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Background: Dementia, including Alzheimer’s disease (AD), is one of the serious diseases at advanced age, and its early detection is important for maintaining quality of life (QOL). Objective: In this study, we sought novel biomarkers for dementia in urine. Methods: Samples of urine were collected from 57 control subjects without dementia, 62 mild cognitive impairment (MCI) patients, and 42 AD patients. Mini-Mental State Examination (MMSE) was evaluated when subjects were examined by medical doctors. Urinary amino acid (lysine)-conjugated acrolein (AC-Acro) was measured using N ɛ-(3-formyl-3, 4-dehydropiperidine) lysine (FDP-Lys) ELISA kit, and taurine content was measured using a taurine assay kit. Values were normalized by creatinine content which was measured with the colorimetric assay kit. Results: We found that urinary amino acid (lysine)-conjugated acrolein (AC-Acro) and taurine negatively correlated with MMSE score and are significantly lower in dementia patients compared to the normal subjects. When AC-Acro and taurine were evaluated together with age using an artificial neural network model, median relative risk values for subjects with AD, subjects with mild cognitive impairment, and control subjects were 0.96, 0.53, and 0.06, respectively. Conclusion: Since urine is relatively easy to collect, our findings provide a novel biomarker for dementia without invasiveness.

Brain stroke is a major cause of being bedridden for elderly people, and preventing stroke is important for maintaining quality of life (QOL). Acrolein is a highly reactive aldehyde and causes tissue damage during stroke. Decreasing acrolein toxicity ameliorates tissue injury during brain stroke. In this study, we tried to identify food components which decrease acrolein toxicity. We found that 2-furanmethanethiol, cysteine methyl and ethyl esters, alliin, lysine and taurine decreased acrolein toxicity. These compounds neutralized acrolein by direct interaction. However, the interaction between acrolein and taurine was not so strong. Approximately 30 mM taurine was necessary to interact with 10 µM acrolein, and 2 g/kg taurine was necessary to decrease the size of mouse brain infarction. Taurine also slightly increased polyamine contents, which are involved in decrease in the acrolein toxicity. Mitochondrial potential damage by acrolein was also protected by taurine. Our results indicate that daily intake of foods containing 2-furanmethanethiol, cysteine methyl and ethylesters, alliin, lysine and taurine may prevent severe injury in brain stroke and improve the quality of life for elderly people.

In the three domains of life, three biosynthetic pathways are known for putrescine. The first route is conversion of ornithine to putrescine by ornithine decarboxylase (ODC: SpeC), the second route is the conversion of arginine to agmatine by arginine decarboxylase (ADC: SpeA), followed by the conversion of agmatine to putrescine by agmatine ureohydrolase (AUH: SpeB), and the third route is the conversion of agmatine to N-carbamoylputrescine by agmatine deiminase (agmatine iminohydrolase, AIH), followed by the conversion of N-carbamoylputrescine to putrescine by N-carbamoylputrescine amidohydrolase (NCPAH). An extreme thermophile, Thermus thermophilus produces putrescine, although this bacterium lacks homologs for putrescine synthesizing pathways, such as ODC, AUH, AIH and NCPAH. To identify genes involved in putrescine biosynthesis in T. thermophilus, putrescine biosynthesis was examined by disruption of a predicted gene for agmatinase (agmatine ureohydrolase), or by using purified enzyme. It was found that arginase (TTHA1496) showed an agmatinase activity utilizing agmatine as a substrate. These results indicate that this bacterium can use arginase for putrescine biosynthesis. Arginase is a major contributor to putrescine biosynthesis under physiological conditions. The presence of an alternative pathway for converting agmatine into putrescine is functionally important for polyamine metabolism supporting survival at extreme environments.

An extreme thermophile

We have found recently that dendritic spine extension is inhibited through acrolein conjugation with α- and β-tubulin proteins during brain infarction. In this current study, we looked for other acrolein-conjugated proteins in the 100,000g precipitate fraction, to clarify how cytoskeleton structure is modified by acrolein. Acrolein-conjugated proteins were sought from acrolein-treated mouse FM3A and Neuro2a cells and from tissues isolated from mouse brain infarction. It was found that vimentin was conjugated with acrolein, and the conjugated amino acid residue was Cys328, which is the only Cys residue in vimentin. It was also found that Cys207, 257, 285, and Lys118 in actin, another cytoskeleton protein, were conjugated with acrolein. The structure and localization of vimentin and actin filaments were changed greatly in infarct brain in photochemically induced thrombosis model mice and in acrolein-treated Neuro2a cells. In addition, degradation of cytoskeleton proteins was accelerated in the order vimentin > tubulin > actin in mouse brain infarction. These findings indicate that a dysfunction of the cytoskeleton by acrolein is strongly involved in the tissue damage during brain infarction, together with the apoptosis caused by glyceraldehyde-3-phosphate dehydrogenase and protein degradation by matrix metalloproteinase-9.

