N1-Acetyl-5-methoxykynuramine, which decreases in the hippocampus with aging, improves long-term memory via CaMKII/CREB phosphorylation.

Melatonin is a molecule ubiquitous in nature and involved in several physiological functions. In the brain, melatonin is converted to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and then to N1-acetyl-5-methoxykynuramine (AMK), which has been reported to strongly enhance long-term object memory formation. However, the synthesis of AMK in brain tissues and the underlying mechanisms regarding memory formation remain largely unknown. In the present study, young and old individuals from a melatonin-producing strain, C3H/He mice, were employed. The amount of AMK in the pineal gland and plasma was very low compared with those of melatonin at night; conversely, in the hippocampus, the amount of AMK was higher than that of melatonin. Indoleamine 2, 3-dioxygenase (Ido) mRNA was expressed in multiple brain tissues, whereas tryptophan 2,3-dioxygenase (Tdo) mRNA was expressed only in the hippocampus, and its lysate had melatonin to AFMK conversion activity, which was blocked by the TDO inhibitor. The expression levels of phosphorylated cAMP response element binding protein (CREB) and PSD-95 in whole hippocampal tissue were significantly increased with AMK treatment. Before increasing in the whole tissue, CREB phosphorylation was significantly enhanced in the nuclear fraction. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that downregulated genes in hippocampus of old C3H/He mice were more enriched for long-term potentiation (LTP) pathway. Gene set enrichment analysis showed that LTP and neuroactive receptor interaction gene sets were enriched in hippocampus of old mice. In addition, Ido1 and Tdo mRNA expression was significantly decreased in the hippocampus of old mice compared with young mice, and the decrease in Tdo mRNA was more pronounced than Ido1. Furthermore, there was a higher decrease in AMK levels, which was less than 1/10 that of young mice, than in melatonin levels in the hippocampus of old mice. In conclusion, we first demonstrated the Tdo-related melatonin to AMK metabolism in the hippocampus and suggest a novel mechanism of AMK involved in LTP and memory formation. These results support AMK as a potential therapeutic agent to prevent memory decline.

Effects of endogenous and exogenous N-acetyl-5-methoxy kynuramine on object recognition memory in male C3H mice.

Prevention of dementia is important, because it is a leading cause of disability in elderly people. We previously reported that acute intraperitoneal treatment with N-acetyl-5-methoxy kynuramine (AMK), a melatonin (MEL) metabolite, enhanced long-term object recognition memory in ICR mice, a MEL deficient strain. Despite the presumable availability of AMK for dementia, its effects on cognitive performance have not been elucidated. It is unclear whether endogenous AMK is responsible for modulating long-term memory performance. To address this question, we assessed the effects of endogenous AMK on learning and memory using an object recognition test. C3H mice, a MEL-proficient strain, showed peak MEL levels at zeitgeber times (ZT) 19 and 22. Object recognition memory at ZT20 was superior to that at ZT8. Norharmane (NHM, 100 mg/kg), an indoleamine-2,3-dioxygenase (IDO) inhibitor, prevented the transformation of MEL to AMK, thereby suppressing AMK synthesis at ZT20. NHM (100 mg/kg) and another IDO inhibitor, 1-methyl-L-tryptophan (1-MT, 100 mg/kg), disrupted elevated cognitive performance at ZT20. These data imply that endogenous AMK may play a physiological role in the modulation of cognitive function. We also investigated the effects of pharmacological doses of MEL and AMK on object recognition memory in young C3H mice. MEL administration of 0.1 mg/kg, but not 0.01 mg/kg, enhanced object recognition memory, whereas 0.01 and 1 mg/kg AMK enhanced object recognition memory. Administration of 0.1 and 1 mg/kg AMK also enhanced object recognition memory in old C3H mice. These findings in MEL-proficient mice should be confirmed in other learning and memory tests before encouraging the clinical use of AMK.

Measurement of MAO Enzymatic Activity by Spectrophotometric Direct Assays.

