Transepithelial transport of putrescine across monolayers of the human intestinal epithelial cell line, Caco-2

AIM To study the transepithelial transport characteristics of the polyamine putrescine in human intestinal Caco-2 cell monolayers to elucidate the mechanisms of the putrescine intestinal absorption.METHODS The transepithelial transport and the cellular accumulation of putrescine was measured using Caco 2 cell monolayers grown on permeable filters.RESULTS Transepithelial transport of putrescine in physiological concentrations (>0.5 mM)from the apical to basolateral side was linear. Intracellular accumulation of putrescine was higher in confluent than in fully differentiated Caco-2 cells, but still negligible (less than 0.5%) of the overall transport across the monolayers in apical-to-basolateral direction. EGF enhanced putrescine accumulation in Caco-2 cells by four-fold, as well as putrescine conversion to spermidine and spermine by enhancing the activity of Sadenosylmethionine decarboxylase. However,EGF did not have any significant influence on putrescine flux across the Caco-2 cell monolayers. Excretion of putrescine from Caco-2cells into the basolateral medium did not exceed 50 picomoles, while putrescine passive flux from the apical to the basolateral chamber,contributed hundreds of micromoles polyamines to the basolateral chamber.CONCLUSION Transepithelial transport of putrescine across Caco-2 cell monolayers occurs in passive diffusion, and is not influenced when epithelial cells are stimulated to proliferate by a potent mitogen such as EGF.

Putrescine Plays a Positive Role in Salt-Tolerance Mechanisms by Reducing Oxidative Damage in Roots of Vegetable Soybean

Polyamines play important roles in plant tolerance to environmental stress. With the aim of investigating the possible involvement of putrescine （Put） in salt-tolerance mechanisms in vegetable soybean roots, exogenous Put （10 mmol L＂） and its biosynthetic inhibitor D-arginine （D-Arg） （0.5 mmol L-1） were added to nutrient solution when vegetable soybean （Glycine max L. cv. Huning 95-1） seedlings were exposed to 100 mmol L^-11 sodium chloride （NaCl）. The results showed that Put ameliorated but D-Arg aggravated the detrimental effects of NaCl on plant growth and biomass production. Under NaCl stress, levels of free, soluble conjugated and insoluble bound types of Put in roots of vegetable soybean were reduced, whereas those of free, soluble conjugated, and insoluble bound types of spermidine （Spd） and spermine （Spm） were increased. Exogenous Put eliminated the decrease in Put but promoted the increase of Spd and Spm. However, these changes could be reversed by D-Arg. Under NaCl stress, activities of arginine decarboxylase （ADC）, S-adenosylmethionine decarboxylase （SAMDC）, diamine oxidase （DAO）, and polyamine oxidase （PAO） were induced, with exogenous Put promoting and D-Arg reversing these changes. Furthermore, NaCl stress decreased activities of antioxidant enzymes. Exogenous Put alleviated but D-Arg exaggerated these effects of NaCl stress, resulting in the same changes in membrane damage and reactive oxygen species （ROS） production. These results indicated that Put plays a positive role in vegetable soybean roots by activating antioxidant enzymes and thereby attenuating oxidative damage.

Putrescine mitigates intestinal atrophy through suppressing inflammatory response in weanling piglets

Background: Polyamines 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% or0.3% putrescine dihydrochloride, whereas porcine intestinal epithelial cells(IPEC-J2) were challenged with lipopolysaccharide(LPS) in the presence of 200 μmol/L putrescine.Results: Dietary 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 m RNA 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.Conclusions: Dietary 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.

