The underappreciated diversity of bile acid modifications.

The repertoire of modifications to bile acids and related steroidal lipids by host and microbial metabolism remains incompletely characterized. To address this knowledge gap, we created a reusable resource of tandem mass spectrometry (MS/MS) spectra by filtering 1.2 billion publicly available MS/MS spectra for bile-acid-selective ion patterns. Thousands of modifications are distributed throughout animal and human bodies as well as microbial cultures. We employed this MS/MS library to identify polyamine bile amidates, prevalent in carnivores. They are present in humans, and their levels alter with a diet change from a Mediterranean to a typical American diet. This work highlights the existence of many more bile acid modifications than previously recognized and the value of leveraging public large-scale untargeted metabolomics data to discover metabolites. The availability of a modification-centric bile acid MS/MS library will inform future studies investigating bile acid roles in health and disease.

Neofunctionalization of S-adenosylmethionine decarboxylase into pyruvoyl-dependent L-ornithine and L-arginine decarboxylases is widespread in bacteria and archaea.

S-adenosylmethionine decarboxylase (AdoMetDC/SpeD) is a key polyamine biosynthetic enzyme required for conversion of putrescine to spermidine. Autocatalytic self-processing of the AdoMetDC/SpeD proenzyme generates a pyruvoyl cofactor from an internal serine. Recently, we discovered that diverse bacteriophages encode AdoMetDC/SpeD homologs that lack AdoMetDC activity and instead decarboxylate L-ornithine or L-arginine. We reasoned that neofunctionalized AdoMetDC/SpeD homologs were unlikely to have emerged in bacteriophages and were probably acquired from ancestral bacterial hosts. To test this hypothesis, we sought to identify candidate AdoMetDC/SpeD homologs encoding L-ornithine and L-arginine decarboxylases in bacteria and archaea. We searched for the anomalous presence of AdoMetDC/SpeD homologs in the absence of its obligatory partner enzyme spermidine synthase, or the presence of two AdoMetDC/SpeD homologs encoded in the same genome. Biochemical characterization of candidate neofunctionalized genes confirmed lack of AdoMetDC activity, and functional presence of L-ornithine or L-arginine decarboxylase activity in proteins from phyla Actinomycetota, Armatimonadota, Planctomycetota, Melainabacteria, Perigrinibacteria, Atribacteria, Chloroflexota, Sumerlaeota, Omnitrophota, Lentisphaerota, and Euryarchaeota, the bacterial candidate phyla radiation and DPANN archaea, and the δ-Proteobacteria class. Phylogenetic analysis indicated that L-arginine decarboxylases emerged at least three times from AdoMetDC/SpeD, whereas L-ornithine decarboxylases arose only once, potentially from the AdoMetDC/SpeD-derived L-arginine decarboxylases, revealing unsuspected polyamine metabolic plasticity. Horizontal transfer of the neofunctionalized genes appears to be the more prevalent mode of dissemination. We identified fusion proteins of bona fide AdoMetDC/SpeD with homologous L-ornithine decarboxylases that possess two, unprecedented internal protein-derived pyruvoyl cofactors. These fusion proteins suggest a plausible model for the evolution of the eukaryotic AdoMetDC.

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Long-term reversal of chronic pain behavior in rodents through elevation of spinal agmatine.

Chronic pain remains a significant burden worldwide, and treatments are often limited by safety or efficacy. The decarboxylated form of L-arginine, agmatine, antagonizes N-methyl-d-aspartate receptors, inhibits nitric oxide synthase, and reverses behavioral neuroplasticity. We hypothesized that expressing the proposed synthetic enzyme for agmatine in the sensory pathway could reduce chronic pain without motor deficits. Intrathecal delivery of an adeno-associated viral (AAV) vector carrying the gene for arginine decarboxylase (ADC) prevented the development of chronic neuropathic pain as induced by spared nerve injury in mice and rats and persistently reversed established hypersensitivity 266 days post-injury. Spinal long-term potentiation was inhibited by both exogenous agmatine and AAV-human ADC (hADC) vector pre-treatment but was enhanced in rats treated with anti-agmatine immunoneutralizing antibodies. These data suggest that endogenous agmatine modulates the neuroplasticity associated with chronic pain. Development of approaches to access this inhibitory control of neuroplasticity associated with chronic pain may yield important non-opioid pain-relieving options.

