Biodegradation of the cyanobacterial toxin anatoxin-a by a Bacillus subtilis strain isolated from a eutrophic lake in Saudi Arabia.

Anatoxin-a (ATX-a) is a neurotoxin produced by some species of cyanobacteria. Due to its water solubility and stability in natural water, it could pose health risks to human, animals, and plants. Conventional water treatment techniques are not only insufficient for the removal of ATX-a, but they also result in cell lysis and toxin release. The elimination of this toxin through biodegradation may be a promising strategy. This study examines for the first time the biodegradation of ATX-a to a non-toxic metabolite (Epoxy-ATX-a) by a strain of Bacillus that has a history of dealing with toxic cyanobacteria in a eutrophic lake. The Bacillus strain AMRI-03 thrived without lag phase in a lake water containing ATX-a. The strain displayed fast degradation of ATX-a, depending on initial toxin concentration. At the highest initial concentrations (50 & 100 µg L- 1), total ATX-a degradation took place in 4 days, but it took 6 & 7 days at lower concentrations (20, 10, and 1 µg L- 1, respectively). The ATX-a biodegradation rate was also influenced by the initial toxin concentration, reaching its maximum value (12.5 µg L- 1 day- 1) at the highest initial toxin concentrations (50 & 100 µg L- 1). Temperature and pH also had an impact on the rate of ATX-a biodegradation, with the highest rates occurring at 25 and 30 ºC and pH 7 and 8. This nontoxic bacterial strain could be immobilized within a biofilm on sand filters and/or sludge for the degradation and removal of ATX-a and other cyanotoxins during water treatment processes, following the establishment of mesocosm experiments to assess the potential effects of this bacterium on water quality.

Engineering cellular metabolite transport for biosynthesis of computationally predicted tropane alkaloid derivatives in yeast.

Microbial biosynthesis of plant natural products (PNPs) can facilitate access to valuable medicinal compounds and derivatives. Such efforts are challenged by metabolite transport limitations, which arise when complex plant pathways distributed across organelles and tissues are reconstructed in unicellular hosts without concomitant transport machinery. We recently reported an engineered yeast platform for production of the tropane alkaloid (TA) drugs hyoscyamine and scopolamine, in which product accumulation is limited by vacuolar transport. Here, we demonstrate that alleviation of transport limitations at multiple steps in an engineered pathway enables increased production of TAs and screening of useful derivatives. We first show that supervised classifier models trained on a tissue-delineated transcriptome from the TA-producing plant Atropa belladonna can predict TA transporters with greater efficacy than conventional regression- and clustering-based approaches. We demonstrate that two of the identified transporters, AbPUP1 and AbLP1, increase TA production in engineered yeast by facilitating vacuolar export and cellular reuptake of littorine and hyoscyamine. We incorporate four different plant transporters, cofactor regeneration mechanisms, and optimized growth conditions into our yeast platform to achieve improvements in de novo hyoscyamine and scopolamine production of over 100-fold (480 µg/L) and 7-fold (172 µg/L). Finally, we leverage computational tools for biosynthetic pathway prediction to produce two different classes of TA derivatives, nortropane alkaloids and tropane N-oxides, from simple precursors. Our work highlights the importance of cellular transport optimization in recapitulating complex PNP biosyntheses in microbial hosts and illustrates the utility of computational methods for gene discovery and expansion of heterologous biosynthetic diversity.

A core root bacteria contribute to plant growth and anisodine accumulation of Anisodus tanguticus.

BACKGROUND: Although it is well recognized that core root microorganisms contribute to plant health and productivity, little is known about their role to the accumulation of secondary metabolites. The roots of Anisodus tanguticus, a traditional herbal medication utilized by Tibetan medicine, are rich in tropane alkaloids. We collected wild A. tanguticus populations throughout a 1500 km transect on the Qinghai-Tibetan Plateau. RESULTS: Our results showed that despite sampling at a distance of 1500 km, the root of A. tanguticus selectively recruits core root bacteria. We obtained 102 root bacterial core OTUs, and although their number only accounted for 2.99% of the total, their relative abundance accounted for 73% of the total. Spearman correlation and random forest analyses revealed that the composition of core root microbiomes was related to anisodine contents, aboveground biomass and nitrogen contents of Anisodus tanguticus. Among them, the main role is played by Rhizobacter, Variovorax, Polaromonas, and Mycobacterium genus that are significantly enriched in roots. Functional prediction by FAPROTAX showed that nitrogen-cycling microorganisms and pathogenic bacteria are strongly associated with anisodine contents, aboveground biomass and nitrogen contents of Anisodus tanguticus. CONCLUSIONS: Our findings show that the root selectively recruits core root bacteria and revealed that the core microbiomes and microbial functions potentially contributed to the anisodine contents, aboveground biomass and nitrogen contents of the plant. This work may increase our understanding of the interactions between microorganisms and plants and improve our ability to manage root microbiota to promote sustainable production of herbal medicines.

