Polyamines Putrescine Research Articles

Polyamines in cancer: integrating organismal metabolism and antitumour immunity.

The natural mammalian polyamines putrescine, spermidine and spermine are essential for both normal and neoplastic cell function and replication. Dysregulation of metabolism of polyamines and their requirements is common in many cancers. Both clinical and experimental depletion of polyamines have demonstrated their metabolism to be a rational target for therapy; however, the mechanisms through which polyamines can establish a tumour-permissive microenvironment are only now emerging. Recent data indicate that polyamines can play a major role in regulating the antitumour immune response, thus likely contributing to the existence of immunologically 'cold' tumours that do not respond to immune checkpoint blockade. Additionally, the interplay between the microbiota and associated tissues creates a tumour microenvironment in which polyamine metabolism, content and function can all be dramatically altered on the basis of microbiota composition, dietary polyamine availability and tissue response to its surrounding microenvironment. The goal of this Perspective is to introduce the reader to the many ways in which polyamines, polyamine metabolism, the microbiota and the diet interconnect to establish a tumour microenvironment that facilitates the initiation and progression of cancer. It also details ways in which polyamine metabolism and function can be successfully targeted for therapeutic benefit, including specifically enhancing the antitumour immune response.

Pilot Study on Acute Effects of Pharmacological Intraperitoneal L-Homoarginine on Homeostasis of Lysine and Other Amino Acids in a Rat Model of Isoprenaline-Induced Takotsubo Cardiomyopathy.

L-Arginine:glycine amidinotransferase (AGAT) catalyzes the formation of L-homoarginine (hArg) and L-ornithine (Orn) from L-arginine (Arg) and L-lysine (Lys): Arg + Lys ↔ hArg + Orn; equilibrium constant KhArg. AGAT also catalyzes the formation of guanidinoacetate (GAA) and Orn from Arg and glycine (Gly): Arg + Gly ↔ GAA + Orn; equilibrium constant KGAA. In humans, pharmacological hArg is metabolized to Lys. Low circulating and low excretory concentrations of hArg are associated with worse outcomes and mortality in the renal and cardiovascular systems. The metabolism and pharmacology of hArg have been little investigated. In the present study, we investigated the effects of pharmacological hArg (i.p., 0, 20, 220, 440 mg/kg at time point 0 min) on amino acids homeostasis in a rat model of isoprenaline-induced takotsubo cardiomyopathy (i.p., 50 mg/kg at time point 15 min). We measured by gas chromatography-mass spectrometry free and proteinic amino acids, as well as the polyamines putrescine and spermidine in the heart, lung, kidney, and liver of ten rats sacrificed at various time points (range, 0 to 126 min). hArg administration resulted in multiple changes in the tissue contents of several free and proteinic amino acids, as well as in the putrescine-spermidine molar ratio, an indicator of polyamines catabolism. Our results suggest that Lys and Arg are major metabolites of pharmacological hArg. Kidneys and heart seem to play a major metabolic role for hArg. Circulating Lys does not change over time, yet there is a considerable interchange of free Lys between organs, notably kidney and heart, during the presence of isoprenaline in the rats (time range, 15 to 90 min). Antidromic changes were observed for KhArg and KGAA, notably in the heart in this time window. Our study shows for the first time that free hArg and sarcosine (N-methylglycine) are positively associated with each other. The acute effects of high-dosed hArg administration and isoprenaline on various amino acids and on AGAT-catalyzed reaction in the heart, lung, kidney, and liver are detailed and discussed.

Role of Polyamines and Hypusine in β Cells and Diabetes Pathogenesis.

The polyamines—putrescine, spermidine, and spermine—are polycationic, low molecular weight amines with cellular functions primarily related to mRNA translation and cell proliferation. Polyamines partly exert their effects via the hypusine pathway, wherein the polyamine spermidine provides the aminobutyl moiety to allow posttranslational modification of the translation factor eIF5A with the rare amino acid hypusine (hydroxy putrescine lysine). The “hypusinated” eIF5A (eIF5Ahyp) is considered to be the active form of the translation factor necessary for the translation of mRNAs associated with stress and inflammation. Recently, it has been demonstrated that activity of the polyamines-hypusine circuit in insulin-producing islet β cells contributes to diabetes pathogenesis under conditions of inflammation. Elevated levels of polyamines are reported in both exocrine and endocrine cells of the pancreas, which may contribute to endoplasmic reticulum stress, oxidative stress, inflammatory response, and autophagy. In this review, we have summarized the existing research on polyamine-hypusine metabolism in the context of β-cell function and diabetes pathogenesis.