Tyramine-induced gastrointestinal dysregulation is attenuated via estradiol associated mechanisms in a zebrafish larval model.

Development of targeted therapeutics to alleviate gastrointestinal (GI) inflammation and its debilitating consequences are required. In this context, the trace aminergic system may link together sex, diet and inflammation. Utilising a zebrafish larval model of GI inflammation, the current study aimed to investigate mechanisms by which excess amounts of trace amines (TAs) may influence GI health. In addition, we probed the potential role of 17ß-estradiol (E2) and its receptors, given the known female-predominance of many GI disorders. To assess GI functionality and integrity, live imaging techniques (neutral red staining) and post-mortem immunofluorescent staining of tight junction proteins (occludin and ZO-1) were analyzed respectively. In addition, behavioural assays, as an indication of overall wellbeing, as well as whole body H2O2 and prostaglandin E2 assays were performed to inform on oxidative and inflammatory status. Excess ß-phenethylamine (PEA), tryptamine (TRP) and ρ-tyramine (TYR) resulted in adverse GI and systemic effects. In this regard, clear beneficial effects of E2 to modulate the effects of PEA, TRP and TYR was evident. Moreover, agmatine displayed potential protective effects on GI epithelium and whole body oxidative status, however, potential to induce systemic inflammation suggests the importance of dosage and administration optimisation. Taken together, TYR seems like the most prominent TA to have damaging GI effects, feasibly exacerbating GI inflammation. In this context, the relative lack of E2 may provide mechanistic insights into the reported female-predominance of GI disorders. Moreover, an effective therapeutic in this context may be required to maintain GI TA load despite fluctuating E2 levels.

Agmatine prevents the manifestation of impulsive burying and depression-like behaviour in progesterone withdrawn female rats.

Premenstrual dysphoric disorder (PMDD) is characterized by various physical and affective symptoms, including anxiety, irritability, anhedonia, social withdrawal, and depression. The present study investigated the role of the agmatinergic system in animal model of progesterone withdrawal in female rats. Chronic progesterone exposure of female rats for 21 days and its abrupt withdrawal showed enhanced marble burying, increased immobility time, and reduced no. of entries in open arm as compared to control animals. The progesterone withdrawal-induced enhanced marble burying anxiety and immobility time was significantly attenuated by agmatine (5-20 mg/kg, i.p.), and its endogenous modulators like L-arginine (100 mg/kg, i.p.), amino-guanidine (25 mg/kg, i.p.) and arcaine (50 mg/kg, i.p.) by their once-daily administration from day 14-day 21 of the protocol. We have also analysed the levels of agmatine, progesterone, and inflammatory cytokines in the hippocampal region of progesterone withdrawn rats. There was a significant decline in agmatine and progesterone levels and an elevation in cytokine levels in the hippocampal region of progesterone withdrawn rats compared to the control animals. In conclusion, the present studies suggest the importance of the endogenous agmatinergic system in progesterone withdrawal-induced anxiety-like and depression-like behaviour. The data also projects agmatine as a potential therapeutic target for the premenstrual dysphoric disorder.

Agmatine-IRF2BP2 interaction induces M2 phenotype of microglia by increasing IRF2-KLF4 signaling.

BACKGROUND: Following central nervous system (CNS) injury, the investigation for neuroinflammation is vital because of its pleiotropic role in both acute injury and long-term recovery. Agmatine (Agm) is well known for its neuroprotective effects and anti-neuroinflammatory properties. However, Agm's mechanism for neuroprotection is still unclear. We screened target proteins that bind to Agm using a protein microarray; the results showed that Agm strongly binds to interferon regulatory factor 2 binding protein (IRF2BP2), which partakes in the inflammatory response. Based on these prior data, we attempted to elucidate the mechanism by which the combination of Agm and IRF2BP2 induces a neuroprotective phenotype of microglia. METHODS: To confirm the relationship between Agm and IRF2BP2 in neuroinflammation, we used microglia cell-line (BV2) and treated with lipopolysaccharide from Escherichia coli 0111:B4 (LPS; 20 ng/mL, 24 h) and interleukin (IL)-4 (20 ng/mL, 24 h). Although Agm bound to IRF2BP2, it failed to enhance IRF2BP2 expression in BV2. Therefore, we shifted our focus onto interferon regulatory factor 2 (IRF2), which is a transcription factor and interacts with IRF2BP2. RESULTS: IRF2 was highly expressed in BV2 after LPS treatment but not after IL-4 treatment. When Agm bound to IRF2BP2 following Agm treatment, the free IRF2 translocated to the nucleus of BV2. The translocated IRF2 activated the transcription of Kruppel-like factor 4 (KLF4), causing KLF4 to be induced in BV2. The expression of KLF4 increased the CD206-positive cells in BV2. CONCLUSIONS: Taken together, unbound IRF2, resulting from the competitive binding of Agm to IRF2BP2, may provide neuroprotection against neuroinflammation via an anti-inflammatory mechanism of microglia involving the expression of KLF4.