An extreme thermophile, Thermus thermophilus grows at an optimum temperature of around 70°C and produces 16 different polyamines including long-chain and branched-chain polyamines. We found that the composition of polyamines in the thermophile cells changes with culture temperature. Long-chain and branched-chain polyamines (unusual polyamines) were increased in the cells grown at high temperature such as 80°C, but they were minor components in the cells grown at relatively lower temperature such as 60°C. The effects of polyamines on cell growth were studied using T. thermophilus HB8 ΔspeA deficient in arginine decarboxylase. Cell growth of this mutant strain was significantly decreased at 70°C. This mutant strain cannot produce polyamines and grows poorly at 75°C. It was also determined whether polyamines are directly involved in protecting DNA from DNA double-strand breaks (DSBs) induced by heat. Polyamines protected DNA against double-strand breaks. Therefore, polyamines play essential roles in cell growth at extremely high temperature through maintaining a functional conformation of DNA against DSBs and depurination.

It was found recently that acrolein (CH2=CH-CHO), mainly produced from spermine, is more toxic than ROS (reactive oxygen species, O2-·, H2O2, and ·OH). In this review, we describe how the seriousness of brain infarction, dementia, renal failure, and Sjӧgren's syndrome is correlated with acrolein. In brain infarction and dementia, it was possible to identify incipient patients with high sensitivity and specificity by measuring protein-conjugated acrolein (PC-Acro) in plasma together with IL-6 and CRP in brain infarction and Aβ40/42 in dementia. The level of PC-Acro in plasma and saliva correlated with the seriousness of renal failure and Sjӧgren's syndrome, respectively. Thus, development of acrolein scavenger medicines containing SH-group such as N-acetylcysteine derivatives is important to maintain QOL (quality of life) of the elderly.

Among low molecular weight substances, polyamines (spermidine, spermine and their precursor putrescine) are present in eukaryotic cells at the mM level together with ATP and glutathione. It is expected therefore that polyamines play important roles in cell proliferation and viability. Polyamines mainly exist as a polyamine-RNA complex and regulate protein synthesis. It was found that polyamines enhance translation from inefficient mRNAs. The detailed mechanisms of polyamine stimulation of specific kinds of protein syntheses and the physiological functions of these proteins are described in this review. Spermine is metabolized into acrolein (CH2 = CH–CHO) and hydrogen peroxide (H2O2) by spermine oxidase. Although it is thought that cell damage is mainly caused by reactive oxygen species (O2−, H2O2, and •OH), it was found that acrolein is much more toxic than H2O2. Accordingly, the level of acrolein produced becomes a useful biomarker for several tissue-damage diseases like brain stroke. Thus, the mechanisms of cell toxicity caused by acrolein are described in this review.

Polyamines stimulate the synthesis of specific proteins at the level of translation, and the genes encoding these proteins are termed as the “polyamine modulon”. The circadian clock generates daily rhythms in mammalian physiology and behavior. We investigated the role of polyamines in the circadian rhythm using control and polyamine-reduced NIH3T3 cells. The intracellular polyamines exhibited a rhythm with a period of about 24 h. In the polyamine-reduced NIH3T3 cells, the circadian period of circadian clock genes was lengthened and the synthesis of BMAL1 and REV-ERBα was significantly reduced at the translation level. Thus, the mechanism of polyamine stimulation of these protein syntheses was analyzed using NIH3T3 cells transiently transfected with genes encoding enhanced green fluorescent protein (EGFP) fusion mRNA with normal or mutated 5′-untranslated region (5′-UTR) of Bmal1 or Rev-erbα mRNA. It was found that polyamines stimulated BMAL1 and REV-ERBα synthesis through the enhancement of ribosomal shunting during the ribosome shunting within the 5′-UTR of mRNAs. Accordingly, the genes encoding Bmal1 and Rev-erbα were identified as the members of “polyamine modulon”, and these two proteins are significantly involved in the circadian rhythm control.