Physiological and Proteomic Analyses Reveal Effects of Putrescine-Alleviated Aluminum Toxicity in Rice Roots

The effects of putrescine on improving rice growth under aluminum(Al)toxicity conditions have been previously demonstrated,however,the underlying mechanism remains unclear.In this study,treatment with 50 pmol/L Al significantly decreased rice root growth and whole rice dry weight,inhibited Ca2+uptake,decreased ATP syn thesis,and in creased Al,H2O2 and malon dialdehyde(MDA)con tents,whereas the application of putrescine mitigated these negative effects.Putrescine increased root growth and total dry weight of rice,reduced total Al content,decreased H2O2 and MDA contents by increasing antioxidant enzyme(superoxide dismutase,peroxidase,catalase and glutathione S・transferase)activities,increased Ca2+uptake and energy product!oru Proteomic analyses using data-independent acquisition successfully identified 7934 proteins,and 59 representative proteins exhibiting fold-change values higher than 1.5 were randomly selected.From the results of the proteomic and biochemical analyses,we found that putrescine significantly inhibited ethylene biosynthesis and phosphorus uptake in rice roots,increased pectin methylation,decreased pectin content and apoplastic Al deposit!on in rice roots.Putrescine also alleviated Al toxicity by repairing damaged DNA and increasing the proteins involved in maintaining plasma membra ne integrity and normal cell proliferation.These fin dings improve our understanding of how putrescine affects the rice response to Al toxicity,which will facilitate further studies on environmental protection,crop safety,in novations in rice performance and real-world producti on.

Putrescine Enhances Seed Germination Tolerance to Heat Stress in Arabidopsis thaliana

Putrescine(Put)as the compound of plant polyamines is catalyzed by arginine decarboxylase(ADC),which is encoded by two members,ADC1 and ADC2 in Arabidopsis,and ADC2 is mainly responsible for Put biosynthesis.Accumulated evidence demonstrates the important function of Put in plant growth and development,but its role in regulating seed germination under high temperature(HT)has not been reported yet.SOMNUS(SOM)is the negative regulator for seed germination thermoinhibition by altering downstream gibberellin(GA)and abscisic acid(ABA)metabolism.In this study,we found exogenous application of Put obviously alleviated the inhibition effect of HT on seed germination.Whereas pharmacological inhibition of endogenous Put level reduced seed germination under HT.Consistently,HT induced the rapid accumulation of Put level,and the adc2 mutant defi-ciency in Put biosynthesis also showed more sensitivity to HT stress.Furthermore,we found that the Put signal suppressed the expression of SOM and changed the transcriptional patterns of genes associated with GA/ABA metabolism.Genetic analysis also revealed SOM was epistatic to ADC2 to alter GA/ABA metabolism.Collectively,our finding reveals the novel function of Put in controlling seed germination under HT through SOM,and provides the possibility to develop Put as the innovational regulator for uniform seed germination under HT stress.

Effect of Putrescine on Low-Temperature Acclimation in Chlamydomonas reinhardtii

Putrescine is reported to be necessary for cold acclimation under low-temperature stress.In this study,the effect of low-temperature on some physiological and biochemical parameters has been investigated using the green algae Chlamydomonas reinhardtii.The lipid peroxidation rate,amount of Rubisco protein,activities of antioxidant enzymes and gene expression of polyamine biosynthesis(odc2,and spd1),heat shock proteins(hsp70c,hsp90a,and hsp90c),and PSII repair mechanisms(psba,rep27,and tba1)were determined to understand the low-temperature response.Exogenous putrescine application significantly increased Rubisco protein concentration and catalase enzyme activities under low-temperature stress.Moreover,real-time RT-PCR results and gene expression analysis showed that polyamine metabolism induced gene expression at low-temperatures in the first 24 h.In the same way,the gene expression of heat shock proteins(hsp70c,hsp90a,and hsp90c)decreased under low-temperature treatment for 72 h;however,application of putrescine enhanced the gene expression in the first 24 h.The results obtained indicated that molecular response in the first 24 h could be important for cold acclimation.The psba and tba1 expressions were reduced under low-temperatures depending on the exposure time.In contrast,the exogenous putrescine enhanced the expression level of the psba response to low-temperature at 24 and 72 h.The results obtained in this study indicate that putrescine could play a role in the PS II repair mechanisms under low-temperature stress.