A variant of guanidine-IV riboswitches exhibits evidence of a distinct ligand specificity.

Riboswitches are regulatory RNAs that specifically bind a small molecule or ion. Like metabolite-binding proteins, riboswitches can evolve new ligand specificities, and some examples of this phenomenon have been validated. As part of work based on comparative genomics to discover novel riboswitches, we encountered a candidate riboswitch with striking similarities to the recently identified guanidine-IV riboswitch. This candidate riboswitch, the Gd4v motif, is predicted in four distinct bacterial phyla, thus almost as widespread as the guanidine-IV riboswitch. Bioinformatic and experimental analysis suggest that the Gd4v motif is a riboswitch that binds a ligand other than guanidine. It is found associated with gene classes that differ from genes regulated by confirmed guanidine riboswitches. In inline-probing assays, we showed that free guanidine binds only weakly to one of the tested sequences of the variant. Further tested compounds did not show binding, attenuation of transcription termination, or activation of a genetic reporter construct. We characterized an N-acetyltransferase frequently associated with the Gd4v motif and compared its substrate preference to an N-acetyltransferase that occurs under control of guanidine-IV riboswitches. The substrates of this Gd4v-motif-associated enzyme did not show activity for Gd4v RNA binding or transcription termination. Hence, the ligand of the candidate riboswitch motif remains unidentified. The variant RNA motif is predominantly found in gut metagenome sequences, hinting at a ligand that is highly relevant in this environment. This finding is a first step to determining the identity of this unknown ligand, and understanding how guanidine-IV-riboswitch-like structures can evolve to bind different ligands.

Cholestasis-Associated Pulmonary Inflammation, Oxidative Stress, and Tissue Fibrosis: The Protective Role of the Biogenic Amine Agmatine.

INTRODUCTION: Cholestasis is the stoppage of bile flow, leading to the accumulation of potentially cytotoxic bile components in the liver. These cytotoxic molecules affect many organs. Cholestasis-induced lung injury is a severe complication that could lead to tissue fibrosis and respiratory distress. Substantial evidence indicates the role of oxidative stress and inflammatory response in the pathogenesis of cholestasis-associated pulmonary damage. Agmatine (AGM; 1-amino-4-guanidinobutane) is a biogenic amine endogenously synthesized in the human body. This amine provides potent anti-inflammatory and antioxidant properties. METHODS: In the current study, a series (six C57BL/6J male mice/group) of bile duct-ligated (BDL) animals were monitored at scheduled intervals (7, 14, and 28 days after the BDL operation) to ensure inflammatory response in their lung tissue (by analyzing their bronchoalveolar lavage fluid [BALF]). It was found that the level of inflammatory cells, pro-inflammatory cytokines, and IgG in the BALF reached their maximum level on day 28 after the BDL surgery. Therefore, other research groups were selected as follows: 1) Sham-operated (2.5 mL/kg normal saline, i.p., for 28 consecutive days), 2) BDL, 3) BDL + AGM (1 mg/kg/day, i.p., for 28 consecutive days), and 4) BDL + AGM (10 mg/kg/day, i.p., for 28 consecutive days). Then, the BALF was monitored at scheduled time intervals (7, 14, and 28 days post-BDL). RESULTS: It was found that pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß), bile acids, bilirubin, and inflammatory cells (monocytes, neutrophils, and lymphocytes) were significantly increased in the BALF of BDL mice. Moreover, biomarkers of oxidative stress were significantly increased in the pulmonary tissue of cholestatic animals. Lung tissue histopathological changes, tissue collagen deposition, and increased TGF-ß were also detected. It was found that AGM significantly ameliorated cholestasis-induced lung injury. CONCLUSION: The effects of AGM on inflammatory indicators, oxidative stress biomarkers, and tissue fibrosis seem to play a pivotal role in its protective properties.