MAO activity measurement can be monitored by direct peroxidase-free assays following different spectroscopy methods. Typically, these are assays that follow the conversion of different MAO substrates into its corresponding products monitored in either absorbance or fluorescence. Herein, we describe the assays for enzyme activity assessment with MAO B and particularly the MAO A substrate kynuramine, as well as the MAO B substrate benzylamine. Moreover, we also describe MAO activity determination using the tertiary amine substrate allyl amine 1-methyl-4-(1-methyl-1 H-pyrrol-2-yl)-1,2,3,6-tetrahydropyridine (MMTP). These are very useful methods for the investigation of MAO inhibitory activity by molecules known to be HRP-interfering. In the present chapter we demonstrate the application of these methods in MAO activity and Michaelis-Menten curve determinations as well as inhibitory activity experiments.

Assay of MAO Inhibition by Chromatographic Techniques (HPLC/HPLC-MS).

Monoamine oxidase (MAO) enzymes (MAO A and B) catalyze the oxidative deamination of biogenic amines, neurotransmitters, and xenobiotic amines and contribute to the regulation of the content of these active substances in mammalian organisms. The oxidation of biogenic amines by MAO produces hydrogen peroxide (H2O2) and aldehydes that represent risk factors for oxidative injury. The inhibitors of MAO are useful as antidepressants and neuroprotective agents. Usually, the assays of MAO determine amine deamination products or measure the H2O2 released by using direct spectrophotometric or fluorimetric methods. Direct methods are more prone to interferences and can afford inaccurate results. Those limitations can be avoided by using chromatographic techniques. This work describes a chromatographic method to assay MAO A and MAO B activity by using kynuramine as a nonselective substrate and the subsequent analysis of 4-hydroxyquinoline by RP-HPLC-DAD-fluorescence and mass spectrometry (MS). Alternatively, the assay uses the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin as a substrate of MAO that is oxidized (bioactivated) to neurotoxic pyridinium cations which are analyzed by HPLC. These methods are applied to assess the inhibition of MAO by bioactive ß-carboline alkaloids occurring in foods, plants, and biological systems.

Melatonin and cortisol as components of the cutaneous stress response system in fish: Response to oxidative stress.

The skin being a passive biological barrier that defends the organism against harmful external factors is also a site of action of the system responding to stress. It appears that melatonin (Mel) and its biologically active metabolite AFMK (N1-acetyl-N2-formyl-5-methoxykynuramine), both known as effective antioxidants, together with cortisol, set up a local (cutaneous) stress response system (CSRS) of fish, similar to that of mammals. Herein we comment on recent studies on CSRS in fish and show the response of three-spined stickleback skin to oxidative stress induced by potassium dichromate. Our study indicates that exposure of the three-spined stickleback to K2Cr2O7 affects Mel and cortisol levels and pigment dispersion in melanophores in the skin. In our opinion, an increased concentration of Mel and cortisol in the skin may be the strategy to cope with oxidative stress, where both components act locally to prevent damage caused by active oxygen molecules. Furthermore, the pigment dispersion may be a valuable, easy-to-observe mark of oxidative stress, useful in the evaluation of fish welfare.

Oxidative Cyclization of Kynuramine and Ynones Enabling Collective Syntheses of Pyridoacridine Alkaloids.

A cerium(III)-catalyzed oxidative cyclization of kynuramine and ynones has been reported as a key reaction in the total synthesis of marine pentacyclic pyridoacridine alkaloids featuring different ring connectivity patterns. The formation of tricyclic benzonaphthyridine rings was identified in the oxidative process. By combining with an intramolecular acylation and the chemoselective late-stage functionalization of pyridine rings, different approaches with 4-10 steps have been designed to accomplish the synthesis of alkaloids demethyldeoxyamphimedine (1), amphimedine (2), meridine (3), isocystodamine (4), N-methylisocystodamine (5), N-hydroxymethylisocystodamine (6), 9-hydroxyisoacididemin (7), neolabuanine A (8), and ecionine A (9).

Melatonin, Its Metabolites and Their Interference with Reactive Nitrogen Compounds.

