OBJECTIVES: To observe the therapeutic effect of spermine in neonatal mouse models of severe hand, foot and mouth disease (HFMD) caused by enterovirus 71 (EV71) infection and explore its therapeutic mechanism in light of regulation of macrophage GBP5/NLRP3 inflammasome pathway. METHODS: Neonatal BALB/c mice (3-5 days old) were divided into control group, EV71 infection group and Spermine treatment group. The mice in the latter two groups received an intraperitoneal injection of 50 μL EV71 suspension (1×10⁶ TCID50 of EV71), followed 3 days later by intraperitoneal injection of 50 μL PBS or 100 μmol/L spermine. GBP5, NLRP3, CXCL10, and TNFSF10 expressions in heart, liver, lung and kidney tissues of the mice were detected using Western blotting and qPCR, and tissue pathologies and macrophage infiltration were assessed with HE staining and immunohistochemistry. In cultured THP-1 and RAW264.7 cells, the effects of EV71 infection, GBP5 siRNA transfection and treatment with spermine or eflornithine on GBP5, NLRP3, CXCL10, and TNFSF10 mRNA expressions were investigated using qPCR. RESULTS: In the neonatal mice, EV71 infection resulted in multiple organ damage, macrophage infiltration and activation of the GBP5/NLRP3 pathway, and spermine treatment significantly improved tissue injuries, reduced macrophage infiltration, and down-regulated the expressions of GBP5, NLRP3 and the inflammatory factors in the infected mice. In THP-1 and RAW264.7 cells, EV71 infection caused significant upregulation of GBP5, NLRP3, CXCL10, and TNFSF10 expressions, which were obviously lowered by spermine treatment. In THP-1 cells, treatment with eflornithine significantly suppressed the reduction of GBP5, NLRP3, CXCL10, and TNFSF10 expressions induced by GBP5 siRNA transfection. CONCLUSIONS Spermine suppressed EV71 infection-induced inflammatory responses by inhibiting GBP5-mediated NLRP3 inflammasome activation, suggesting a new strategy for treatment of severe HFMD.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein ; Mice ; Macrophages/metabolism* ; Enterovirus A, Human ; Mice, Inbred BALB C ; Inflammasomes/metabolism* ; Spermine/therapeutic use* ; Animals, Newborn ; Humans ; Enterovirus Infections ; Hand, Foot and Mouth Disease/drug therapy* ; RAW 264.7 Cells ; Chemokine CXCL10/metabolism*

1, 5-diaminopentane, also known as cadaverine, is an important raw material for the production of biopolyamide. It can be polymerized with dicarboxylic acid to produce biopolyamide PA5X whose performances are comparable to that of the petroleum-based polyamide materials. Notably, biopolyamide uses renewable resources such as starch, cellulose and vegetable oil as substrate. The production process does not cause pollution to the environment, which is in line with the green and sustainable development strategy. The biosynthesis of 1, 5-diaminopentane mainly includes two methods: the de novo microbial synthesis and the whole cell catalysis. Lysine decarboxylase as the key enzyme for 1, 5-diaminopentane production, mainly includes an inducible lysine decarboxylase CadA and a constituent lysine decarboxylase LdcC. Lysine decarboxylase is a folded type Ⅰ pyridoxal-5' phosphate (PLP) dependent enzyme, which displays low activity and unstable structure, and is susceptible to deactivation by environmental factors in practical applications. Therefore, improving the catalytic activity and stability of lysine decarboxylase has become a research focus in this field, and molecular engineering and immobilization are the mainly approaches. Here, the mechanism, molecular engineering and immobilization strategies of lysine decarboxylase were reviewed, and the further strategies for improving its activity and stability were also prospected, with the aim to achieve efficient production of 1, 5-diaminopentane.
Escherichia coli/metabolism* ; Carboxy-Lyases/metabolism* ; Catalysis ; Cadaverine/metabolism*

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition mechanisms.
Bridged-Ring Compounds ; Imidazoles ; Ornithine ; Ornithine Decarboxylase ; Ornithine Decarboxylase Inhibitors ; Putrescine