Background One of the main causes of death in psychiatric patients is cardiovascular diseases which are closely related with lifestyle-related diseases. Psychiatric disorders include schizophrenia and mood disorders, whose symptoms and treatment medicines are different, suggesting that they might have different metabolic disorders. Thus, we studied the differences of lifestyle-related diseases between schizophrenia and mood disorders in Japan. Methods This cross-sectional study was performed from 2015 to 2017. Study participants were 189 Japanese hospitalized patients (144 schizophrenia group, 45 mood disorders group) in the department of psychiatry at Kohnodai hospital. We examined physical disorders, metabolic status of glucose and lipid, estimated glomerular filtration rate (eGFR) and brain magnetic resonance imaging. We compared these data between schizophrenia and mood disorders groups using analysis of covariance or logistic regression analysis. In comparisons between inpatients with schizophrenia or mood disorders group and the standard, we quoted ‘The National Health and Nutrition Survey in Japan 2015’ by Ministry of Health, Labor and Welfare as the standard. Results eGFR and prevalence of smoking were significantly lower in patients with mood disorder group than those with schizophrenia group by adjustment for age. In comparisons between patients with schizophrenia group or mood disorders group and each standard, the ratio of silent brain infarction (SBI) and cerebral infarction were significantly high in both groups. Schizophrenia group showed significantly higher prevalence of diabetes, low high-density lipoprotein (HDL) cholesterolemia, metabolic syndrome and smoking than the standard. Mood disorders group had significantly high prevalence of low HDL-cholesterolemia compared with the standard. Fasting blood glucose and HbA1c were significantly higher in schizophrenia group and female mood disorders group than the standard. Female mood disorders group had significantly decreased eGFR with increased ratio of eGFR Conclusions Participants of both groups had increased ratio of SBI and cerebral infarction, accompanied with glucose and lipid disorders. Compared with schizophrenia group, mood disorders group showed significantly low eGFR and prevalence of smoking.

We previously observed an inverse correlation between stroke and urinary 3-hydroxypropyl mercapturic acid (3-HPMA), an acrolein-glutathione metabolite, through its measurement by liquid chromatography with tandem mass spectrometry (LC-MS/MS). However, the cost of equipment for LC-MS/MS and its maintenance fee is very expensive and a cost-efficient method is required. In this study, we have developed a sensitive enzyme-linked immunosorbent assay (ELISA) system to measure 3-HPMA using a chicken antibody recognizing 3-HPMA-conjugated chicken albumin as antigen. Linearity to measure 3-HPMA was obtained from 0 to 10 μM, indicating that this ELISA system is useful for measurement of urine 3-HPMA. It was confirmed that 3-HPMA in urine of stroke patients decreased significantly compared with that of control subjects using the ELISA system. Using the ELISA kit, it became possible to evaluate the risk of brain stroke by not only plasma but also by urine. These results confirm that shortage of glutathione to detoxify acrolein is one of the major causes of stroke incidence. Our method contributes to maintenance of quality of life (QOL) of the elderly.

Infiltration of peripheral immune cells after blood-brain barrier dysfunction causes severe inflammation after a stroke. Although the endothelial glycocalyx, a network of membrane-bound glycoproteins and proteoglycans that covers the lumen of endothelial cells, functions as a barrier to circulating cells, the relationship between stroke severity and glycocalyx dysfunction remains unclear. In this study, glycosaminoglycans, a component of the endothelial glycocalyx, were studied in the context of ischemic stroke using a photochemically induced thrombosis mouse model. Decreased levels of heparan sulfate and chondroitin sulfate and increased activity of hyaluronidase 1 and heparanase (HPSE) were observed in ischemic brain tissues. HPSE expression in cerebral vessels increased after stroke onset and infarct volume greatly decreased after co-administration of N-acetylcysteine + glycosaminoglycan oligosaccharides as compared with N-acetylcysteine administration alone. These results suggest that the endothelial glycocalyx was injured after the onset of stroke. Interestingly, scission activity of proHPSE produced by immortalized endothelial cells and HEK293 cells transfected with hHPSE1 cDNA were activated by acrolein (ACR) exposure. We identified the ACR-modified amino acid residues of proHPSE using nano LC-MS/MS, suggesting that ACR modification of Lys139 (6-kDa linker), Lys107, and Lys161, located in the immediate vicinity of the 6-kDa linker, at least in part is attributed to the activation of proHPSE. Because proHPSE, but not HPSE, localizes outside cells by binding with heparan sulfate proteoglycans, ACR-modified proHPSE represents a promising target to protect the endothelial glycocalyx.