The Evolutionary Pattern of Cocaine and Hyoscyamine Biosynthesis Provides Strategies To Produce Tropane Alkaloids.

Cocaine and hyoscyamine are two tropane alkaloids (TA) from Erythroxylaceae and Solanaceae, respectively. These famous compounds possess anticholinergic properties that can be used to treat neuromuscular disorders. While the hyoscyamine biosynthetic pathway has been fully elucidated allowing its de novo synthesis in yeast, the cocaine pathway remained only partially elucidated. Recently, the Huang research group has completed the cocaine biosynthetic route by characterizing its two missing enzymes. This allowed the whole pathway to be transferring into Nicotiana benthamiana to achieve cocaine production. Here, besides highlighting the impact of this discovery, we discuss how TA biosynthesis evolved via the recruitment of two distinct and convergent pathways in Erythroxylaceae and Solanaceae. Finally, while enriching our knowledge on TA biosynthesis, this diversification of the molecular actors involved in cocaine and hyoscyamine biosynthesis opens perspectives in metabolic engineering by exploring enzyme biochemical plasticity that can ease and shorten TA pathway reconstitution in heterologous organisms.

Redirecting tropane alkaloid metabolism reveals pyrrolidine alkaloid diversity in Atropa belladonna.

Plant-specialized metabolism is complex, with frequent examples of highly branched biosynthetic pathways, and shared chemical intermediates. As such, many plant-specialized metabolic networks are poorly characterized. The N-methyl Δ1 -pyrrolinium cation is a simple pyrrolidine alkaloid and precursor of pharmacologically important tropane alkaloids. Silencing of pyrrolidine ketide synthase (AbPyKS) in the roots of Atropa belladonna (Deadly Nightshade) reduces tropane alkaloid abundance and causes high N-methyl Δ1 -pyrrolinium cation accumulation. The consequences of this metabolic shift on alkaloid metabolism are unknown. In this study, we utilized discovery metabolomics coupled with AbPyKS silencing to reveal major changes in the root alkaloid metabolome of A. belladonna. We discovered and annotated almost 40 pyrrolidine alkaloids that increase when AbPyKS activity is reduced. Suppression of phenyllactate biosynthesis, combined with metabolic engineering in planta, and chemical synthesis indicates several of these pyrrolidines share a core structure formed through the nonenzymatic Mannich-like decarboxylative condensation of the N-methyl Δ1 -pyrrolinium cation with 2-O-malonylphenyllactate. Decoration of this core scaffold through hydroxylation and glycosylation leads to mono- and dipyrrolidine alkaloid diversity. This study reveals the previously unknown complexity of the A. belladonna root metabolome and creates a foundation for future investigation into the biosynthesis, function, and potential utility of these novel alkaloids.

Nigrosome 1 visibility and its association with nigrostriatal dopaminergic loss in Parkinson's disease.