Exogenous Putrescine Increases Heat Tolerance in Tomato Seedlings by Regulating Chlorophyll Metabolism and Enhancing Antioxidant Defense Efficiency

Crops around the world are facing a diversity of environmental problems, of which high temperatures are proving to be the most serious threat to crops. Polyamine putrescine (Put) acts as a master growth regulator that contributes to optimal plant growth and development and increased stress tolerance. Here, the current study aimed to elucidate how Put functions in regulating chlorophyll (Chl) metabolism, oxidative stress, and antioxidant defense, as well as to characterize the expression of genes related to heat stress in tomato seedlings under such stress. The results revealed that Put treatment significantly attenuates heat-induced damage by promoting biomass production, increasing photosynthetic efficiency, and inhibiting excessive production of oxidative stress markers. Heat stress markedly decreased the Chl content in the tomato leaf and accelerated the leaf yellowing process. However, Put-treated tomato seedlings showed a higher Chl content, which could be associated with the functions of Put in elevating PBGD activity (Chl biosynthesis enzyme) and suppressing the activity of the Chl catabolic enzyme (Chlase and MDCase). Under high-temperature stress, the expression levels of the gene encoding factors involved in Chl biosynthesis and Chl catabolism were significantly down- and upregulated, respectively, and this trend was reversed in Put-treated heat-stressed seedlings. In addition, exogenous application of Put boosted the activity of antioxidant enzymes, along with the levels of expression of their encoding genes, only in plants that were heat stressed. Furthermore, the expression levels of heat-shock-related genes (HSP90, HSP70, and HsfA1) were elevated in Put-treated, high-temperature-stressed tomato seedlings. Taken together, our results indicate that Put treatment significantly increases the heat tolerance of tomato seedlings, by elevating Chl concentrations and suppressing Chl catabolic enzyme activity, modulating endogenous free PA content, increasing antioxidant defense efficiency, and upregulating the expression of heat-shock-related genes.

A Second Gamma-Glutamylpolyamine Synthetase, GlnA2, Is Involved in Polyamine Catabolism in Streptomyces coelicolor.

Streptomyces coelicolor is a soil bacterium living in a habitat with very changeable nutrient availability. This organism possesses a complex nitrogen metabolism and is able to utilize the polyamines putrescine, cadaverine, spermidine, and spermine and the monoamine ethanolamine. We demonstrated that GlnA2 (SCO2241) facilitates S. coelicolor to survive under high toxic polyamine concentrations. GlnA2 is a gamma-glutamylpolyamine synthetase, an enzyme catalyzing the first step in polyamine catabolism. The role of GlnA2 was confirmed in phenotypical studies with a glnA2 deletion mutant as well as in transcriptional and biochemical analyses. Among all GS-like enzymes in S. coelicolor, GlnA2 possesses the highest specificity towards short-chain polyamines (putrescine and cadaverine), while its functional homolog GlnA3 (SCO6962) prefers long-chain polyamines (spermidine and spermine) and GlnA4 (SCO1613) accepts only monoamines. The genome-wide RNAseq analysis in the presence of the polyamines putrescine, cadaverine, spermidine, or spermine revealed indication of the occurrence of different routes for polyamine catabolism in S. coelicolor involving GlnA2 and GlnA3. Furthermore, GlnA2 and GlnA3 are differently regulated. From our results, we can propose a complemented model of polyamine catabolism in S. coelicolor, which involves the gamma-glutamylation pathway as well as other alternative utilization pathways.

Expression of polyamines and its association with GnRH-I in the hypothalamus during aging in rodent model.