Preventive putative effect of agmatine on cognitive and molecular outcomes in ventral tegmental area of male offspring following physical and psychological prenatal stress.

Prenatal stress (PS) results from a maternal experience of stressful events during pregnancy, which has been associated with an increased risk of behavioral disorders including substance abuse and anxiety in the offspring. PS is known to result in heightened dopamine release in the ventral tegmental area (VTA), in part through the effects of corticotropin-releasing hormone, which directly excites dopaminergic cells. It has recently been suggested that agmatine plays a role in modulating anxiety-like behaviors. In this study, we investigated whether agmatine could reduce negative cognitive outcomes in male mice prenatally exposed to psychological/physical stress, and whether this could be associated with molecular changes in VTA. Agmatine (37.5 mg/kg) was administrated 30 min prior to PS induction in pregnant Swiss mice. Male offspring were evaluated in a series of behavioral and molecular assays. Findings demonstrated that agmatine reduced the impairment in locomotor activity induced by both psychological and physical PS. Agmatine also decreased heightened conditioned place preference to morphine seen in PS offspring. Moreover, agmatine ameliorated the anxiety-like behavior and drug-seeking behavior induced by PS in the male offspring. Molecular effects were seen in VTA as the enhanced brain-derived neurotrophic factor (BDNF) induced by PS in the VTA was reduced by agmatine. Behavioral tests indicate that agmatine exerts a protective effect on PS-induced impairments in male offspring, which could be due in part to agmatine-associated molecular alterations in the VTA. Taken together, our data suggest that prenatal treatment with agmatine exerts protective effect against negative consequences of PS on the development of affective circuits in the offspring.

Possible involvement of GABAergic system on central amygdala Mediated anxiolytic effect of agmatine in rats.

OBJECTIVES: To study the pharmacological interactions between agmatine and gamma aminobutyric acid (GABA) modulatory agents in the regulation of anxiety-like behavior in rats. MATERIALS AND METHODS: Male Wistar rats were treated drugs per se or in combination and 15 min after last injection were subjected to elevated plus-maze (EPM) test. Anxiety-like behavior was evaluated by measuring behavioral conventional readout, open arm activity (duration and/or entries) for 5-minute duration. RESULTS: Acute intra-central amygdala (CeA) injection of agmatine (0.1-0.6 µmol/site/rat), muscimol (0.25-1 nmol/site/rat), diazepam (5-20 µg/site/rat) and allopregnanolone (2-8 µg/site/rat) increased open arm entries of the rats in EPM suggesting anxiolytic effect in dose dependent manner. Moreover, the anxiolytic effect at subeffective dose of agmatine (0.1 µmol/site/rat) was potentiated by subeffective dose of muscimol (0.25 nmol/site/rat), diazepam (5 µg/site/rat) and allopregnanolone (4 µg/site/rat). Whereas, pretreatment with GABAA receptor antagonist, bicuculline (10 ng/site/rat) blocked the anxiolytic effect of agmatine and its synergistic effect of agmatine plus muscimol. Similarly, benzodiazepine (BZD) receptor antagonist, flumazenil (15 µg/site/rat) and GABA allosteric modulator antagonist, RO 15-45 13 (10 µg/site/rat) reduced the anxiolytic effect of agmatine, given alone and with diazepam and allopregnanolone, respectively. CONCLUSION: These results indicated that anxiolytic effect of agmatine is medicated via GABAergic mechanisms, probably conciliated by the GABAA receptor subtypes. Modulation of interplay between agmatine and GABAA receptor activity might be a pertinent solution for the regulation of anxiety.

The hyperthermic response to intra-preoptic area administration of agmatine in male rats.