Influence of Putrescine on Enzymes of Ammonium Assimilation in Maize Seedling

The effect of different concentrations of putrescine on biochemical changes in root and shoot of six days old maize seedlings in terms of enzymes of ammonium assimilation were examined. The results revealed that glutamate dehydrogenase (GDH) activity was enhanced at lower concentration of putrescine but at higher concentration, the activity of this enzyme was declined. Glutamine synthetase (GS) activity decreased with increase in concentration of putrescine and it was highest at 1000 μm concentration. Howe ver, glutamate synthase (GOGAT) activity increased with increase in concentration of putrescine upto 100 μm in root and upto 50 μm in shoot and further increase in concentration resulted in decline of enzymatic activity. Protein and total nitrogen content increased upto 10 μm concentration of putrescine and it decreased further with increase in concentration both in root and shoot of maize seedling.

Cadaverine and putrescine exposure influence carbon and nitrogen cycling genes in water and sediment of the Yellow River

The response patterns of microbial functional genes involved in biogeochemical cycles to cadaver decay is a central topic of recent environmental sciences.However,the response mechanisms and pathways of the functional genes associated with the carbon(C)and nitrogen(N)cycling to cadaveric substances such as cadaverine and putrescine remain unclear.This study explored the variation of functional genes associated with C fixation,C degradation and N cycling and their influencing factors under cadaverine,putrescine and mixed treatments.Our results showed only putrescine significantly increased the alpha diversity of C fixation genes,while reducing the alpha diversity of N cycling genes in sediment.For the C cycling,the mixed treatment significantly decreased the total abundance of reductive acetyl-CoA pathway genes(i.e.,acsB and acsE)and lig gene linked to lignin degradation in water,while only significantly increasing the hydroxypropionate-hydroxybutylate cycle(i.e.,accA)gene abundance in sediment.For the N cycling,mixed treatment significantly decreased the abundance of the nitrification(i.e.,amoB),denitrification(i.e.,nirS3)genes in water and the assimilation pathway gene(i.e.,gdhA)in sediment.Environmental factors(i.e.,total carbon and total nitrogen)were all negatively associated with the genes of C and N cycling.Therefore,cadaverine and putrescine exposure may inhibit the pathway in C fixation and N cycling,while promoting C degradation.These findings can offer some new insight for the management of amine pollution caused by animal cadavers.

ART1 and putrescine contribute to rice aluminum resistance via OsMYB30 in cell wall modification

Cell wall is the first physical barrier to aluminum(Al)toxicity.Modification of cell wall properties to change its binding capacity to Al is one of the major strategies for plant Al resistance;nevertheless,how it is regulated in rice remains largely unknown.In this study,we show that exogenous application of putrescines(Put)could significantly restore the Al resistance of art1,a rice mutant lacking the central regulator Al RESISTANCE TRANSCRIPTION FACTOR 1(ART1),and reduce its Al accumulation particularly in the cell wall of root tips.Based on RNA-sequencing,yeast-onehybrid and electrophoresis mobility shift assays,we identified an R2R3 MYB transcription factor OsMYB30 as the novel target in both ART1-dependent and Put-promoted Al resistance.Furthermore,transient dual-luciferase assay showed that ART1 directly inhibited the expression of OsMYB30,and in turn repressed Os4CL5-dependent 4-coumaric acid accumulation,hence reducing the Al-binding capacity of cell wall and enhancing Al resistance.Additionally,Put repressed OsMYB30 expression by eliminating Alinduced H2O2accumulation,while exogenous H2O2promoted OsMYB30 expression.We concluded that ART1 confers Put-promoted Al resistance via repression of OsMYB30-regulated modification of cell wall properties in rice.