GnRH-I and GnIH are the key neuropeptides that regulate the hypothalamic-pituitary-gonadal axis in mammals during aging. Polyamines are important aliphatic amines that are expressed in the brain and show variation with aging. The present study demonstrates evidence of variation in the level of expression of polyamines, GnRH-I and GnIH in the hypothalamus of female mice during aging. The study also suggests regulatory effects of polyamines over expression of the hypothalamic GnRH-I. The study shows a significant positive correlation between polyamines, its associated factors and GnRH-I along with significant negative correlation between polyamines, its associated factors and GnIH. This is the first study to report the effect of polyamines along with lactate or TNF-α or both on GnRH-I expression in GT1-7 cell line. TNF-α and lactate significantly decreased hypothalamic GnRH-I mRNA expression in GT1-7 cells when treated for 24h. Polyamines (putrescine and agmatine) in contrast, significantly increased GnRH-I mRNA expression in GT1-7 cells when treated for 24h. Also, polyamines increased GnRH-I mRNA expression when treated in presence of TNF-α or lactate thereby suggesting its neuro-protective role.This study also found 3809 differentially expressed genes through RNA-seq done between the hypothalamic GT1-7 cells treated with putrescine only versus TNF-α and putrescine. The present study suggests for the first time that putrescine treatment to TNFα-primed GT1-7 cells upregulates GnRH-I expression via regulation of several pathways such as calcium ion pathway, estrogen signaling, clock genes as well as regulating other metabolic process like neuronal differentiation and neurulation.

Pharmacological targeting of polyamine and hypusine biosynthesis reduces tumour activity of endometrial cancer

Endometrial cancer (EC) is a common and deadly cancer in women and novel therapeutic approaches are urgently needed. Polyamines (putrescine, spermidine, spermine) are critical for mammalian cell proliferation and MYC coordinately regulates polyamine metabolism through ornithine decarboxylase (ODC). ODC is a MYC target gene and rate-limiting enzyme of polyamine biosynthesis and the FDA-approved anti-protozoan drug α-difluoromethylornithine (DFMO) inhibits ODC activity and induces polyamine depletion that leads to tumour growth arrest. Spermidine is required for the hypusine-dependent activation of eukaryotic translation initiation factors 5A1 (eIF5A1) and 5A2 (eIF5A2) and connects the MYC/ODC-induced deregulation of spermidine to eIF5A1/2 protein translation, which is increased during cancer cell proliferation. We show that eIF5A1 is significantly upregulated in EC cells compared to control cells (p=.000038) and that combined pharmacological targeting of ODC and eIF5A hypusination with cytostatic drugs DFMO and N1-guanyl-1,7-diaminoheptane (GC7), respectively, reduces eIF5A1 activation and synergistically induces apoptosis in EC cells. In vivo, DFMO/GC7 suppressed xenografted EC tumour growth in mice more potently than each drug alone compared to control (p=.002) and decreased putrescine (p=.045) and spermidine levels in tumour tissues. Our data suggest DFMO and GC7 combination therapy may be useful in the treatment or prevention of EC.

The effect of putrescine on the lysozyme activity and structure: Spectroscopic approaches and molecular dynamic simulation

The present research addressed the influence of polyamine (putrescine) on the compound as well as function of lysozyme; accordingly, UV- Visible, fluorescence spectroscopy and simulation method were applied to fulfill this goal. Lysozyme's structural variability was examined at various putrescine ‌concentrations; also, the putrescine binding to lysozyme was addressed using spectrofluorescence, circular dichroism (CD) and UV-Vis measurements. The obtained results indicated that with raising the putrescine concentration, the intrinsic quenching fluorescence of lysozyme was decreased based on the static mechanism. Analysis of thermodynamic parameters also indicated that van der Waals as well as hydrogen bond forces served a fundamental role in determining the resulting stability; this was in agreement with modeling studies. Measurement of UV absorption spectroscopy, fluorescence spectroscopy, and circular dichroism spectroscopy also demonstrated that lysozyme's second and tertiary structures were altered in a putrescine concentration-dependent manner. Putrescine inhibited lysozyme's enzymatic activity, displaying its affinity with the lysozyme's active site. Further, molecular simulation conducted revealed that putrescine could have spontaneous binding to lysozyme, changing its structure, thus further emphasizing the experimental results.