Melatonin and several of its metabolites are interfering with reactive nitrogen. With the notion of prevailing melatonin formation in tissues that exceeds by far the quantities in blood, metabolites come into focus that are poorly found in the circulation. Apart from their antioxidant actions, both melatonin and N1-acetyl-5-methoxykynuramine (AMK) downregulate inducible and inhibit neuronal NO synthases, and additionally scavenge NO. However, the NO adduct of melatonin redonates NO, whereas AMK forms with NO a stable product. Many other melatonin metabolites formed in oxidative processes also contain nitrosylatable sites. Moreover, AMK readily scavenges products of the CO2-adduct of peroxynitrite such as carbonate radicals and NO2. Protein AMKylation seems to be involved in protective actions.

The melatonin metabolite N1-acetyl-5-methoxykynuramine facilitates long-term object memory in young and aging mice.

Melatonin (MEL) has been reported to enhance cognitive processes, making it a potential treatment for cognitive decline. However, the role of MEL's metabolites, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5-methoxykynuramine (AMK), in these effects are unknown. The current study directly investigated the acute effects of systemic MEL, AFMK, and AMK on novel object recognition. We also analyzed MEL, AFMK, and AMK levels in hippocampus and temporal lobe containing the perirhinal cortex following systemic MEL and AMK treatment. AMK administered post-training had a more potent effect on object memory than MEL and AFMK. AMK was also able to rescue age-associated declines in memory impairments when object memory was tested up to 4 days following training. Results from administering AMK at varying times around the training trial and the metabolism time course in brain tissue suggest that AMK's memory-enhancing effects reflect memory consolidation. Furthermore, inhibiting the MEL-to-AMK metabolic pathway disrupted object memory at 24 hours post-training, suggesting that endogenous AMK might play an important role in long-term memory formation. This is the first study to report that AMK facilitates long-term object memory performance in mice, and that MEL crosses the blood-brain barrier and is immediately converted to AMK in brain tissue. Overall, these results support AMK as a potential therapeutic agent to improve or prevent memory decline.

Melatonin and its metabolite N(1)-acetyl-N(1)-formyl-5-methoxykynuramine improve learning and memory impairment related to Alzheimer's disease in rats.

The aim of this study was to investigate the effect of melatonin (MT) and its metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) on Alzheimer-like learning and memory impairment in rats intracerebroventricularly injected with streptozotocin (STZ). The results showed that the escape latency of the STZ group was longer than that of the control (CON), MT, and AFMK groups. Increased levels of hyperphosphorylated tau, neurofilament proteins, and malondialdehyde and decreased superoxide dismutase levels were observed in the brains of the rats from the STZ group compared with the brains of the rats from the CON, MT, AFMK high and low group. These results suggest that exogenous MT and AFMK can improve memory impairment and downregulate AD-like hyperphosphorylation induced by STZ, most likely through their antioxidation function. Meanwhile, we found that an equal dose of AFMK had a stronger effect than that of MT. Our results indicate that MT and its metabolite AFMK represent novel treatment strategies for Alzheimer's disease.

ß-N-Oxalyl-L-α,ß-diaminopropionic acid from Panax notoginseng plays a major role in the treatment of type 2 diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is one of the most serious and dangerous chronic complications of diabetes mellitus.Panax notoginseng has been widely used with great efficacy in the long-term treatment of kidney disease. However, the mechanism by which it exerts its effects has not been fully elucidated. AIM: We sought to identify the major components ofPanax notoginseng that are effective in reducing the symptoms of DN in vitro and in vivo. METHODS: Inhibition of cell proliferation and collagen secretion were used to screen the ten most highly concentrated components ofPanax notoginseng. The STZ-induced DN rat model on a high-fat-high-glucose diet was used to investigate the renal protective effect of Panax notoginseng and dencichine and their underlying molecular mechanisms. RESULTS: Among the ten components analysed, dencichine (ß-N-oxalyl-L-α,ß-diaminopropionic acid) was the most protective against DN. Dencichine andPanax notoginseng attenuated glucose and lipid metabolic disorders in STZ-induced DN rats on a high-fat-high-glucose diet. In the untreated DN rats, we observed albuminuria, renal failure, and pathological changes. However, treatment with dencichine and Panax notoginseng alleviated these symptoms. We also observed that dencichine suppressed the expression of TGF-ß1 and Smad2/3, which mediates mesangial cell proliferation and extracellular matrix (ECM) accumulation in the glomerulus, and enhanced the expression of Smad7, the endogenous inhibitor of the TGF-ß1/Smad signalling pathway. CONCLUSION: From these results, we concluded that dencichine is the main compound inPanax notoginseng that is responsible for alleviating renal injury in the experimental DN model. Its mechanism may be related to the reduction of the deposition of ECM in glomeruli and inhibition of the epithelial mesenchymal transformation (EMT) by inhibition of the TGF-ß1/Smad signalling pathway.