Serum biochemical parameters and amino acids metabolism are altered in piglets by early-weaning and proline and putrescine supplementations

The study was to investigate the effect of early-weaning stress and proline(Pro)and putrescine(Put)supplementations on serum biochemical parameters and amino acids(AA)metabolism in suckling and post-weaning pigs.Blood and small intestinal mucosa were harvested from suckling piglets at 1,7,14,and 21 d of age and piglets on d 1,3,5,and 7 after weaning at 14 d of age,as well as from piglets received oral administration of Pro and Put from 1 to 14 d old.In suckling piglets,the serum glucose,albumin and total cholesterol levels were increased(P<0.05)with increasing age,whereas the serum globulin,urea nitrogen(BUN),alkaline phosphatase(ALP)and aspartate aminotransferase(AST)levels were lowered(P<0.05).The concentrations of most serum AA and the AA transporters related gene expressions were highest in 7-d-old piglets(P<0.05),whereas the phosphorylation status of the mammalian target of the rapamycin(mTOR)signaling pathway in the small intestine increased in piglets from 1 to 21 d old(P<0.05).Weaning at 14 d old increased(P<0.05)the BUN and triglycerides levels in serum,as well as jejunal solute carrier family 7 member 6(SLC7A6),ileal SLC36A1 and SLC1A1 mRNA abundances at d 1 or 3 post-weaning.Weaning also inhibited(P<0.05)the phosphorylation levels of mTOR and its down-stream ribosomal protein S6 kinase 1(S6K1)and 4E-binding protein-1(4EBP1)in the small intestine of weanling pigs.Oral administration of Put and Pro decreased(P<0.05)serum ALP levels and increased(P<0.05)intestinal SLC36A1 and SLC1A1 mRNA abundances and mTOR pathway phosphorylation levels in post-weaning pigs.Pro but not Put treatment enhanced(P<0.05)serum Pro,arginine(Arg)and glutamine(Gln)concentrations of weaning-pigs.These findings indicated that early-weaning dramati-cally altered the biochemical blood metabolites,AA profile and intestinal mTOR pathway activity,and Pro and Put supplementations improved the AA metabolism and transportation as well as activated the intestinal mTOR pathway in weanling-pigs.Our study has an important implication for the broad application of Pro and Put in the weaning transition of piglets.

Interacted Effect of Arbuscular Mycorrhizal Fungi and Polyamines on Root System Architecture of Citrus Seedlings

Either arbuscular mycorrhizal fungi （AMF） or polyamines （PAs） may change root system architecture （RSA） of plants, whereas the interaction of AMF and PAs on RSA remains unclear. In the present study, we studied the interaction between AMF （Paraglomus occultum） and exogenous PAs, including putrescine （Put）, spermidine （Spd） and spermine （Spin） on mycorrhizal development of different parts of root system, plant growth, RSA and carbohydrate concentrations of 6-m-old citrus （Citrus tangerine Hort. ex Tanaka） seedlings. After 14 wk of PAs application, PA-treated mycorrhizal seedlings exhibited better mycorrhizal colonization and numbers of vesicles, arbuscules, and entry points, and the best mycorrhizal status of taproot, first-, second-, and third-order lateral roots was respectively found in mycorrhizal seedlings supplied with Put, Spd and Spm, suggesting that PAs might act as a regulated factor of mycorrhizal development through transformation of root sucrose more into glucose for sustaining mycorrhizal development. AMF usually notably increases RSA traits （taproot length, total length, average diameter, projected area, surface area, volume, and number of first-, second-, and third-order lateral roots） of only PA-treated seedlings. Among the three PA species, greater positive effects on RSA change and plant biomass increment of the seedlings generally rank as Spd〉Spm〉Put, irrespective of whether or not AMF colonization. PAs significantly changed the RSA traits in mycorrhizal but not in non-mycorrhizal seedlings. It suggests that the application of PAs （especially Spd） to AMF plants would optimize RSA of citrus seedlings, thus increasing plant growth （shoot and root dry weight）.

Synthesis of New Benzoimidazole Derivatives

The reactions of cryptotanshinone and tanshinone IIA with cadaverine and putrescine were investigated. Six new compounds, four with imidazole functional groups and two with oxazole groups, were obtained. The possible reaction mechanism was proposed.