Climate change influences foliar nutrition and metabolism of red maple (Acer rubrum) trees in a northern hardwood forest

AbstractMean annual air temperatures are projected to increase, while the winter snowpack is expected to shrink in depth and duration for many mid‐ and high‐latitude temperate forest ecosystems over the next several decades. Together, these changes will lead to warmer growing season soil temperatures and an increased frequency of soil freeze–thaw cycles (FTCs) in winter. We took advantage of the Climate Change Across Seasons Experiment (CCASE) at the Hubbard Brook Experimental Forest in the White Mountains of New Hampshire, USA, to determine how these changes in soil temperature affect foliar nitrogen (N) and carbon metabolism of red maple (Acer rubrum) trees in 2015 and 2017. Earlier work from this study revealed a similar increase in foliar N concentrations with growing season soil warming, with or without the occurrence of soil FTCs in winter. However, these changes in soil warming could differentially affect the availability of cellular nutrients, concentrations of primary and secondary metabolites, and the rates of photosynthesis that are all responsive to climate change. We found that foliar concentrations of phosphorus (P), potassium (K), N, spermine (a polyamine), amino acids (alanine, histidine, and phenylalanine), chlorophyll, carotenoids, sucrose, and rates of photosynthesis increased with growing season soil warming. Despite similar concentrations of foliar N with soil warming with and without soil FTCs in winter, winter soil FTCs affected other foliar metabolic responses. The combination of growing season soil warming and winter soil FTCs led to increased concentrations of two polyamines (putrescine and spermine) and amino acids (alanine, proline, aspartic acid, γ‐aminobutyric acid, valine, leucine, and isoleucine). Treatment‐specific metabolic changes indicated that while responses to growing season warming were more connected to their role as growth modulators, soil warming + FTC treatment‐related effects revealed their dual role in growth and stress tolerance. Together, the results of this study demonstrate that growing season soil warming has multiple positive effects on foliar N and cellular metabolism in trees and that some of these foliar responses are further modified by the addition of stress from winter soil FTCs.

Progesterone and interferon tau regulate expression of polyamine enzymes during the ovine peri-implantation period†.

Progesterone (P4) and interferon tau (IFNT) are important for establishment and maintenance of pregnancy in ruminants. Agmatine and polyamines (putrescine, spermidine, and spermine) have important roles in the survival, growth, and development of mammalian conceptuses. This study tested the hypothesis that P4 and/or IFNT stimulate the expression of genes and proteins involved in the metabolism and transport of polyamines in the ovine endometrium. Rambouillet ewes (n = 24) were surgically fitted with intrauterine catheters on Day 7 of the estrous cycle. They received daily intramuscular injections of 50mg P4 in corn oil vehicle and/or 75-mg progesterone receptor antagonist (RU486) in corn oil vehicle from Days 8-15, and twice daily intrauterine injections (25μg/uterine horn/day) of either control serum proteins (CX) or IFNT from Days 11-15, resulting in four treatment groups: (i) P4 + CX; (ii) P4 + IFNT; (iii) RU486 + P4 + CX; or (iv) RU486 + P4 + IFNT. On Day 16, ewes were hysterectomized. The total amounts of arginine, citrulline, ornithine, agmatine, and putrescine in uterine flushings were affected (P < 0.05) by P4 and/or IFNT. P4 increased endometrial expression of SLC22A2 (P < 0.01) and SLC22A3 (P < 0.05) mRNAs. IFNT affected endometrial expression of MAT2B (P < 0.001), SAT1 (P < 0.01), and SMOX (P < 0.05) mRNAs, independent of P4. IFNT increased the abundance of SRM protein in uterine luminal (LE), superficial glandular (sGE), and glandular epithelia (GE), as well as MAT2B protein in uterine LE and sGE. These results indicate that P4 and IFNT act synergistically to regulate the expression of key genes required for cell-specific metabolism and transport of polyamines in the ovine endometrium during the peri-implantation period of pregnancy.

Critical Role of Astrocytic Polyamine and GABA Metabolism in Epileptogenesis.