BACKGROUND: Nigrosome 1 (NG1), a small cluster of dopaminergic cells in the substantia nigra and visible in the susceptibility map-weighted magnetic resonance image (SMwI), is severely affected in Parkinson's disease (PD). However, the degree of nigrostriatal degeneration according to the visibility of NG1 has not yet been well elucidated. METHODS: We consecutively recruited 138 PD and 78 non-neurodegenerative disease (non-ND) patients, who underwent both 18 F-FP-CIT positron emission tomography (PET) and SMwI. Three neurologists and one radiologist evaluated the visibility of NG1 in SMwI. The participants were thereby grouped into visible, intermediate, and non-visible groups. Nigrostriatal dopaminergic input was calculated using the specific binding ratio (SBR) of the 18 F-FP-CIT PET. We determined the threshold of regional SBR for discriminating NG1 visibility and the probability for NG1 visibility according to regional SBR. RESULTS: Visual rating of NG1 showed excellent interobserver agreements as well as high sensitivity and specificity to differentiate the PD group from the non-ND group. NG1 was visible in seven patients (5.1%) in the PD group, who had relatively short disease duration or less severe loss of striatal dopamine. The threshold of putaminal SBR reduction on the more affected side for the disappearance of NG1 was 45.5%, and the probability for NG1 visibility dropped to 50% after the reduction of putaminal SBR to 41% from the normal mean. CONCLUSIONS: Almost half loss of nigrostriatal dopaminergic input is required to dissipate the hyperintensity of NG1 on SMwI, suggesting its utility in diagnosing PD only after the onset of the motor symptoms.

Engineering tropane alkaloid production and glyphosate resistance by overexpressing AbCaM1 and G2-EPSPS in Atropa belladonna.

Atropa belladonna is an important industrial crop for producing anticholinergic tropane alkaloids (TAs). Using glyphosate as selection pressure, transgenic homozygous plants of A. belladonna are generated, in which a novel calmodulin gene (AbCaM1) and a reported EPSPS gene (G2-EPSPS) are co-overexpressed. AbCaM1 is highly expressed in secondary roots of A. belladonna and has calcium-binding activity. Three transgenic homozygous lines were generated and their glyphosate tolerance and TAs' production were evaluated in the field. Transgenic homozygous lines produced TAs at much higher levels than wild-type plants. In the leaves of T2GC02, T2GC05, and T2GC06, the hyoscyamine content was 8.95-, 10.61-, and 9.96 mg/g DW, the scopolamine content was 1.34-, 1.50- and 0.86 mg/g DW, respectively. Wild-type plants of A. belladonna produced hyoscyamine and scopolamine respectively at the levels of 2.45 mg/g DW and 0.30 mg/g DW in leaves. Gene expression analysis indicated that AbCaM1 significantly up-regulated seven key TA biosynthesis genes. Transgenic homozygous lines could tolerate a commercial recommended dose of glyphosate in the field. In summary, new varieties of A. belladonna not only produce pharmaceutical TAs at high levels but tolerate glyphosate, facilitating industrial production of TAs and weed management at a much lower cost.

Gastrointestinal Symptoms and Dopamine Transporter Asymmetry in Early Parkinson's Disease.

BACKGROUND: The neurophysiological correlates of gastrointestinal symptoms (GISs) in Parkinson's disease (PD) are not well understood. It has been proposed that in patients with a gastrointestinal origin of PD dopaminergic neurodegeneration would be more symmetric. OBJECTIVES: The aim is to assess the associations between GISs and asymmetry of nigrostriatal dopaminergic neurodegeneration in PD. METHODS: Ninety PD patients were assessed using motor and GIS scales and 123 I-FP-CIT SPECT. We calculated the asymmetry index and the predominant side of motor symptoms and dopamine transporter (DAT) imaging defect and assessed their association with GISs. RESULTS: There were no significant differences in GISs between symmetric and asymmetric dopaminergic defect. Left predominant defect was related to more GIS and higher constipation scores. CONCLUSIONS: GISs were associated with left predominant reduction in putaminal DAT binding but not asymmetry per se. It remains open whether left-sided DAT deficit is related to more pronounced GI involvement or symptom perception in PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

Striatal dopamine transporter imaging in Parkinson's disease drug-naïve patients: focus on sexual dysfunction.