Agmatine is an endogenous biogenic amine that exerts various effects on the central nervous system. The hypothalamic preoptic area (POA, thermoregulatory command center) has high agmatine immunoreactivity. In this study, in conscious and anesthetized male rats, agmatine microinjection into the POA induced hyperthermic responses associated with increased heat production and locomotor activity. Intra-POA administration of agmatine increased the locomotor activity, the brown adipose tissue temperature and rectum temperature, and induced shivering as demonstrated by increased neck muscle electromyographic activity. However, intra-POA administration of agmatine almost had no impact on the tail temperature of anesthetized rats. Furthermore, there were regional differences in the response to agmatine in the POA. The most effective sites for the microinjection of agmatine to elicit hyperthermic responses were localized in the medial preoptic area (MPA). Agmatine microinjection into the median preoptic nucleus (MnPO) and lateral preoptic nucleus (LPO) had a minimal effect on the mean core temperature. Analysis of the in vitro discharge activity of POA neurons in brain slices when perfused with agmatine showed that agmatine inhibited most warm-sensitive but not temperature-insensitive neurons in the MPA. However, regardless of thermosensitivity, the majority of MnPO and LPO neurons were not responsive to agmatine. The results demonstrated that agmatine injection into the POA of male rats, especially the MPA, induced hyperthermic responses, which may be associated with increased BAT thermogenesis, shivering and locomotor activity by inhibiting warm-sensitive neurons.

Modulating Substrate Specificity of Rhizobium sp. Histamine Dehydrogenase through Protein Engineering for Food Quality Applications.

Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class.

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.

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 50 mg 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 (25 µg/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.

High-level production of the agmatine in engineered Corynebacterium crenatum with the inhibition-releasing arginine decarboxylase.

BACKGROUND: Agmatine is a member of biogenic amines and is an important medicine which is widely used to regulate body balance and neuroprotective effects. At present, the industrial production of agmatine mainly depends on the chemical method, but it is often accompanied by problems including cumbersome processes, harsh reaction conditions, toxic substances production and heavy environmental pollution. Therefore, to tackle the above issues, arginine decarboxylase was overexpressed heterologously and rationally designed in Corynebacterium crenatum to produce agmatine from glucose by one-step fermentation. RESULTS: In this study, we report the development in the Generally Regarded as Safe (GRAS) L-arginine-overproducing C. crenatum for high-titer agmatine biosynthesis through overexpressing arginine decarboxylase based on metabolic engineering. Then, arginine decarboxylase was mutated to release feedback inhibition and improve catalytic activity. Subsequently, the specific enzyme activity and half-inhibitory concentration of I534D mutant were increased 35.7% and 48.1%, respectively. The agmatine production of the whole-cell bioconversion with AGM3 was increased by 19.3% than the AGM2. Finally, 45.26 g/L agmatine with the yield of 0.31 g/g glucose was achieved by one-step fermentation of the engineered C. crenatum with overexpression of speAI534D. CONCLUSIONS: The engineered C. crenatum strain AGM3 in this work was proved as an efficient microbial cell factory for the industrial fermentative production of agmatine. Based on the insights from this work, further producing other valuable biochemicals derived from L-arginine by Corynebacterium crenatum is feasible.

New Insights into the Determinants of Specificity in Human Type I Arginase: Generation of a Mutant That Is Only Active with Agmatine as Substrate.

Arginase catalyzes the hydrolysis of L-arginine into L-ornithine and urea. This enzyme has several analogies with agmatinase, which catalyzes the hydrolysis of agmatine into putrescine and urea. However, this contrasts with the highlighted specificity that each one presents for their respective substrate. A comparison of available crystal structures for arginases reveals an important difference in the extension of two loops located in the entrance of the active site. The first, denominated loop A (I129-L140) contains the residues that interact with the alpha carboxyl group or arginine of arginase, and the loop B (D181-P184) contains the residues that interact with the alpha amino group of arginine. In this work, to determine the importance of these loops in the specificity of arginase, single, double, and triple arginase mutants in these loops were constructed, as well as chimeras between type I human arginase and E. coli agmatinase. In previous studies, the substitution of N130D in arginase (in loop A) generated a species capable of hydrolyzing arginine and agmatine. Now, the specificity of arginase is completely altered, generating a chimeric species that is only active with agmatine as a substrate, by substituting I129T, N130Y, and T131A together with the elimination of residues P132, L133, and T134. In addition, Quantum Mechanic/Molecular Mechanic (QM/MM) calculations were carried out to study the accommodation of the substrates in in the active site of this chimera. With these results it is concluded that this loop is decisive to discriminate the type of substrate susceptible to be hydrolyzed by arginase. Evidence was also obtained to define the loop B as a structural determinant for substrate affinity. Concretely, the double mutation D181T and V182E generate an enzyme with an essentially unaltered kcat value, but with a significantly increased Km value for arginine and a significant decrease in affinity for its product ornithine.