Some enzymes belonging to the multicopper oxidase (MCO) family can degrade the hazardous biogenic amine (BA) present in food. However, the oxidation of MCO in the process of degrading BAs may reduce its activity and stability, resulting in decreased catalytic efficiency. In this work, an MCO from Lactobacillus fermentum (MCOF) was fused with a Bacillus subtilis catalase (CAT) using different strategies and the fusion enzymes were respectively expressed in Escherichia coli BL21(DE3). The tolerance of eight fused MCOFs to H2O2 increased by 51%-68%, and the stability of CAT&MCOF increased by 17%, compared to the wild type MCOF. Using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate, the substrate affinity (Km), the catalytic efficiency (kcat/Km) and the molar specific activity of CAT&MCOF increased by 1.0-fold, 1.7-fold and 1.2-fold than those of MCOF, respectively. The stability of CAT&MCOF under acidic conditions (pH 2.5-4.5) and moderate temperatures (35-55 °C) also improved. Moreover, the degradation rates of putrescine, cadaverine and histamine catalyzed by CAT&MCOF reached 31.7%, 36.0% and 57.8%, respectively, which increased by 132.5%, 45.7% and 38.9% compared to that of MCOF. The improvement on the stability and catalytic efficiency of MCOF by fusion expression with CAT provides a good example for improving the applicability of enzymes through molecular modifications.
Biogenic Amines ; Cadaverine ; Catalase/genetics* ; Escherichia coli/genetics* ; Hydrogen Peroxide

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.
Amino Acids ; Binding Sites ; Calcium ; Cytosol ; Glutamic Acid ; Myocytes, Smooth Muscle ; Permeability ; Polyamines ; Receptors, Muscarinic ; Spermine ; Transient Receptor Potential Channels

Despite considerable efforts to prevent and treat cardiovascular disease (CVD), it has become the leading cause of death worldwide. Cardiac mitochondria are crucial cell organelles responsible for creating energy-rich ATP and mitochondrial dysfunction is the root cause for developing heart failure. Therefore, maintenance of mitochondrial quality control (MQC) is an essential process for cardiovascular homeostasis and cardiac health. In this review, we describe the major mechanisms of MQC system, such as mitochondrial unfolded protein response and mitophagy. Moreover, we describe the results of MQC failure in cardiac mitochondria. Furthermore, we discuss the prospects of 2 drug candidates, urolithin A and spermidine, for restoring mitochondrial homeostasis to treat CVD.
Adenosine Triphosphate ; Cardiovascular Diseases ; Cause of Death ; Heart Failure ; Heart ; Homeostasis ; Mitochondria ; Mitochondrial Degradation ; Organelles ; Quality Control ; Spermidine ; Unfolded Protein Response

Biogenic amines (BAs) are low molecular weight organic compounds that present in fermented foods. Large amount of ingested biogenic amines can cause allergy or significant symptoms. Reduction of BAs by enzymatic reaction in fermented foods is one of the most efficient methods for removal of biohazard compounds and assurance food safety. In this study, the multicopper oxidase (MCO) gene in the genome of Lactobacillus fermentum was successfully cloned in Escherichia coli BL21 and expressed at 484 U/L. The recombinant MCO was purified by the immobilized metal affinity chromatography method. The optimal reaction temperature and pH for this enzyme was detected to be 50 °C and 3.5. The Km and Vmax values of the recombinant MCO was determined to be 1.30 mmol/L and 7.67×10⁻² mmol/(L·min). Moreover, this MCO dramatically degrades histamine and tyramine by 51.6% and 40.9%, and can degrade other BAs including tryptamine, phenylethylamine, putrescine, cadaverine and spermidine, and was found to be tolerant to 18% (W/V) NaCl. The recombinant MCO is also capable of degrading BAs in soy sauce. The degradation rate of total BAs in soy sauce reaches 10.6% though a relatively low level of enzyme (500 U/L) is used. Multicopper oxidase has the potential to degrade biogenic amines in fermented foods, which lays a foundation for the further application of this kind of food enzymes.
Biogenic Amines ; Cadaverine ; Escherichia coli ; Lactobacillus fermentum ; Oxidoreductases