INTRODUCTION: Dopamine is involved in sexual behavior, but dopaminergic imaging studies establishing the relationship between nigrostriatal dopaminergic degeneration and sexual dysfunction (SD) in Parkinson's disease (PD) are lacking. METHODS: We retrospectively analyzed clinical and 123I-FP-CIT SPECT data of 43 drug-naïve PD patients. Based on the sexual function domain of the Non-Motor Symptoms Scale (NMSS), we identified 23 patients with sexual concerns (WSC), reporting a score ≥ 2 due to hyposexuality, and 20 patients without sexual concerns (NoSC). Dopamine transporter (DAT) uptake was assessed through semi-quantitative analysis in the most and least affected putamen (maP, laP), and most and least affected caudate (maC, laC). Total putamen-to-caudate ratio and total striatal binding ratio (tSBR) were also quantified. RESULTS: WSC and NoSC had similar demographic and disease-related characteristics. WSC displayed lower uptake values in maC (p = 0.016), maP (p = 0.004), laC (p = 0.019), laP (p = 0.009), and tSBR (p = 0.006). Pearson correlation analysis revealed, in the WSC group, moderate inverse correlations between the log-transformed SD scores and the uptake in maP (r = - 0.473, p = 0.023), maC (r = - 0.428, p = 0.042), laP (r = -0.437, p = 0.037), and tSBR (r = - 0.460, p = 0.027). After controlling in a two-way ANCOVA model for age and sex, between-group differences,between WSC and NoSC remained statistically significant only for dopaminergic denervation in maP [F(1,38) = 7.478, p = 0.009)], laP [F(1,38) = 4.684, p = 0.037)], and tSBR [F(1,38) = 5.069, p = 0.030]. CONCLUSION: To the best of our knowledge, this is the first study reporting the relationship between the severity of SD and specific patterns of nigrostriatal dopaminergic denervation (especially involving both putamina) in newly diagnosed drug-naïve PD patients.

Tropane alkaloid biosynthesis: a centennial review.

Covering: 1917 to 2020Tropane alkaloids (TAs) are a remarkable class of plant secondary metabolites, which are characterized by an 8-azabicyclo[3.2.1]octane (nortropane) ring. Members of this class, such as hyoscyamine, scopolamine, and cocaine, are well known for their long history as poisons, hallucinogens, and anaesthetic agents. Since the structure of the tropane ring system was first elucidated in 1901, organic chemists and biochemists have been interested in how these mysterious tropane alkaloids are assembled in vitro and in vivo. However, it was only in 2020 that the complete biosynthetic route of hyoscyamine and scopolamine was clarified, and their de novo production in yeast was also achieved. The aim of this review is to present the innovative ideas and results in exploring the story of tropane alkaloid biosynthesis in plants from 1917 to 2020. This review also highlights that Robinson's classic synthesis of tropinone, which is one hundred years old, is biomimetic, and underscores the importance of total synthesis in the study of natural product biosynthesis.

Biosynthesis of Anatoxins in Cyanobacteria: Identification of the Carboxy-anatoxins as the Penultimate Biosynthetic Intermediates.

Anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a are potent cyanobacterial neurotoxins. They are biosynthesized in cyanobacteria from proline and acetate by a pathway involving three polyketide synthases. We report the identification of carboxy-anatoxin-a, carboxy-homoanatoxin-a, and carboxy-dihydroanatoxin-a in acidic extracts of Cuspidothrix issatschenkoi CHARLIE-1, Oscillatoria sp. PCC 6506, and Cylindrospermum stagnale PCC 7417, respectively, using liquid chromatography coupled to mass spectrometry. The structure of these carboxy derivatives was confirmed by mass spectrometry and by isotopic incorporation experiments using labeled proline and acetate. Each of these three cyanobacteria only produce one carboxy-anatoxin, suggesting that these metabolites are the product of the hydrolysis by AnaA, the type II thioesterase, of the thioesters bound to AnaG, the last polyketide synthase of the pathway. By measuring the rate of isotopic incorporation of labeled proline into carboxy-homoanatoxin-a and homoanatoxin-a produced by Oscillatoria sp. PCC 6506, we show that carboxy-homoanatoxin-a is the intracellular precursor of homoanatoxin-a, and that homoanatoxin-a is then excreted into the extracellular medium. The transformation of carboxy-homoanatoxin-a into homoanatoxin-a is a very slow two-step process, with accumulation of carboxy-homoanatoxin-a, suggesting that the decarboxylation is spontaneous and not enzymatically catalyzed. However, an unidentified and extracellular catalyst accelerates the decarboxylation when the cell extracts are prepared at neutral pH.

The utility of the combined use of 123I-FP-CIT SPECT and neuromelanin MRI in differentiating Parkinson's disease from other parkinsonian syndromes.