Selectivity of Dietary Phenolics for Inhibition of Human Monoamine Oxidases A and B.

Monoamine oxidases (MAOs) regulate local levels of neurotransmitters such as dopamine, norepinephrine, and serotonin and thus have been targeted by drugs for the treatment of certain CNS disorders. However, recent studies have shown that these enzymes are upregulated with age in nervous and cardiac tissues and may be involved in degeneration of these tissues, since their metabolic mechanism releases hydrogen peroxide leading to oxidative stress. Thus, targeting these enzymes may be a potential anti-aging strategy. The purpose of this study was to compare the MAO inhibition and selectivity of selected dietary phenolic compounds, using a previously validated assay that would avoid interference from the compounds. Kynuramine metabolism by human recombinant MAO-A and MAO-B leads to formation of 4-hydroxyquinoline, with Vmax values of 10.2±0.2 and 7.35±0.69 nmol/mg/min, respectively, and Km values of 23.1±0.8 µM and 18.0±2.3 µM, respectively. For oral dosing and interactions with the gastrointestinal tract, curcumin, guaiacol, isoeugenol, pterostilbene, resveratrol, and zingerone were tested at their highest expected luminal concentrations from an oral dose. Each of these significantly inhibited both enzymes except for zingerone, which only inhibited MAO-A. The IC50 values were determined, and selectivity indices (MAO-A/MAO-B IC50 ratios) were calculated. Resveratrol and isoeugenol were selective for MAO-A, with IC50 values of 0.313±0.008 and 3.72±0.20 µM and selectivity indices of 50.5 and 27.4, respectively. Pterostilbene was selective for MAO-B, with IC50 of 0.138±0.013 µM and selectivity index of 0.0103. The inhibition of resveratrol (MAO-A) and pterostilbene (MAO-B) was consistent with competitive time-independent mechanisms. Resveratrol 4'-glucoside was the only compound which inhibited MAO-A, but itself, resveratrol 3-glucoside, and pterostilbene 4'-glucoside failed to inhibit MAO-B. Additional studies are needed to establish the effects of these compounds on MAO-A and/or MAO-B in humans.

Melatonin and its metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (afmk) enhance chemosensitivity to gemcitabine in pancreatic carcinoma cells (PANC-1).

BACKGROUND: Gemcitabine is a standard chemotherapeutic agent for patients suffering from pancreatic cancer. However, the applied therapy is not effective due to the resistance of tumor cells to cytostatics, caused by inefficiency of the apoptotic mechanisms. Herein, we present the hypothesis that melatonin and its metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) modify the effect of gemcitabine on PANC-1 cells and that this phenomenon is dependent on the modulation of apoptosis. METHODS: PANC-1 cells have been incubated with melatonin, AFMK or gemcitabine alone or in combination to determine the cytotoxity and proliferative effects. In subsequent part of the study, cells were harvested, the proteins were isolated and analyzed employing immunoprecipitation/immunoblotting. RESULTS: Incubation of PANC-1 cells with gemcitabine resulted in upregulation of pro-apoptotic bax and caspases proteins expression, downregulation of anti-apoptotic Bcl-2, heat shock proteins (HSPs) and modulation of cellular inhibitors of apoptosis (IAPs). Both melatonin and AFMK administered to PANC-1 in combination with gemcitabine inhibited the production of HSP70 and cIAP-2 as compared to the results obtained with gemcitabine alone. These changes were accompanied by upregulation of Bax/Bcl-2 ratio and reduction of procaspases-9 and -3 abundance, followed by an increase in the formation of active caspase of PANC-1 cells with combination of gemcitabine plus low doses of melatonin or AFMK led to enhanced cytotoxicity and resulted in the inhibition of PANC-1 cells growth as compared to effects of gemcitabine alone. CONCLUSION: Melatonin and AFMK could improve the anti-tumor effect of gemcitabine in PANC-1 cells presumably through the modulation of apoptotic pathway.

