and mass spectrometry in LVS culture supernatants, while citryl-ornithine was detected as the siderophore intermediate in the culture supernatant of the ΔfslC mutant. Our findings support a three-step pathway for rhizoferrin production in Francisella; unlike the fungus Rhizopus delemar, where putrescine functions as a primary precursor for rhizoferrin, biosynthesis in Francisella preferentially starts with ornithine as the substrate for FslA-mediated condensation with citrate. Decarboxylation of this citryl ornithine intermediate by FslC is necessary for a second condensation reaction with citrate to produce rhizoferrin.

Putrescine biosynthesis and export genes are essential for normal growth of avian pathogenic Escherichia coli

BackgroundAvian pathogenic Escherichia coli (APEC) is the infectious agent of a wide variety of avian diseases, which causes substantial economic losses to the poultry industry worldwide. Polyamines contribute to the optimal synthesis of nucleic acids and proteins in bacteria. The objectives of this study were to investigate; i) whether APEC E. coli encodes the same systems for biosynthesis and uptake as described for E. coli K12 and ii) the role of polyamines during in vitro growth of an avian pathogenic E. coli strain (WT-ST117- O83:H4T).ResultsFollowing whole genome sequencing, polyamine biosynthesis and export genes present in E. coli MG1655 (K-12) were found to be identical in WT-ST117. Defined mutants were constructed in putrescine and spermidine biosynthesis pathways (ΔspeB, ΔspeC, ΔspeF, ΔspeB/C and ΔspeD/E), and in polyamines transport systems (ΔpotE, ΔyeeF, ΔpotABCD and ΔpotFGHI). Contrary to what was observed for MG1655, the ΔpotE-ST117 mutant was growth attenuated, regardless of putrescine supplementation. The addition of spermidine or orthinine restored the growth to the level of WT-ST117. Growth attenuation after induction of membrane stress by SDS suggested that PotE is involved in protection against this stress. The ΔspeB/C-ST117 mutant was also growth attenuated in minimal medium. The addition of putrescine or spermidine to the media restored growth rate to the wild type level. The remaining biosynthesis and transport mutants showed a growth similar to that of WT-ST117. Analysis by Ultra-High Performance Liquid Chromatography revealed that the ΔspeB/C mutant was putrescine-deficient, despite that the gene speF, which is also involved in the synthesis of putrescine, was expressed.ConclusionsDeletion of the putrescine transport system, PotE, or the putrescine biosynthesis pathway genes speB/C affected in vitro growth of APEC (ST117- O83:H4) strain, but not E. coli MG1655, despite the high similarity of the genetic make-up of biosynthesis and transport genes. Therefore, blocking these metabolic reactions may be a suitable way to prevent APEC growth in the host without disturbing the commensal E. coli population.