BACKGROUND: Neuromelanin magnetic resonance imaging (NmMRI) and 123I-FP-CIT dopamine transporter single photon emission computed tomography (DAT-SPECT) provide specific information that distinguishes Parkinson's disease (PD) from non-degenerative parkinsonian syndrome (NDPS). PURPOSE: To determine whether a multiparametric scoring system (MSS) could improve accuracy compared to each parameter of DAT-SPECT and NmMRI in differentiating PD from NDPS. MATERIAL AND METHODS: A total of 49 patients, including 14 with NDPS, 30 with PD, and five with atypical parkinsonian disorder (APD) underwent both NmMRI and DAT-SPECT and were evaluated. The average (Ave) and the asymmetry index (AI) were calculated in the substantia nigra compacta area (SNc-area), SNc midbrain-tegmentum contrast ratio (SNc-CR), and specific binding ratio (SBR). Cut-off values were determined, using receiver operating characteristic (ROC) analysis, for the differentiation of PD from NDPS on the statistically significant parameters. All cases were scored as either 1 (PD) or 0 (NDPS) for each parameter according to its threshold. These individual scores were totaled for each case, yielding a combined score for each case to obtain a cut-off value for the MSS. RESULTS: The Ave-SNc-area, Ave-SNc-CR, and Ave-SBR in PD were significantly lower than those in NDPS. The AI-SNc-area and AI-SBR in PD were significantly higher than those in NDPS. Of the five parameters, the highest accuracy was 93% for the Ave-SNc-area. For the MSS, a cut-off value of 3 was the accuracy of 96%. Besides, no significant difference was observed between PD and APD on all parameters. CONCLUSION: An MSS has comparable or better accuracy compared to each parameter of DAT-SPECT and NmMRI in distinguishing PD from NDPS.

Localization and Organization of Scopolamine Biosynthesis in Duboisia myoporoides R. Br.

Tropane alkaloids (TAs), especially hyoscyamine and scopolamine, are important precursors for anticholinergic and antispasmodic drugs. Hyoscyamine and scopolamine are currently obtained at commercial scale from hybrid crosses of Duboisia myoporoides × Duboisia leichhardtii plants. In this study, we present a global investigation of the localization and organization of TA biosynthesis in a Duboisia myoporoides R. Br. wild-type line. The tissue-specific spatial distribution of TAs within D. myoporoides is presented, including quantification of the TAs littorine, 6-hydroxy hyoscyamine, hyoscyamine, scopolamine and, additionally, hyoscyamine aldehyde as well as scopolamine glucoside. Scopolamine (14.77 ± 5.03 mg g-1), and to a lesser extent hyoscyamine (3.01 ± 1.54 mg g-1) as well as 6-hydroxy hyoscyamine (4.35 ± 1.18 mg g-1), are accumulated in leaves during plant development, with the highest concentration of total TAs detected in 6-month-old plants. Littorine, an early precursor in TA biosynthesis, was present only in the roots (0.46 ± 0.07 mg g-1). During development, the spatial distribution of all investigated alkaloids changed due to secondary growth in the roots. Transcripts of pmt, tr-I and cyp80f1 genes, involved in early stages of TA biosynthesis, were found to be most abundant in the roots. In contrast, the transcript encoding hyoscyamine 6ß-hydroxylase (h6h) was highest in the leaves of 3-month-old plants. This investigation presents the spatial distribution of biochemical components as well as gene expression profiles of genetic factors known to participate in TA biosynthesis in D. myoporoides. The results of this investigation may aid in future breeding or genetic enhancement strategies aimed at increasing the yields of TAs in these medicinally valuable plant species.

Does smoking impact dopamine neuronal loss in de novo Parkinson disease?

This study analyzed data from dopamine transporter (DAT) positron emission tomographic scans of 282 male patients with de novo Parkinson disease to investigate whether smoking impacts striatal dopamine neuronal degeneration. Mean DAT activity in the posterior (p = 0.016) and ventral putamen (p = 0.028) was higher in 44 current smokers in comparison to 105 ex-smokers and 133 never-smokers. The severity of baseline motor deficits and the longitudinal increases in levodopa-equivalent doses during follow-up were similar among the 3 groups. These results suggest that current smoking, but not past smoking, protects dopamine neuronal degeneration in the sensorimotor striatum with no additional clinical benefits. Ann Neurol 2017;82:850-854.

Measurement of [123I]FP-CIT binding to the dopamine transporter (DAT) in healthy mouse striatum using dedicated small animal SPECT imaging: feasibility, optimization and validation.