Agmatine Mitigates Inflammation-Related Oxidative Stress in BV-2 Cells by Inducing a Pre-Adaptive Response.

Neuroinflammation and microglial activation, common components of most neurodegenerative diseases, can be imitated in vitro by challenging microglia cells with Lps. We here aimed to evaluate the effects of agmatine pretreatment on Lps-induced oxidative stress in a mouse microglial BV-2 cell line. Our findings show that agmatine suppresses nitrosative and oxidative burst in Lps-stimulated microglia by reducing iNOS and XO activity and decreasing O2- levels, arresting lipid peroxidation, increasing total glutathione content, and preserving GR and CAT activity. In accordance with these results, agmatine suppresses inflammatory NF-kB, and stimulates antioxidant Nrf2 pathway, resulting in decreased TNF, IL-1 beta, and IL-6 release, and reduced iNOS and COX-2 levels. Together with increased ARG1, CD206 and HO-1 levels, our results imply that, in inflammatory conditions, agmatine pushes microglia towards an anti-inflammatory phenotype. Interestingly, we also discovered that agmatine alone increases lipid peroxidation end product levels, induces Nrf2 activation, increases total glutathione content, and GPx activity. Thus, we hypothesize that some of the effects of agmatine, observed in activated microglia, may be mediated by induced oxidative stress and adaptive response, prior to Lps stimulation.

Quantitative Metabolomics to Explore the Role of Plasma Polyamines in Colorectal Cancer.

Colorectal cancer (CRC) is one of the major public health and socio-economic problems, which management demands the development of non-invasive screening tests. Assessment of circulating polyamines could be a valuable tool, although analytical problems still preclude its clinical practice. We exploited ultra-high-resolution liquid chromatography and mass spectrometry, as a highly sensitive and innovative method, to profile eleven polyamines, including spermine and spermidine with their acetylated forms. These data together with an evaluation of the inflammatory indexes might represent suitable biomarkers for the identification of CRC patients. The statistical models revealed good discrimination in distinguishing CRC patients from healthy subjects. The plasma assessment of ornithine and acetylspermine, as well as lymphocyte/platelet ratio, revealed helpful information on the progression of CRC. The combined profiles of circulating polyamines and inflammatory indexes, together with the application of an innovative technology, could represent a valuable tool for discriminating patients from different clinical groups.

Neural Stem Cells Overexpressing Arginine Decarboxylase Improve Functional Recovery from Spinal Cord Injury in a Mouse Model.

Current therapeutic strategies for spinal cord injury (SCI) cannot fully facilitate neural regeneration or improve function. Arginine decarboxylase (ADC) synthesizes agmatine, an endogenous primary amine with neuroprotective effects. Transfection of human ADC (hADC) gene exerts protective effects after injury in murine brain-derived neural precursor cells (mNPCs). Following from these findings, we investigated the effects of hADC-mNPC transplantation in SCI model mice. Mice with experimentally damaged spinal cords were divided into three groups, separately transplanted with fluorescently labeled (1) control mNPCs, (2) retroviral vector (pLXSN)-infected mNPCs (pLXSN-mNPCs), and (3) hADC-mNPCs. Behavioral comparisons between groups were conducted weekly up to 6 weeks after SCI, and urine volume was measured up to 2 weeks after SCI. A subset of animals was euthanized each week after cell transplantation for molecular and histological analyses. The transplantation groups experienced significantly improved behavioral function, with the best recovery occurring in hADC-mNPC mice. Transplanting hADC-mNPCs improved neurological outcomes, induced oligodendrocyte differentiation and remyelination, increased neural lineage differentiation, and decreased glial scar formation. Moreover, locomotor and bladder function were both rehabilitated. These beneficial effects are likely related to differential BMP-2/4/7 expression in neuronal cells, providing an empirical basis for gene therapy as a curative SCI treatment option.