Synthesis and in vitro Evaluation of 2-heteroarylidene-1-tetralone Derivatives as Monoamine Oxidase Inhibitors.

The present study investigates the human monoamine oxidase (MAO) inhibition properties of a series of twelve 2-heteroarylidene-1-tetralone derivatives. Also included are related cyclohexylmethylidene, cyclopentylmethylidene and benzylidene substituted 1-tetralones. These compounds are related to the 2-benzylidene-1-indanone class of compounds which has previously been shown to inhibit the MAOs, with specificity for the MAO-B isoform. The target compounds were synthesised by the Claisen-Schmidt condensation between 7-methoxy-1-tetralone or 1-tetralone, and various aldehydes, under acid (hydrochloric acid) or base (potassium hydroxide) catalysis. The results of the MAO inhibition studies showed that the 2-heteroarylidene-1-tetralone and related derivatives are in most instances more selective inhibitors of the MAO-B isoform compared to MAO-A. (2E)-2-Benzylidene-7-methoxy-3,4-dihydronaphthalen-1(2 H)-one (IC50=0.707 µM) was found to be the most potent MAO-B inhibitor, while the most potent MAO-A inhibitor was (2E)-2-[(2-chloropyridin-3-yl)methylidene]-7-methoxy-3,4-dihydronaphthalen-1(2 H)-one (IC50=1.37 µM). The effect of the heteroaromatic substituent on MAO-B inhibition activity, in decreasing order was found to be: cyclohexyl, phenyl>thiophene>pyridine, furane, pyrrole, cyclopentyl. This study concludes that, although some 2-heteroarylidene-1-tetralone derivatives are good potency MAO inhibitors, in general their inhibition potencies, particularly for MAO-B, are lower than structurally related chalcones and 1-indanone derivatives that were previously studied.

Evaluation of melatonin and AFMK levels in women with breast cancer.

PURPOSE: Changes in the circadian rhythm may contribute to the development of cancer and are correlated with the high risk of breast cancer (BC) in night workers. Melatonin is a hormone synthesized by the pineal gland at night in the absence of light. Levels of melatonin and the metabolite of oxidative metabolism AFMK (acetyl-N-formyl-5-methoxykynurenamine), are suggested as potential biomarkers of BC risk. The aims of this study were to evaluate levels of melatonin and AFMK in women recently diagnosed with BC, women under adjuvant chemotherapy, and night-shift nurses, and compare them with healthy women to evaluate the relation of these compounds with BC risk. METHODS: Blood samples were collected from 47 women with BC, 9 healthy women, 10 healthy night shift nurses, and 6 patients under adjuvant chemotherapy. Compound levels were measured by mass spectrometry. RESULTS AND CONCLUSIONS: Our results showed that women with BC had lower levels of melatonin compared to control group women, and even lower in night-shift nurses and in patients under adjuvant chemotherapy. There was no significant difference of AFMK levels between the groups. In addition to this, high levels of melatonin and AFMK were related to patients with metastasis, and high levels of AFMK were related to the presence of lymph node-positive, tumor > 20 mm and patients who sleep with light at night. Our results showed a reduction of melatonin levels in BC patients, suggesting a relation with the disease, and in addition, point to the importance of melatonin supplementation in women that work at night to reduce the BC risk.

Cortisol and melatonin in the cutaneous stress response system of fish.