BACKGROUND: In-vivo imaging of dopamine transporter (DAT), a reliable marker of degeneration of nigrostriatal dopaminergic innervation, has gained increasing interest in preclinical neurodegenerative research for studying disease mechanisms and testing new therapeutic strategies. We assessed the feasibility and the reliability of in vivo and ex vivo quantification of Methyl (3S,4S,5R)-8-(3-fluoropropyl)-3-(4-iodophenyl)-8-azabicyclo[3.2.1]octane-4-carboxylate ([123I]FP-CIT) binding to striatal DAT sites in mouse brain. METHODS: Dedicated small animal single-photon emission computed tomography (SPECT) images of [123I]FP-CIT binding were obtained in 3 groups of healthy mice: untreated (N.=6), pre-treated with lugol solution (N.=4), and pre-treated with selective dopamine transporter uptake inhibitor GBR12909 (N.=4). Ex-vivo autoradiography studies were performed at the end of SPECT studies with phosphor image system in 4 out of the 6 untreated mice and in all mice pre-treated with lugol. Regions of interest were defined over the striatum. The specific binding (SB) was calculated using the cerebral cortex as reference region. RESULTS: SPECT images in untreated mice showed high [123I]FP-CIT uptake in the striatum and extracerebral regions. Lugol pretreatment improved striatal images quality decreasing salivary and thyroid glands uptake. SB was higher (P<0.0001) in mice pre-treated with lugol (5.97±0.60) than in untreated mice (2.25±0.28). Autoradiography showed similar SB findings in untreated (2.27±0.33) and lugol-treated (4.27±0.57) mice (P<0.0001). In-vivo striatal 123I-FP-CIT SB and ex-vivo striatal 123I-FP-CIT SB were significantly correlated (r=0.87; P<0.0001). SPECT competition studies showed a significant (P<0.0001) reduction of [123I]FP-CIT SB in the striatum after GBR12909. CONCLUSIONS: We demonstrated the feasibility of [123I]FP-CIT imaging of the normal mouse brain using small-animal SPECT without pinhole collimators. The reliability of quantitative measurement of striatal [123I]FP-CIT SB is supported by competition studies showing measurable inhibition of uptake induced by GBR12909 and by the strong correlation between in vivo and ex vivo striatal [123I]FP-CIT SB. Our data also demonstrate that pre-treatment with lugol might improve striatal [123I]FP-CIT SB in mice.

In Vivo Use of 1D and 2D 1H NMR to Examine the Glycosylation of Scopoletin in Duboisia myoporoides Cell Suspensions.

Cell suspensions initiated from Duboisia myoporoides-a shrub belonging to the Solanaceae family and being a rich source of tropane alkaloids-previously showed their ability to glycosylate scopoletin into scopolin, which represent coumarins showing health benefits. To investigate the time course of this glycosylation reaction, an in vivo NMR approach was developed using a perfusion system in an 8-mm NMR tube and 1H NMR with 1D and 2D (TOCSY and NOESY) experiments. The time course of metabolic changes could therefore be followed without any labeling.

Novel Microbial Sources of Tropane Alkaloids: First Report of Production by Endophytic Fungi Isolated from Datura metel L.

Eighteen endophytic fungi were isolated from various tissues of Datura metel and genes encoding for putrescine N-methyltransferase (PMT), tropinone reductase 1 (TR1) and hyoscyamine 6ß-hydroxylase (H6H) were used as molecular markers for PCR-based screening approach for tropane alkaloids (TAs) producing endophytic fungi. These fungi were identified taxonomically by sequence analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) and also based on morphological characteristics of the fungal spore as Colletotrichum boninense, Phomopsis sp., Fusarium solani, Colletotrichum incarnatum, Colletotrichum siamense and Colletotrichum gloeosporioides. The production of TAs hyoscyamine and scopolamine by the fungi has been ascertained using chromatography and spectroscopy methods by comparison with the standards. Among the fungi, the highest yields of hyoscyamine (3.9 mg/L) and scopolamine (4.1 mg/L) were found in C. incarnatum culture. This is the first report of endophytic fungi possess the PMT, TR1 and H6H genes and produces TAs. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for TAs economically.

Sodium nitroprusside stimulated production of tropane alkaloids and antioxidant enzymes activity in hairy root culture of Hyoscyamus reticulatus L.