It is well known that schizophrenic patients have high incidence of metabolic syndrome and life-style related diseases. There are reports that the rates of these diseases are increased more in outpatients than inpatients, but are also reports that the rates are not different between both patient groups. These differences might be related to the length of hospitalization. Hospitalization of Japanese psychiatric patients is about 300 days, much longer than western countries (below 50 days). Therefore, we investigated lipid and glucose metabolism of schizophrenic patients transferred from hospitalization to outpatients at Kohnodai hospital with a mean of 80 days hospitalization period to clarify metabolic characteristics in Japanese patients. Study participants were 144 schizophrenia inpatients and 109 outpatients at Kohnodai Hospital. These 109 outpatients were followed for approximately 2 years, without changes of administrated drugs, and from 144 inpatients. Data from outpatients were obtained at 6 months, 1 year and 2 years after their discharge. Outpatients 2 years after discharge had significantly higher levels of total cholesterol, triglyceride and non-high density lipoprotein (non-HDL) cholesterol than inpatients, accompanied with an increase of body weight. Serum HDL-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) levels had no significant difference between both groups. These lipids and glucose levels also showed the same tendency in outpatients 0.5 year and 1 year after discharge as those after 2 years. We found that schizophrenic patients in our study appeared to have changes of lipid metabolism 2 years after their discharge, but no significant changes of glucose metabolism, such as FPG and HbA1c.

Polyamines regulate gene expression in Escherichia coli by translationally stimulating mRNAs encoding global transcription factors. In this study, we focused on histone acetylation, one of the mechanisms of epigenetic regulation of gene expression, to attempt to clarify the role of polyamines in the regulation of gene expression in eukaryotes. We found that activities of histone acetyltransferases in both the nucleus and cytoplasm decreased significantly in polyamine-reduced mouse mammary carcinoma FM3A cells. Although protein levels of histones H3 and H4 did not change in control and polyamine-reduced cells, acetylation of histones H3 and H4 was greatly decreased in the polyamine-reduced cells. Next, we used control and polyamine-reduced cells to identify histone acetyltransferases whose synthesis is stimulated by polyamines. We found that polyamines stimulate the translation of histone acetyltransferases GCN5 and HAT1. Accordingly, GCN5- and HAT1-catalyzed acetylation of specific lysine residues on histones H3 and H4 was stimulated by polyamines. Consistent with these findings, transcription of genes required for cell proliferation was enhanced by polyamines. These results indicate that polyamines regulate gene expression by enhancing the expression of the histone acetyltransferases GCN5 and HAT1 at the level of translation. Mechanistically, polyamines enhanced the interaction of microRNA-7648-5p (miR-7648-5p) with the 5′-UTR of GCN5 mRNA, resulting in stimulation of translation due to the destabilization of the double-stranded RNA (dsRNA) between the 5′-UTR and the ORF of GCN5 mRNA. Because HAT1 mRNA has a short 5′-UTR, polyamines may enhance initiation complex formation directly on this mRNA.

Excessive accumulation of polyamines causes cytotoxicity, including inhibition of cell growth and a decrease in viability. We investigated the mechanism of cytotoxicity caused by spermidine accumulation under various conditions using an Escherichia coli strain deficient in spermidine acetyltransferase (SAT), a key catabolic enzyme in controlling polyamine levels. Due to the excessive accumulation of polyamines by the addition of exogenous spermidine to the growth medium, cell growth and viability were markedly decreased through translational repression of specific proteins [RMF (ribosome modulation factor) and Fis (rRNA transcription factor) etc.] encoded by members of polyamine modulon, which are essential for cell growth and viability. In particular, synthesis of proteins that have unusual locations of the Shine&ndash, Dalgarno (SD) sequence in their mRNAs was inhibited. In order to elucidate the molecular mechanism of cytotoxicity by the excessive accumulation of spermidine, the spermidine-dependent structural change of the bulged-out region in the mRNA at the initiation site of the rmf mRNA was examined using NMR analysis. It was suggested that the structure of the mRNA bulged-out region is affected by excess spermidine, so the SD sequence of the rmf mRNA cannot approach initiation codon AUG.

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Mast cells have secretory granules containing chemical mediators such as histamine and play important roles in the immune system. Polyamines are essential factors for cellular processes such as gene expression and translation. It has been reported that secretory granules contain both histamine and polyamines, which have similar chemical structures and are produced from the metabolism of cationic amino acids. We investigated the effect of polyamine depletion on mast cells using bone marrow-derived mast cells (BMMCs). Polyamine depletion was induced using α-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. DFMO treatment resulted in a significant reduction of cell number and abnormal secretory granules in BMMCs. Moreover, the cells showed a 2.3-fold increase in intracellular histamine and up-regulation of histidine decarboxylase (HDC) at the transcriptional level during BMMC differentiation. Levels of the transcription factor kruppel-like factor 4 (KLF4) greatly decreased upon DFMO treatment; however, Klf4 mRNA was expressed at levels similar to controls. We determined the translational regulation of KLF4 using reporter genes encoding Klf4-luc2 fusion mRNA, for transfecting NIH3T3 cells, and performed in vitro translation. We found that the efficiency of KLF4 synthesis in response to DFMO treatment was enhanced by the existence of a GC-rich 5'-untranslated region (5'-UTR) on Klf4 mRNA, regardless of the recognition of the initiation codon. Taken together, these results indicate that the enhancement of histamine synthesis by DFMO depends on the up-regulation of Hdc expression, achieved by removal of transcriptional suppression of KLF4, during differentiation.

Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the aging process, are not fully understood. We found that 4S/6S ratio and molecular weight of CS were decreased in polyamine-depleted cells. In addition, decreased levels of chondroitin synthase 1 (CHSY1) and chondroitin 4-O-sulfotransferase 2 proteins were also observed on polyamine depletion. Interestingly, the translation initiation of CHSY1 was suppressed by a highly structured sequence (positions −202 to −117 relative to the initiation codon) containing RNA G-quadruplex (G4) structures in 5′-untranslated region. The formation of the G4s was influenced by the neighboring sequences to the G4s and polyamine stimulation of CHSY1 synthesis disappeared when the formation of the G4s was inhibited by site-directed mutagenesis. These results suggest that the destabilization of G4 structures by polyamines stimulates CHSY1 synthesis and, at least in part, contribute to the maturation of CS chains.

Wound healing is a dynamic process involving gene-expression changes that drive re-epithelialization. Here, we describe an essential role for polyamine regulator AMD1 in driving cell migration at the wound edge. The polyamines, putrescine, spermidine, and spermine are small cationic molecules that play essential roles in many cellular processes. We demonstrate that AMD1 is rapidly upregulated following wounding in human skin biopsies. Knockdown of AMD1 with small hairpin RNAs causes a delay in cell migration that is rescued by the addition of spermine. We further show that spermine can promote cell migration in keratinocytes and in human ex vivo wounds, where it significantly increases epithelial tongue migration. Knockdown of AMD1 prevents the upregulation of urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor on wounding and results in a failure in actin cytoskeletal reorganization at the wound edge. We demonstrate that keratinocytes respond to wounding by modulating polyamine regulator AMD1 in order to regulate downstream gene expression and promote cell migration. This article highlights a previously unreported role for the regulation of polyamine levels and ratios in cellular behavior and fate. ASTAR (Agency for Sci., Tech. and Research, S’pore)

Background and Purpose— We recently found that acrolein (CH 2 =CH-CHO) is more strongly involved in brain infarction compared with reactive oxygen species. In this study, we looked for acrolein scavengers with less side effects. Methods— Photochemically induced thrombosis model mice were prepared by injection of Rose Bengal. Effects of N -acetylcysteine (NAC) derivatives on brain infarction were evaluated using the public domain National Institutes of Health image program. Results— NAC, NAC ethyl ester, and NAC benzyl ester (150 mg/kg) were administered intraperitoneally at the time of induction of ischemia, or these NAC derivatives (50 mg/kg) were administered 3× at 24-h intervals before induction of ischemia and 1 more administration at the time of induction of ischemia. The size of brain infarction decreased in the order NAC benzyl ester>NAC ethyl ester>NAC in both experimental conditions. Detoxification of acrolein occurred through conjugation of acrolein with glutathione, which was catalyzed by glutathione S -transferases, rather than direct conjugation between acrolein and NAC derivatives. The level of glutathione S -transferases at the locus of brain infarction was in the order of administration of NAC benzyl ester>NAC ethyl ester>NAC>no NAC derivatives, suggesting that NAC derivatives stabilize glutathione S -transferases. Conclusions— The results indicate that detoxification of acrolein by NAC derivatives is caused through glutathione conjugation with acrolein catalyzed by glutathione S -transferases, which can be stabilized by NAC derivatives. This is a new concept of acrolein detoxification by NAC derivatives.

Depurination is accelerated by heat and reactive oxygen species under physiological conditions. We previously reported that polyamines are involved in mitigation of heat shock and oxidative stresses through stimulation of the synthesis of heat shock and antioxidant proteins. This time, we investigated whether polyamines are directly involved in protecting nucleic acids from thermal depurination induced by high temperature. The suppressing efficiencies of depurination of DNA by spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg2+, were approximately 50%, 60% and 80%, respectively. Mg2+ also protected nucleic acids against depurination but to a lesser degree than polyamines. Longer unusual polyamines were more effective at protecting DNA against depurination compared to standard polyamines. The tRNA depurination suppressing efficiencies of spermine, caldopentamine and caldohexamine in the presence of 1 mM Mg2+, were approximately 60%, 70% and 80%, respectively. Standard polyamines protected tRNA and ribosomes more effectively than DNA against thermal depurination. Branched polyamines such as mitsubishine and tetrakis(3-aminopropyl)ammonium also protected RNA more effectively than DNA against depurination. These results suggest that the suppressing effect of depurination of nucleic acids (DNA and RNA) depends on the types of polyamines: i.e. to maintain functional conformation of nucleic acids at high temperature, longer and branched polyamines play important roles in protecting nucleic acids from depurination compared to standard polyamines and Mg2+.