Accumulating evidence indicate that astrocytes are essential players of the excitatory and inhibitory signaling during normal and epileptiform activity via uptake and release of gliotransmitters, ions, and other substances. Polyamines can be regarded as gliotransmitters since they are almost exclusively stored in astrocytes and can be released by various mechanisms. The polyamine putrescine (PUT) is utilized to synthesize GABA, which can also be released from astrocytes and provide tonic inhibition on neurons. The polyamine spermine (SPM), synthesized form PUT through spermidine (SPD), is known to unblock astrocytic Cx43 gap junction channels and therefore facilitate astrocytic synchronization. In addition, SPM released from astrocytes may also modulate neuronal NMDA, AMPA, and kainate receptors. As a consequence, astrocytic polyamines possess the capability to significantly modulate epileptiform activity. In this study, we investigated different steps in polyamine metabolism and coupled GABA release to assess their potential to control seizure generation and maintenance in two different epilepsy models: the low-[Mg2+] model of temporal lobe epilepsy in vitro and in the WAG/Rij rat model of absence epilepsy in vivo. We show that SPM is a gliotransmitter that is released from astrocytes and significantly contributes to network excitation. Importantly, we found that inhibition of SPD synthesis completely prevented seizure generation in WAG/Rij rats. We hypothesize that this antiepileptic effect is attributed to the subsequent enhancement of PUT to GABA conversion in astrocytes, leading to GABA release through GAT-2/3 transporters. This interpretation is supported by the observation that antiepileptic potential of the Food and Drug Administration (FDA)-approved drug levetiracetam can be diminished by specifically blocking astrocytic GAT-2/3 with SNAP-5114, suggesting that levetiracetam exerts its effect by increasing surface expression of GAT-2/3. Our findings conclusively suggest that the major pathway through which astrocytic polyamines contribute to epileptiform activity is the production of GABA. Modulation of astrocytic polyamine levels, therefore, may serve for a more effective antiepileptic drug development in the future.

Potential significance of aliphatic polyamines, α-synucleins and Helicobacter Pylori in diagnostics and prognosis of some malignant tumors.

Polyamines (putrescine, spermidine and spermine) as well as γ- and α-synucleins are currently the subject of a special comprehensive study due to their direct influence on the formation of neoplastic processes. This review article presents informative data on the role of aliphatic polyamines (putrescine, spermidine, spermine) in the induction of a number of oncological diseases with their localization in the digestive and urinary systems. Aspects related to the synthesis and aggregation of γ-synucleins into malignantly reborn cells have been discussed. The article provides information on the possible prevention of further growth of malignant cells, by inhibiting enzymatic processes responsible for the polyamine synthesis. In case of prolonged persistence of Helicobacter Pylori in the stomach, an enhanced synthesis of polyamines and cumulation in situ of oligo- and aggregated gamma synucleins occurs in mucous membrane cells. It is possible that all of the abovementioned factors in total directly and/or indirectly have a stimulating effect on the processes of the malignancy of gastric mucosal cells. The subject of special discussion was the latest literature data, according to which, under the conditions of Helicobacter Pylori persistence in the digestive tract organs, in the gastric mucosal cells an enhanced synthesis of putrescine occurs, high concentrations of which can have a co-carcinogenic effect on normally functioning target cells. From a qualitatively new point of view, the fact of cumulation in malignantly reborn cells of an aggregated gamma synuclein, which was found during Helicobacter Pylori infection, should be considered.

Efficient Plant Regeneration from Explants of Compact Oregano (&amp;lt;i&amp;gt;Origanum compactum&amp;lt;/i&amp;gt; Bentham)

Origanum compactum Benth., a species endemic to Morocco characterized by its biological activities, is overexploited because of its commercial value and threatened with extinction. Accordingly, measures for its conservation are needed. Micropropagation serves as a solution for the protection and the domestication of this species. In this investigation, we established a protocol for vegetative multiplication in vitro of Origanum compactum by the axillary bud technique. Six culture media (SH, SD, N30K, MS, MSm, B5) were tested to determine the most suitable mineral medium for growth and development of explants. Four cytokinins: Kinetin, Zeatin, BAP (6-Benzylaminopurine), 2ip (2-Isopentenyladenine) and three compounds with cytokinin activity: Adénine, 1,3-Diphenylurea (DPU) and Thidiazuron (TDZ) at five concentrations (0.44, 1.33, 2.22, 3.11, 4.44 μM) were tested on budding, growth, hyperhydria and rooting. Then three auxins, Indole-3-acetic acid (IAA), Naphthalene acetic acid (NAA) and Indole butyric acid (IBA) at four concentrations (1.14, 2.85, 4.56, 6.27 μM) in presence of 2.22 μM BAP were evaluated. The combination of Gibberellic acid (0.29, 1.5, 2.60, 2.89 μM GA3) and three polyamines (Putrescine, Spermidine, Spermine) at five concentrations (1.134, 3.402, 5.67, 7.938, 11.34 μM) with cytokinins and auxins were considered. Our results show that Margara medium is the most efficient and BAP at 2.22 μM is the best cytokinin for development of the aerial parts, with a regeneration rate of 88, 90%; rhizogenesis is successful with the combination of 6.27 μM IAA and 2.22 μM BAP. Moreover, the integration of 2.89 μM GA3 with 2.22 μM BAP and 6.27 μM IAA promotes vitroplant growth, bud and shoot multiplication and elongation of the aerial part. The addition of polyamines with 2.22 μM BAP and 6.27 μM IAA does not improve the root part, but Spermine at 5.67 μM promotes bud and shoot multiplication with a high percentage of regeneration, and spermidine at the same concentration gives long explants. Finally, plantlets with good root development were successfully acclimatized to natural conditions and served as a source to establish in vitro culture again.