Spermidine is a naturally occurring polyamine compound that has recently emerged with anti-aging properties and suppresses inflammation and oxidation. However, its mechanisms of action on anti-inflammatory and antioxidant effects have not been fully elucidated. In this study, the potential of spermidine for reducing pro-inflammatory and oxidative effects in lipopolysaccharide (LPS)-stimulated macrophages and zebrafish was explored. Our data indicate that spermidine significantly inhibited the production of pro-inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2), and cytokines including tumor necrosis factor-α and interleukin-1β in RAW 264.7 macrophages without any significant cytotoxicity. The protective effects of spermidine accompanied by a marked suppression in their regulatory gene expression at the transcription levels. Spermidine also attenuated the nuclear translocation of NF-κB p65 subunit and reduced LPS-induced intracellular accumulation of reactive oxygen species (ROS) in RAW 264.7 macrophages. Moreover, spermidine prevented the LPS-induced NO production and ROS accumulation in zebrafish larvae and was found to be associated with a diminished recruitment of neutrophils and macrophages. Although more work is needed to fully understand the critical role of spermidine on the inhibition of inflammation-associated migration of immune cells, our findings clearly demonstrate that spermidine may be a potential therapeutic intervention for the treatment of inflammatory and oxidative disorders.
Antioxidants ; Cytokines ; Dinoprostone ; Genes, Regulator ; Inflammation* ; Larva ; Macrophages* ; Necrosis ; Neutrophils ; Nitric Oxide ; Oxidative Stress* ; Reactive Oxygen Species ; Spermidine* ; Zebrafish*

The hallmark of cisplatin-induced acute kidney injury is the necrotic cell death in the kidney proximal tubules. However, an effective approach to limit cisplatin nephrotoxicity remains unknown. Spermidine is a polyamine that protects against oxidative stress and necrosis in aged yeasts, and the present study found that exogenous spermidine markedly attenuated tubular necrosis and kidney dysfunction, but not apoptosis, during cisplatin nephrotoxicity. In addition, exogenous spermidine potently inhibited oxidative/nitrative DNA damage, poly(ADP-ribose) polymerase 1 (PARP1) activation and ATP depletion after cisplatin injection. Conversely, inhibition of ornithine decarboxylase (ODC) via siRNA transfection in vivo significantly increased DNA damage, PARP1 activation and ATP depletion, resulting in acceleration of tubular necrosis and kidney dysfunction. Finally, exogenous spermidine removed severe cisplatin injury induced by ODC inhibition. In conclusion, these data suggest that spermidine protects kidneys against cisplatin injury through DNA damage and tubular necrosis, and this finding provides a novel target to prevent acute kidney injury including nephrotoxicity.
Acceleration ; Acute Kidney Injury ; Adenosine Triphosphate ; Apoptosis ; Cell Death ; Cisplatin* ; DNA Damage ; Kidney ; Lipid Peroxidation ; Necrosis* ; Ornithine Decarboxylase ; Oxidative Stress ; Poly(ADP-ribose) Polymerases ; RNA, Small Interfering ; Spermidine* ; Transfection ; Yeasts

Kidney ischemia and reperfusion injury (IRI) is associated with a high mortality rate, which is attributed to tubular oxidative and nitrative stresses; however, an effective approach to limit IRI remains elusive. Spermidine, a naturally occurring polyamine, protects yeast cells against aging through the inhibition of oxidative stress and necrosis. In the present study, spermidine supplementation markedly attenuated histological damage and kidney dysfunction during IRI. In addition, exogenous spermidine potently inhibited poly(ADP-ribose) polymerase 1 (PARP1) activation and DNA nitrative/oxidative stress following IRI. Conversely, inhibition of ornithine decarboxylase (ODC) via siRNA transfection in vivo significantly enhanced DNA nitration, PARP1 activation, and functional damage during IRI. Finally, in ODC knockdown kidneys, PARP1 inhibition attenuated histological and functional damage induced by IRI, but not DNA nitrative stress. In conclusion, these data suggest that spermidine protects kidneys against IRI through blocking DNA nitration and PARP1 activation and this finding provides a novel target for prevention of acute kidney injury including IRI.
Acute Kidney Injury ; Aging ; DNA* ; Ischemia* ; Kidney* ; Mortality ; Necrosis ; Ornithine Decarboxylase ; Oxidative Stress ; Poly(ADP-ribose) Polymerases ; Reperfusion Injury* ; Reperfusion* ; RNA, Small Interfering ; Spermidine* ; Transfection ; Yeasts