Embryonic stem cells have the ability to self‐renew or differentiate and these processes are under tight control. We previously reported that the polyamine regulator AMD1 is critical for embryonic stem cell self‐renewal. The polyamines putrescine, spermidine, and spermine are essential organic cations that play a role in a wide array of cellular processes. Here, we explore the essential role of the polyamines in the promotion of self‐renewal and identify a new stem cell regulator that acts downstream of the polyamines: MINDY1. MINDY1 protein levels are high in embryonic stem cells (ESCs) and are dependent on high polyamine levels. Overexpression of MINDY1 can promote ESC self‐renewal in the absence of the usually essential cytokine Leukemia Inhibitory Factor (LIF). MINDY1 protein is prenylated and this modification is required for its ability to promote self‐renewal. We go on to show that Mindy1 RNA is targeted for repression by mir‐710 during Neural Precursor cell differentiation. Taken together, these data demonstrate that high polyamine levels are required for ESC self‐renewal and that they function, in part, through promotion of high MINDY1 levels. ASTAR (Agency for Sci., Tech. and Research, S’pore) NMRC (Natl Medical Research Council, S’pore) Published version

N-Methyl-d-aspartate (NMDA) receptors have been implicated in learning and memory, and may also play a central role in various conditions leading to neuronal degradation. NMDA receptor antagonists could therefore be of therapeutic benefit for a number of neurological disorders. We have designed hybrid compounds of polyamines and memantine, both of which function as NMDA channel blockers. The triamine derivative with a guanidine moiety showed more potent antagonistic activity than memantine.

Maintaining tissue homeostasis depends on a balance between cell proliferation, differentiation, and apoptosis. Within the epidermis, the levels of the polyamines putrescine, spermidine, and spermine are altered in many different skin conditions, yet their role in epidermal tissue homeostasis is poorly understood. We identify the polyamine regulator, Adenosylmethionine decarboxylase 1 (AMD1), as a crucial regulator of keratinocyte (KC) differentiation. AMD1 protein is upregulated on differentiation and is highly expressed in the suprabasal layers of the human epidermis. During KC differentiation, elevated AMD1 promotes decreased putrescine and increased spermine levels. Knockdown or inhibition of AMD1 results in reduced spermine levels and inhibition of KC differentiation. Supplementing AMD1-knockdown KCs with exogenous spermidine or spermine rescued aberrant differentiation. We show that the polyamine shift is critical for the regulation of key transcription factors and signaling proteins that drive KC differentiation, including KLF4 and ZNF750. These findings show that human KCs use controlled changes in polyamine levels to modulate gene expression to drive cellular behavior changes. Modulation of polyamine levels during epidermal differentiation could impact skin barrier formation or can be used in the treatment of hyperproliferative skin disorders.

Spermine oxidase (SMOX) catalyzes oxidation of spermine to generate spermidine, hydrogen peroxide (H2O2) and 3-aminopropanal, which is spontaneously converted to acrolein. SMOX is induced by a variety of stimuli including bacterial infection, polyamine analogues and acetaldehyde exposure. However, the physiological functions of SMOX are not yet fully understood. We investigated the physiological role of SMOX in liver cells using human hepatocellular carcinoma cell line HepG2. SMOX localized to the bile canalicular lumen, as determined by F-actin staining. Knockdown of SMOX reduced the formation of bile canalicular lumen. We also found that phospho-Akt (phosphorylated protein kinase B) was localized to canalicular lumen. Treatment with Akt inhibitor significantly reduced the formation of bile canalicular lumen. Acrolein scavenger also inhibited the formation of bile canalicular lumen. PTEN, phosphatase and tensin homolog and an inhibitor of Akt, was alkylated in a SMOX-dependent manner. Our results suggest that SMOX plays a central role in the formation of bile canalicular lumen in liver cells by activating Akt pathway through acrolein production.