Micropropagation and Acclimatization of Common Oregano (&amp;lt;i&amp;gt;Origanum vulgare&amp;lt;/i&amp;gt; L. Subsp. &amp;lt;i&amp;gt;vulgare&amp;lt;/i&amp;gt;) by Shoot Tip Culture

Origanum vulgare L. is a commercially valued species with remarkable biological properties. It is subject to over-exploitation practices that seriously threaten its sustainability for future generations. Thus, micropropagation serves as a tool for the protection and domestication of this species. In this study, we established an in vitro vegetative propagation protocol for Origanum vulgare. This is done through the axillary bud technique by carrying out various tests. Six culture media (MS, MSm, N30K, SD, SH and B5) were tested. Therefore, SD was chosen for the following experiments. Seven cytokinins (adenine (Ad), N6-(2-isopentenyl) (2ip), zeatin (Zeat), kinetin (Kin), benzyladenine (BAP), 1,3-diphenylurea (DPU) and thidiazuron (TDZ) at 5 concentrations (0.44, 1.33, 2.22, 3.11 and 4.44 μM/L) were evaluated. Thus, Kin at 3.11 μM allowed high regeneration of vitroplants, optimal elongation, total rooting of explants, maximum bud multiplication, and absence of hyperhydric explants. In fact, the integration of auxins (indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA)) into the culture medium and their combinations with 3.11 μM Kinetin contributed to the optimization of the root part. Thus, it was improved in particular in the case of 3.11 μM Kin and 6.27 μM IBA. Three polyamines (Putrescine, Spermidine and Spermine) at different concentrations (1.134, 3.402, 5.67, 7.938 and 11.34 μM/L) combined at 3.11 μM Kin and 6.27 μM IBA were tested. In fact, 1.304 μM putrescine was considered to be the most suitable for in vitro culture of explants, since it allowed optimal propagation of buds and roots, also a high rate of regeneration and rhizogenesis. GA3 at 1.15 μM combined with 3.11 μM Kin and 6.27 μM IBA permitted maximum bud multiplication. The acclimatization was carried out successfully using vitroplants showing good foliar and root development. Thus, three months after acclimatization, the seedlings were transferred into large pots under natural light and temperature conditions. Almost all acclimatized plants developed flowers in the first year between May and July.

Retraction: Physiological Responses of Pea Plants to Salinity and Gibberellic Acid

Pea is a seed legume. It is rich in cellulose fibre and protein. It is also a significant source of minerals and vitamins. In this paper, we set out to better characterize the physiological responses of Pisum sativum L. to the combined effects of NaCl, 100 mM and gibberellins (GA3). Our analysis revealed that NaCl caused a decrease in growth resulting in a reduction in root elongation, distribution and density, leaf number and leaf area, and a decrease in dry matter of roots and shoots. However, the contribution of GA3 in the salty environment induced an increase in these different parameters suggesting an improving effect of this hormone on growth of pea in presence of salt. NaCl also led to a disturbance of the photosynthetic machinery. Indeed, level of chlorophyll pigments (a and total) and photosynthetic activity were decreased compared to the control plants. However, the exogenous supply of GA3 restored this decrease in net CO2 assimilation, but not in chlorophyll content. Additional analyses were performed on the effect of salinity/GA3 interaction on osmolytes (soluble sugars and starch). Our results showed an increase in sugars and a decrease in starch in the presence of 100 mM NaCl. The salt-GA3 combination resulted in compensation of soluble sugar contents but not of starch contents, suggesting a beneficial effect of GA3 under saline stress conditions. Level of three main polyamines putrescine, spermidine, and spermine increased significantly in all organs of salt-treated plants.