Hyoscyamus reticulatus L. is a herbaceous biennial belonging to the solanaceae family. Hyoscyamine and scopolamine as main tropane alkaloids accumulated in henbane are widely used in medicine to treat diseases such as parkinson's or to calm schizoid patients. Hairy roots media manipulation which uses elicitors to activate defense mechanisms is one of the main strategies for inducing secondary metabolism as well as increasing the production of valuable metabolites. Cotyledon-derived hairy root cultures were transformed by Agrobacterium rhizogenes. Sodium nitroprusside (SNP), a nitric oxide donor), was used in various concentrations (0, 50, 100, 200 and 300 µM) and exposure times (24 and 48 h). Treatment with SNP led to a significant reduction in fresh and dry weight of hairy roots, compared to control cultures. ANOVA results showed that elicitation of hairy root cultures with SNP at different concentrations and exposure times significantly affected the activity of as antioxidant enzymes such as catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). The highest hyoscyamine and scopolamine production (about 1.2-fold and 1.5-fold increases over the control) was observed at 50 and 100 µM SNP at 48 and 24 hours of exposure time, respectively. This is the first report of SNP elicitation effects on the production of tropane alkaloids in hairy root cultures.

Non-statistical 13C Fractionation Distinguishes Co-incident and Divergent Steps in the Biosynthesis of the Alkaloids Nicotine and Tropine.

During the biosynthesis of natural products, isotopic fractionation occurs due to the selectivity of enzymes for the heavier or lighter isotopomers. As only some of the positions in the molecule are implicated in a given reaction mechanism, position-specific fractionation occurs, leading to a non-statistical distribution of isotopes. This can be accessed by isotope ratio monitoring (13)C NMR spectrometry. The solanaceous alkaloids S-(-)-nicotine and hyoscyamine (atropine) are related in having a common intermediate, but downstream enzymatic steps diverge, providing a relevant test case to: (a) elucidate the isotopic affiliation between carbon atoms in the alkaloids and those in the precursors; (b) obtain information about the kinetic isotope effects of as yet undescribed enzymes, thus to make predictions as to their possible mechanism(s). We show that the position-specific (13)C/(12)C ratios in the different moieties of these compounds can satisfactorily be related to their known precursors and to the known kinetic isotope effects of enzymes involved in their biosynthesis, or to similar reaction mechanisms. Thus, the pathway to the common intermediate, N-methyl-Δ(1)-pyrrolinium, is seen to introduce similar isotope distribution patterns in the two alkaloids independent of plant species, whereas the remaining atoms of each target compound, which are of different origins, reflect their specific metabolic ancestry. We further demonstrate that the measured (13)C distribution pattern can be used to deduce aspects of the reaction mechanism of enzymes still to be identified.

Tropane alkaloids as substrates and inhibitors of human organic cation transporters of the SLC22 (OCT) and the SLC47 (MATE) families.

Tropane alkaloids and their derivatives are anticholinergic drugs with narrow therapeutic range. Here we characterize the organic cation transporters from the SLC22 (OCT1, OCT2, and OCT3) and the SLC47 families (MATE1 and MATE2-K) as potential mediators of the renal and extra-renal excretion, the two major roads of elimination of these substances. All analyzed compounds inhibited and the quaternary amine derivatives ipratropium and trospium were strongly transported by OCTs and MATEs. Overexpression of OCTs or MATEs in HEK293 cells resulted in an up to 63-fold increase in the uptake of ipratropium (Km of 0.32 µm to OCT2 and Vmax of 3.34 nmol×mg protein-1×min-1 to MATE1). The transcellular transport of ipratropium was 16-fold higher in OCT2-MATE1 and 10-fold higher in OCT1-MATE1 overexpressing compared to control MDCKII cells. Genetic polymorphisms in OCT1 and OCT2 affected ipratropium uptake and clinically relevant concentration of ondansetron and pyrithiamine inhibited ipratropium uptake via MATEs by more than 90%. This study suggests that OCT1, OCT2 and MATEs may be strongly involved in the renal and extra-renal elimination of ipratropium and other quaternary amine alkaloids. These substances have a notoriously narrow therapeutic range and the drug-drug interactions suggested here should be further critically evaluated in humans.