A three column-switching high-performance liquid chromatography (HPLC) using an electrochemical detector (ECD) equipped with a diamond electrode was established to determine 3-hydroxypropylmercapturic acid (3-HPMA) in urine. An extracted urine sample was consecutively fractionated using a strong anion-exchange column (first column) and a C8 column (second column) via a switching valve before application on an Octa Decyl Silyl (ODS) column (third column), followed by ECD analysis. The% recovery of 3-HPMA standard throughout the three-column process and limit of detection (LOD) were 94±1% and 0.1pmol, respectively. A solid phase extraction step is required for the sensitive analysis of 3-HPMA in urine by column-switching HPLC-ECD despite a decreased% recovery (55%) of urine sample spiked with 100pmol of 3-HPMA. To test the utility of our column-switching HPLC-ECD method, 3-HPMA levels of 27 urine samples were determined, and the correlation between HPLC-ECD and LC-Electrospray ionization (ESI)-MS/MS method was examined. As a result, the median values of μmol 3-HPMA/g Creatinine (Cre) in urine obtained by column-switching HPLC-ECD and LC-MS/MS were 2.19±2.12μmol/g Cre and 2.13±3.38μmol/g Cre, respectively, and the calibration curve (y=1.5171x-1.007) exhibited good linearity within a defined range (r2=0.907). These results indicate that the combination of column-switching HPLC and ECD is a powerful tool for the specific, reliable detection of 3-HPMA in urine.

Objective Measurement of plasma levels of protein-conjugated acrolein (PC-Acro) together with IL-6 and CRP can be used to identify silent brain infarction (SBI) with high sensitivity and specificity. The aim of this study was to determine how these biomarkers vary during stroke. Methods Levels of PC-Acro, IL-6 and CRP in plasma were measured on day 0, 2, 7 and 14 after the onset of ischemic or hemorrhagic stroke. Results After the onset of stroke, the level of PC-Acro in plasma was elevated corresponding to the size of stroke. It returned to near control levels by day 2, and remained similar through day 14. The degree of the decrease in PC-Acro on day 2 was greater when the size of brain infarction or hemorrhage was larger. An increase in IL-6 and CRP occurred after the increase in PC-Acro, and it was well correlated with the size of the injury following infarction or hemorrhage. The results suggest that acrolein becomes a trigger for the production of IL-6 and CRP, as previously observed in a mouse model of stroke and in cell culture systems. The increase in IL-6 and CRP was also correlated with poor outcome judging from mRS. Conclusion The results indicate that the degree of the decrease in PC-Acro and the increase in IL-6 and CRP from day 0 to day 2 was correlated with the size of brain infarction, and the increase in IL-6 and CRP with poor outcome at discharge.

Background We clarified the correlation between brain damage, associated biomarkers and medication in psychiatric patients, because patients with schizophrenia have an increased risk of stroke. Methods The cross-sectional study was performed from January 2013 to December 2015. Study participants were 96 hospitalized patients (41 men and 55 women) in the Department of Psychiatry at Kohnodai Hospital, National Center for Global Health and Medicine, Ichikawa, Chiba, Japan. Patients were classified into schizophrenia (n = 70) and mood disorders (n = 26) by psychiatric diagnoses with DSM-IV-TR criteria. Results The incidence of brain damage [symptomatic and silent brain infarctions (SBIs) and white matter hyperintensity (WMH)] was correlated more with mood disorders than with schizophrenia. It has been previously shown that the concentrations of protein-conjugated acrolein (PC-Acro) and interleukin-6 (IL-6) increased in plasma of brain infarction patients together with C-reactive protein (CRP). The concentration of PC-Acro was significantly higher in patients with mood disorders than in those with schizophrenia. The concentration of IL-6 in both groups was nearly equal to that in the control group, but that of CRP in both groups, especially in mood disorders, was higher than that in the control group. Accordingly, the relative risk value for brain infarction was higher in patients with mood disorders than with schizophrenia. Medication with atypical antipsychotics reduced PC-Acro significantly in all psychiatric patients and reduced IL-6 in mood disorder patients. Conclusion Measurement of 3 biomarkers (CRP, PC-Acro and IL-6) are probably useful for judgement of severity of brain damage and effectiveness of medication in psychiatric patients.

We have recently found that conjugation of acrolein with a 50 kDa protein(s) is strongly associated with tissue damage during brain infarction. In the current study, the identity and function of the 50 kDa protein(s) conjugated with acrolein during brain infarction were investigated. The 50 kDa protein(s) conjugated with acrolein were identified as α- and β-tubulins. Ten cysteine residues in α- and β-tubulins (Cys25, 295, 347 and 376 in α-tubulin and Cys12, 129, 211, 239, 303 and 354 in β-tubulin) were mainly conjugated with acrolein. Since two cysteine residues of α-tubulin (Cys347 and 376) and four cysteine residues of β-tubulin (Cys12, 129, 239 and 354) were located at the interaction site of α- and β-tubulins, association between α- and β-tubulins to form microtubules was strongly inhibited by conjugation with acrolein. Accordingly, dendritic spine extension consisting of microtubules was greatly inhibited in acrolein-treated Neuro2a cells. The results strongly suggest that acrolein contributes to the functional losses in brain signaling through its conjugation with α- and β-tubulins.