A unique aromatic cluster near the active site of H.pylori CPA is essential for catalytic function

Polyamines are essential for cell growth and proliferation. In plants and many bacteria, including Helicobacter pylori, the parent polyamine putrescine is only produced through the metabolism of N-carbamoylputrescine by N-carbamoylputrescine amidase (CPA). Thus, CPA is a crucial intermediate enzyme. Moreover, the absence of CPA in humans makes its presence in H. pylori a potential target for the development of new therapeutics against this pathogen. Despite this enzyme's presence in plants and bacteria, its function is not completely explored. Using structure-guided biochemical and biophysical studies on H. pylori CPA, we discovered an aromatic cluster containing four conserved tryptophans near the catalytic site and elucidated its role. Mutational studies revealed that they are individually vital to enzyme function. Unlike wild-type, which forms a hexamer, the Trp to Ala mutants only formed dimers. Interestingly, two other conserved residues, Gln155 and Asp278, interact with the tryptophan cluster and perform similar roles. Our results indicate that aromatic-aromatic and H-bonding contacts between the residues (Trp156-Trp273, Trp196-Gln155, and Trp153-Asp278) play a crucial role in stimulating activity through hexamer formation. Additionally, Trp156 is essential to generating a catalytically efficient hexamer. These results suggest dual roles for the tryptophans; in hexamer formation and in generating its functionally active form, thereby providing a mechanistic understanding into the role of the cluster. We also elucidated the catalytic roles of Glu43, Lys115, and Cys152, which are present at the active site. Our findings highlight, for the first time, the importance of a tryptophan cluster in H. pylori CPA that can be exploited to design therapeutic inhibitors.

Polyamine Putrescine Regulates Oxidative Stress and Autophagy of Hemocytes Induced by Lipopolysaccharides in Pearl Oyster Pinctada fucata martensii.

The polyamine putrescine (Put) is a ubiquitous small cationic amine. It plays an essential role in controlling the innate immune response. However, little is known about its function in mollusks. In this study, the Put content was observed to increase in the serum of pearl oyster Pinctada fucata martensii after 6 and 24 h of lipopolysaccharide (LPS) stimulation. Activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) increased, and nitric oxide synthase was downregulated in the Put group (i.e., combined treatment with Put and LPS) compared with that in the LPS group (i.e., combined treatment with phosphate-buffered saline and LPS). Furthermore, activities of alkaline phosphatase and acid phosphatase were inhibited after 6 h of LPS stimulation. The expression levels of the nuclear factor kappa B, IκB kinase, Janus kinase, and signal transducer and activator of transcription proteins genes were all significantly suppressed at 12 and 24 h in the Put group. Pseudomonas aeruginosa and Bacillus subtilis grew better after being incubated with the serum from the Put group than that from the LPS group. Additionally, the Put treatment remarkably inhibited the autophagy of hemocytes mediated by the AMP-activated protein kinase-mammalian target of rapamycin-Beclin-1 pathway. This study demonstrated that Put can effectively inhibit the inflammatory response induced by LPS in pearl oysters. These results provide useful information for further exploration of the immunoregulatory functions of polyamines in bivalves and contribute to the development of immunosuppressive agents.

Investigation of the polyamine biosynthetic and transport capability of Streptococcus agalactiae: the non-essential PotABCD transporter.

Polyamines constitute a group of organic polycations positively charged at physiological pH. They are involved in a large variety of biological processes, including the protection against physiological stress. In this study, we show that the genome of Streptococcus agalactiae , a commensal bacterium of the intestine and the vagina and one of the most common agents responsible of neonate infections, does not encode proteins homologous to the specific enzymes involved in the known polyamine synthetic pathways. This lack of biosynthetic capability was verified experimentally by TLC analysis of the intracellular content of S. agalactiae grown in the absence of polyamines. However, similar analyses showed that the polyamines spermidine, spermine and putrescine can be imported from the growth media into the bacteria. We found that all strains of S. agalactiae possess the genes encoding the polyamine ABC transporter PotABCD. We demonstrated that these genes form an operon with folK, a gene involved in folate biosynthesis, murB, a gene involved in peptidoglycan biosynthesis, and with clc, a gene encoding a Cl−/H+ antiporter involved in resistance to acid stress in Escherichia coli . Transcription of the potABCD operon is induced by peroxide-induced oxidative stress but not by acidic stress. Spermidine and spermine were found to be inducers of potABCD transcription at pH 7.4 whereas putrescine induces this expression only during peroxide-induced oxidative stress. Using a deletion mutant of potABCD, we were nevertheless unable to associate phenotypic traits to the PotABCD transporter, probably due to the existence of one or more as yet identified transporters with a redundant action.