The stress hormone cortisol, together with antioxidants, melatonin (Mel) and its biologically active metabolites, 5-methoxykynuramines, including AFMK, set up a local stress response system in mammalian skin. Our in vitro study of the European flounder (Platichthys flesus) was designed to examine whether Mel and AFMK would respond to cortisol while a glucocorticoid is added to the incubation medium. The concentrations of cortisol in the incubation medium mimic plasma cortisol levels seen in fish exposed to different types of stresses such as handling, confinement, high density, food-deprivation or air-exposure. We measured Mel and AFMK in skin explants and culture media using high-performance liquid chromatography (HPLC) with fluorescence detection. We also analysed melanosome response (dispersion/aggregation) in the explants subjected to the different treatments. Cortisol stimulated the release of Mel and AFMK from skin explants in a dose-dependent manner. Melanosome dispersion and a darkening of the skin explants were observed after incubation with cortisol. This study is the first to demonstrate the interrelationship between cortisol and Mel/AFMK in fish skin. Our data strongly suggest that the cutaneous stress response system (CSRS) is present in fish. The question remains whether Mel, AFMK or cortisol are synthetized locally in fish skin and/or transported by the bloodstream. The presence of the CSRS should be taken into account during elaboration of new indicators of fish welfare both in aquaculture and in the wild.

Analysis of monoamine oxidase (MAO) enzymatic activity by high-performance liquid chromatography-diode array detection combined with an assay of oxidation with a peroxidase and its application to MAO inhibitors from foods and plants.

Monoamine oxidase (MAO) enzymes catalyze the oxidative deamination of biogenic amines and neurotransmitters and produce ammonia, aldehydes, and hydrogen peroxide which is involved in oxidative processes. Inhibitors of MAO-A and -B isozymes are useful as antidepressants and neuroprotectants. The assays of MAO usually measure amine oxidation products or hydrogen peroxide by spectrophotometric techniques. Those assays are often compromised by interfering compounds resulting in poor results. This research describes a new method that combines in the same assay the oxidative deamination of kynuramine to 4-hydroxyquinoline analyzed by HPLC-DAD with the oxidation of tetramethylbenzidine (TMB) (or Amplex Rex) by horseradish peroxidase (HRP) in presence of hydrogen peroxide. The new method was applied to study the inhibition of human MAO-A and -B by bioactive compounds including ß-carboline alkaloids and flavonoids occurring in foods and plants. As determined by HPLC-DAD, ß-carbolines, methylene blue, kaempferol and clorgyline inhibited MAO-A and methylene blue, 5-nitroindazole, norharman and deprenyl inhibited MAO-B, and all of them inhibited the oxidation of TMB in the same extent. The flavonoids catechin and cyanidin were not inhibitors of MAO by HPLC-DAD but highly inhibited the oxidation of TMB (or Amplex Red) by peroxidase whereas quercetin and resveratrol were moderate inhibitors of MAO-A by HPLC-DAD, but inhibited the peroxidase assay in a higher level. For some phenolic compounds, using the peroxidase-coupled assay to measure MAO activity led to mistaken results. The new method permits to discern between true inhibitors of MAO from those that are antioxidants and which interfere with peroxidase assays but do not inhibit MAO. For true inhibitors of MAO, inhibition as determined by HPLC-DAD correlated well with inhibition of the oxidation of TMB and this approach can be used to assess the in vitro antioxidant activity (less hydrogen peroxide production) resulting from MAO inhibition.

Three serum metabolite signatures for diagnosing low-grade and high-grade bladder cancer.

To address the shortcomings of cystoscopy and urine cytology for detecting and grading bladder cancer (BC), ultrahigh performance liquid chromatography (UHPLC) coupled with Q-TOF mass spectrometry in conjunction with univariate and multivariate statistical analyses was employed as an alternative method for the diagnosis of BC. A series of differential serum metabolites were further identified for low-grade(LG) and high-grade(HG) BC patients, suggesting metabolic dysfunction in malignant proliferation, immune escape, differentiation, apoptosis and invasion of cancer cells in BC patients. In total, three serum metabolites including inosine, acetyl-N-formyl-5-methoxykynurenamine and PS(O-18:0/0:0) were selected by binary logistic regression analysis, and receiver operating characteristic (ROC) test based on their combined use for HG BC showed that the area under the curve (AUC) was 0.961 in the discovery set and 0.950 in the validation set when compared to LG BC. Likewise, this composite biomarker panel can also differentiate LG BC from healthy controls with the AUC of 0.993 and 0.991 in the discovery and validation set, respectively. This finding suggested that this composite serum metabolite signature was a promising and less invasive classifier for probing and grading BC, which deserved to be further investigated in larger samples.