We have reported that acrolein-conjugated low-density lipoprotein (Acro-LDL) uptake by scavenger receptor class A type 1 (SR-A1) can mediate macrophage foam cell formation. The purpose of this study was to determine which amino acid residues of apoB protein in LDL are conjugated with acrolein. Acro-apoB was prepared by incubation of LDL with acrolein (10 to 60 μM) at 37 °C for 7 days. Identification of acrolein-conjugated amino acid residues in apoB was performed using LC-MS/MS. The levels of acrolein-conjugated amino acid residues of apoB as well as crosslinking apoB increased in proportion to acrolein concentration. The level of LDL uptake by macrophages was parallel with the acrolein-conjugated monomer apoB. Acrolein-conjugated amino acid residues in apoB were C212, K327, K742, K949, K1087, H1923, K2634, K3237 and K3846. The NH2-teriminal four amino acid residues (C212, K327, K742 and K949) were located at the scavenger receptor SR-A1 recognition site, suggesting that these four acrolein-conjugated amino acids are involved in the rapid uptake of Acro-LDL by macrophages. It is proposed that the rapid uptake of LDL by macrophages is dependent on acrolein conjugation of four amino acids residues at the scavenger receptor recognition site of apoB in LDL.

We previously reported that tissue damage during brain infarction was mainly caused by inactivation of proteins by acrolein. This time, it was tested why brain infarction increases in parallel with aging. A mouse model of photochemically induced thrombosis (PIT) was studied using 2, 6, and 12 month-old female C57BL/6 mice. The size of brain infarction in the mouse PIT model increased with aging. The volume of brain infarction in 12 month-old mice was approximately 2-fold larger than that in 2 month-old mice. The larger brain infarction in 12 month-old mice was due to an increase in acrolein based on an increase in the activity of spermine oxidase, together with a decrease in glutathione (GSH), a major acrolein-detoxifying compound in cells, based on the decrease in one of the subunits of glutathione biosynthesizing enzymes, γ-glutamylcysteine ligase modifier subunit, with aging. The results indicate that aggravation of brain infarction with aging was mainly due to the increase in acrolein production and the decrease in GSH in brain.

Background and purpose N -Methyl-d-aspartate (NMDA) receptors have a high permeability to Ca 2+ , contributing to neuronal cell death after stroke. We recently found that acrolein produced from polyamines is a major toxic compound during stroke. Thus, it was determined whether over-accumulation of Ca 2+ increases the production of acrolein from polyamines in a photochemically-induced thrombosis mouse model of stroke and in cell culture systems. Methods A unilateral infarction was induced in mouse brain by photoinduction after injection of Rose Bengal. The volume of the infarction was analyzed using the public domain National Institutes of Health image program. Protein-conjugated acrolein levels at the locus of infarction and in cells were measured by Western blotting. Levels of polyamines were measured by high-performance liquid chromatography. Results When the size of brain infarction was decreased by N 1 , N 4 , N 8 -tribenzylspermidine, a channel blocker of the NMDA receptors, levels of Ca 2+ and protein-conjugated acrolein (PC-Acro) were reduced, while levels of polyamines were increased at the locus of infarction. When cell growth of mouse mammary carcinoma FM3A cells and neuroblastoma Neuro2a cells was inhibited by Ca 2+ , the level of polyamines decreased, while that of PC-Acro increased. It was also shown that Ca 2+ toxicity was decreased in an acrolein toxicity decreasing FM3A mutant cells recently isolated. In addition, 20–40 μM Ca 2+ caused the release of polyamines from ribosomes. The results indicate that acrolein is produced from polyamines released from ribosomes through Ca 2+ increase. Conclusion The results indicate that toxicity of Ca 2+ during brain infarction is correlated with the increase of acrolein.

Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5A resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13A. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT.

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

Polyamines, consisting of putrescine, spermidine and spermine are essential for normal cell growth and viability in eukaryotic cells. Since polyamines are cations, they interact with DNA, ATP, phospholipids, specific kinds of proteins, and especially with RNA. Consequently, the functions of these acidic compounds and some proteins are modified by polyamines. In this review, the functional modifications of these molecules by polyamines are presented. Structural change of specific mRNAs by polyamines causes the stimulation of the synthesis of several different proteins, which are important for cell growth and viability. eIF5 A, the only known protein containing a spermidine derivative, i.e. hypusine, also functions at the level of translation. Experimental results thus far obtained strongly suggest that the most important function of polyamines is at the level of translation.