High Performance Liquid Chromatography Separation and Simultaneous Fluorometric Detection of Trace Amount of Polyamines in Ce

A highly sensitive and precise method for the determination of the polyamines (including putrescine, spermidine and spermine) in cell culture media is described. The samples were concentrated by C18 column and the polyamines were converted to fluorometric derivatives with DNS-Cl. The polyamine derivatives were then completely separated by HPLC and determined by simultaneous fluorometric detection. The CV of intragroup and intergroup were 2. 49 %-4. 26% and 4. 29 %-5. 16 %, respectively. The rate of recovery was 103%- 99%. There was trace amount of polyamines detected by this method in the media of F12, 8900, RPMI1640, DMEM and M199 even without incubation with cells. So this method can be used for detecting the changes of polyamines in a medium before and after incubation with cells. It is helpful for the researches on the regulation of cell proliferation by polyamines.

Changes in the Ovarian Polyamine Content and Seed Set Efficiency of Cotton by the Plant Growth Regulator BM86

Naturally occurring polyamines in plants have been implicated in floral initiation, and fruit development. The plant growth regulator BM86 was formulated to stimulate seed production and fruit growth by enhancing polyamine synthe-sis. The objectives of this study were to determine the effect of BM86 on free polyamine content in cotton (Gos-sypium hirsutum L.) ovaries and on seed set efficiency, and also to investigate genotypic differences in ovarian polyamine content. BM86 was applied at the first flower stage and two weeks later at 2.34 mL/ha. This study showed that application of BM86 had a significant positive effect on ovarian polyamine content of cotton. Putrescine and sper-midine one week after the 1st BM86 application and putrescine two week after the 1st BM86 application, were significantly increased compared to the untreated control. Higher seed set efficiency with the BM86 application was observed when the total number of seeds was used for the calculation. However, when the number of harvestable seeds was used to calculate seed set efficiency BM86 had no significant effect. In addition, application of BM86 did not significantly alter seedcotton yield of the crop. Only small differences in ovarian polyamine content were detected among the genotypes tested, possibly due to the narrow genetic pool of the commercial cotton genotypes used. Application of BM86 can significantly increase cotton seed number by enhancing polyamines biosynthesis, but further research is needed to determine how to capitalize on the increased potential number of harvestable seeds.

RELATION OF POLYAMINE TITER TO THYMUS DEGENERATION IN MICE

There is a growing body of evidence in recent years that aging is closely related to the decline in the function of immune system, particularly the cellular immune system of various organisms. Because of the extraordinary role played by the thymus as a central lymph organ, the investigation on the interrelation between thymus degeneration and aging has naturally received particular attention.

Metagenomes of polyamine-transforming bacterioplankton along a nearshore-open ocean transect

Short-chained aliphatic polyamines (PAs) have recently been recognized as an important carbon, nitrogen, and/or energy source for marine bacterioplankton. To study the genes and taxa involved in the transformations of different PA compounds and their potential variations among marine systems, we collected surface bacterioplankton from nearshore, offshore, and open ocean stations in the Gulf of Mexico and examined their metagenomic responses to additions of single PA model compounds (putrescine, spermidine, or spermine). Genes affiliated with PA uptake and all three known PA degradation pathways, i.e., transamination, γ-glutamylation, and spermidine cleavage, were significantly enriched in most PA-treated metagenomes. In addition, identified PA-transforming taxa were mostly the alpha and gamma classes of Proteobacteria, with less important contributions from members of Betaproteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Planctomycetes. These findings suggest that PA transformations are ubiquitous, have diverse pathways, and are carried out by a broad range of the bacterioplankton taxa in the Gulf of Mexico. Identified PA-transforming bacterial genes and taxa were different among nearshore, offshore, and open ocean sites, but were little different among individual compound-amended metagenomes at any specific site. These observations further indicate that PA-transforming taxa and genes are site-specific and with high similarities among PA compounds.