Polyamine synthesis from arginine and proline in tissues of developing chickens.

Polyamines (putrescine, spermidine, and spermine) are synthesized primarily from ornithine via ornithine decarboxylase (ODC) in mammals. Although avian tissues contain ODC activity, little is known about intracellular sources of ornithine for their polyamine synthesis. This study tested the hypothesis that arginase and proline oxidase contribute to polyamine synthesis in chickens. Kidney, jejunum, leg muscle, and liver from 0-, 7-, 14- and 21-day-old broiler chickens were assayed for the activities of arginase, proline oxidase (POX), ornithine aminotransferase (OAT), and ornithine decarboxylase (ODC). Kidney slices were also used to determine 14C-polyamine synthesis from [U-14C]arginine and [U-14C]proline. Furthermore, these tissues and plasma were analyzed for polyamines. Results indicate that all tissues contained OAT (mitochondrial) and ODC (cytosolic) activities, but arginase and POX activities were only detected in the mitochondria of chicken kidneys. Renal POX and arginase activities were greater at 7days of age compared to newly hatched birds, and declined by Day 14. Renal arginase activity was greater at 21 days compared to 14days of age, but there was no change in renal POX activity during that same period. Concentrations of polyamines in the kidneys and plasma were greater on Day 7 compared to Day 0 and decreased thereafter on Days 14 and 21. Kidney slices readily converted arginine and proline into polyamines, with peak rates being on Day 7. Concentrations of putrescine, spermidine and spermine in the plasma of chickens were about 20- to 100-fold greater than those in mammals. Our results indicate that polyamines are synthesized from arginine and proline in avian kidneys. Unlike mammals, polyamines released from the kidneys are likely the major source of polyamines in the blood and other extra-renal tissues in chickens.

Discovery of ancestral L-ornithine and L-lysine decarboxylases reveals parallel, pseudoconvergent evolution of polyamine biosynthesis

Polyamines are fundamental molecules of life, and their deep evolutionary history is reflected in extensive biosynthetic diversification. The polyamines putrescine, agmatine, and cadaverine are produced by pyridoxal 5′-phosphate-dependent L-ornithine, L-arginine, and L-lysine decarboxylases (ODC, ADC, LDC), respectively, from both the alanine racemase (AR) and aspartate aminotransferase (AAT) folds. Two homologous forms of AAT-fold decarboxylase are present in bacteria: an ancestral form and a derived, acid-inducible extended form containing an N-terminal fusion to the receiver-like domain of a bacterial response regulator. Only ADC was known from the ancestral form and limited to the Firmicutes phylum, whereas extended forms of ADC, ODC, and LDC are present in Proteobacteria and Firmicutes. Here, we report the discovery of ancestral form ODC, LDC, and bifunctional O/LDC and extend the phylogenetic diversity of functionally characterized ancestral ADC, ODC, and LDC to include phyla Fusobacteria, Caldiserica, Nitrospirae, and Euryarchaeota. Using purified recombinant enzymes, we show that these ancestral forms have a nascent ability to decarboxylate kinetically less preferred amino acid substrates with low efficiency, and that product inhibition primarily affects preferred substrates. We also note a correlation between the presence of ancestral ODC and ornithine/arginine auxotrophy and link this with a known symbiotic dependence on exogenous ornithine produced by species using the arginine deiminase system. Finally, we show that ADC, ODC, and LDC activities emerged independently, in parallel, in the homologous AAT-fold ancestral and extended forms. The emergence of the same ODC, ADC, and LDC activities in the nonhomologous AR-fold suggests that polyamine biosynthesis may be inevitable.