Enhanced production of camptothecin and biological preparation of N 1-acetylkynuramine in Camptotheca acuminata cell suspension cultures.

The Camptotheca acuminata cell suspension cultures were established to produce the well-known antitumor monoterpene indole alkaloid camptothecin (CAM). Most CAM was present in the broth of the C. acuminata cell suspension cultures. The CAM production was evidenced to be attenuated when the C. acuminata cell suspension cultures were continuously subcultured and grown under identical axenic conditions. A practical cryopreservation and recovery procedure was established to maintain the C. acuminata cell suspension cultures. Biotic and abiotic elicitors were administrated to the C. acuminata cell suspension cultures to restore and enhance CAM production. Of them, sorbitol, a well-known hyperosmotic stressor, was proven to be the most effective elicitor that stimulates a ∼500-fold increase of CAM production. The committed biosynthetic precursors of CAM, tryptamine and secologanin, were feed to the C. acuminata cell suspension cultures and the CAM production is not remarkably increased. However, N 1-acetylkynuramine (NAK), an important metabolite of kynuramine pathway, was isolated and identified from the cell suspension cultures feeding with tryptamine. The present work provides an efficient method to produce CAM and NAK using the C. acuminata cell suspension cultures. The biotransformation of tryptamine to NAK sheds lights on the biosynthetic formation of the pyrroloquinoline moiety of CAM.

Melatonin metabolite, N(1)-acetyl-N(1)-formyl-5-methoxykynuramine (AFMK), attenuates acute pancreatitis in the rat: in vivo and in vitro studies.

Melatonin protects the pancreas from inflammation and free radical damage but the effect of the melatonin metabolite: N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) on acute pancreatitis is unknown. This study assessed the effects of AFMK on acute pancreatitis (AP) in the rats in vivo and on pancreatic cell line AR42J in vitro. AFMK (5, 10 or 20 mg/kg) was given intraperitoneally to the rats 30 min prior to the induction of AP by subcutaneous caerulein infusion (25 µg/kg). Lipid peroxidation products (MDA + 4-HNE) and the activity of an antioxidant enzyme glutathione peroxidase (GPx) were measured in pancreatic tissue. Blood samples were taken for evaluation of amylase activity and TNF-α concentration. GPx, TNF-α, proapoptotic Bax protein, antiapoptotic Bcl-2 protein and the executor of apoptosis, caspase-3, were determined by Western blot in AR42J cells subjected to AFMK or to melatonin (both used at 10(-12), 10(-10), or 10(-8)M), without or with addition of caerulein (10(-8)M). AP was confirmed by histological examination and by serum increases of amylase and TNF-α (by 800% and 300%, respectively). In AP rats, pancreatic MDA + 4-HNE levels were increased by 300%, whereas GPx was reduced by 50%. AFMK significantly diminished histological manifestations of AP, decreased serum amylase activity and TNF-α concentrations, reduced MDA + 4-HNE levels and augmented GPx in the pancreas of AP rats. In AR42J cells, AFMK combined with caerulein markedly increased protein signals for GPx, Bax, caspase-3 and reduced these for TNF-α and Bcl-2. In conclusion, AFMK significantly attenuated acute pancreatitis in the rat. This may relate to the antioxidative and anti-inflammatory effects of this molecule and possibly to the stimulation of proapoptotic signal transduction pathway.

The antioxidative property of melatonin against brain ischemia.

INTRODUCTION: This review briefly summarizes some of the large amount of data documenting the ability of melatonin to limit molecular and organ tissue damage in neural ischemia-reperfusion injury (stroke), where free radicals are generally considered as being responsible for much of the resulting tissue destruction. AREA COVERED: Melatonin actions that have been identified include its ability to directly neutralize a number of toxic reactants and stimulate antioxidative enzymes. Furthermore, several of its metabolites such as N(1)-acetyl-N(2)-formyl-5- methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMF), are themselves scavengers suggesting that there is a cascade of reactions that greatly increase the efficacy of melatonin. Expert Commentary: However, the mechanisms by which melatonin is protective in such widely diverse areas of the cell and different organs are likely not yet all identified.