Discovering a mitochondrion-localized BAHD acyltransferase involved in calystegine biosynthesis and engineering the production of 3ß-tigloyloxytropane.

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3α- and 3ß-tropanol. Although littorine synthase is revealed to be responsible for 3α-tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3ß-tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna, 3ß-tigloyloxytropane synthase (TS), catalyzes 3ß-tropanol and tigloyl-CoA to form 3ß-tigloyloxytropane, the key intermediate in calystegine biosynthesis and a potential drug for treating neurodegenerative disease. Unlike other cytosolic-localized BAHD acyltransferases, TS is localized to mitochondria. The catalytic mechanism of TS is revealed through molecular docking and site-directed mutagenesis. Subsequently, 3ß-tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3ß-tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3ß-tigloyloxytropane from tiglic acid and 3ß-tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3ß-tigloyloxytropane.

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.

Incidental Finding of Bilateral Thalamic Glioma on 99mTc TRODAT-1 SPECT/CT.

ABSTRACT: 99mTc-TRODAT-1 SPECT/CT has been used to evaluate parkinsonian disorders. We present an interesting case of a 63-year-old woman with progressive tremulousness over the left side of the body for 6 months. Bilateral thalamic glioma with reduced uptake was shown on 99mTc-TRODAT-1 SPECT/CT. Secondary parkinsonism was impressed in this case.

Bupropion Causes Misdiagnosis in Brain Dopamine Transporter Imaging for Parkinsonism.

OBJECTIVE: The objective of this study was to report long-lasting effects of bupropion on brain dopamine transporter (DAT) in a patient with depression and parkinsonism. METHODS: The patient was a 52-year old man who had been treated with 150 mg/d of bupropion for depression. The patient developed cognitive problems, bradykinesia, and reduced stride length for which he was scanned with [I]FP-CIT single photon emission computed tomography after the recommended 1-week discontinuation of bupropion. Levodopa treatment trial was initiated without a response. Eleven months later, the patient was scanned for a second time after a 1-month stoppage of bupropion. RESULTS: The first scan was abnormal with left putamen specific binding ratio of 1.99 (SDs from the reference value mean, -2.40), right putamen of 2.27 (SD, -1.84), left caudate of 2.33 (SD, -2.26), and right caudate of 2.29 (SD, -2.18). The second scan (after 1-month discontinuation) was normal, and specific binding ratios had increased from 5.2% to 31.7% in all striatal regions as compared with the first scan. Brain magnetic resonance imaging and [F]fluorodeoxyglucose positron emission tomography imaging were normal, and there was no levodopa response or other features supporting neurodegenerative parkinsonism. CONCLUSIONS: Bupropion has previously generally been discontinued 1 week prior DAT imaging, which meets the recommended, albeit arbitrary, time interval of 5 plasma clearance half-lives before the scan. One-week discontinuation of bupropion before DAT imaging may be insufficiently short. Our case shows that longer medication washout and rescan may be needed when there is contradiction between the imaging result and clinical outcome in patients with medications affecting DAT binding.

Brain Lymphoma on 99mTc-TRODAT-1 SPECT.

Tc-TRODAT-1, as a tropane-derived compound with highly selective binding to the dopamine transporter, has been extensively used as an in vivo biomarker to evaluate parkinsonism. There have been few reports indicating various etiologies about extrastriatal findings on the Tc-TRODAT-1 SPECT. We herein present an interesting case about the incidental discovery of brain lymphoma with increasing uptake of Tc-TRODAT-1.

Prediction of cognitive worsening in de novo Parkinson's disease: Clinical use of biomarkers.

BACKGROUND: Cognitive impairment is a frequent and disabling feature of Parkinson's disease. Identifying the factors able to predict cognitive worsening since the early stage may improve disease management. The objective of this study was to define the best predictors of future cognitive worsening in a group of patients with newly diagnosed PD and to propose cutoff values potentially useful at the individual level. METHODS: Fifty-four consecutive drug-naive patients with de novo PD were prospectively evaluated by clinical and neuropsychological assessment, resting EEG, and 123 I-FP-CIT-SPECT and clinically classified into mainly motor, diffuse/malignant, and intermediate PD subtypes; they were then followed up for an average of 5 years. Cognitive outcome was defined by identifying cognitively stable or worsened patients. RESULTS: Step-wise logistic regression selected the posterior qEEG mean frequency and 123 I-FP-CIT-SPECT uptake at caudate level (P < 0.0001). The posterior qEEG mean frequency (cut point, 8.3 Hz) and the caudate 123 I-FP-CIT-SPECT uptake (cut point, 2.3, specific to nondisplaceable binding ratio) achieved 82% and 80% of accuracy, respectively, in predicting cognitive outcome. Survival analysis showed decreasing expected time to cognitive worsening associated with scores below the established thresholds for qEEG and 123 I-FP-CIT-SPECT and with the presence of a malignant clinical phenotype. CONCLUSIONS: Resting EEG and 123 I-FP-CIT-SPECT are good predictors of future cognitive worsening, in de novo drug-naive PD patients. Wherever available, these biomarkers could add valuable prognostic information to classification into different clinical phenotypes. © 2017 International Parkinson and Movement Disorder Society.

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.

Genetically engineered hairy root cultures of Hyoscyamus senecionis and H. muticus: ploidy as a promising parameter in the metabolic engineering of tropane alkaloids.

KEY MESSAGE: Tetraploidy improves overexpression of h6h and scopolamine production of H. muticus, while in H. senecionis, pmt overexpression and elicitation can be used as effective methods for increasing tropane alkaloids. The effects of metabolic engineering in a polyploid context were studied by overexpression of h6h in the tetraploid hairy root cultures of H. muticus. Flow cytometry analysis indicated genetic stability in the majority of the clones, while only a few clones showed genetic instability. Among all the diploid and tetraploid clones, the highest level of h6h transgene expression and scopolamine accumulation was interestingly observed in the tetraploid clones of H. muticus. Therefore, metabolic engineering of the tropane biosynthetic pathway in polyploids is suggested as a potential system for increasing the production of tropane alkaloids. Transgenic hairy root cultures of Hyoscyamus senecionis were also established. While overexpression of pmt in H. senecionis was correlated with a sharp increase in hyoscyamine production, the h6h-overexpressing clones were not able to accumulate higher levels of scopolamine than the leaves of intact plants. Applying methyl jasmonate was followed by a sharp increase in the expression of pmt and a drop in the expression of tropinone reductase II (trII) which consequently resulted in the higher biosynthesis of hyoscyamine and total alkaloids in H. senecionis.

Inhibitor mechanisms in the S1 binding site of the dopamine transporter defined by multi-site molecular tethering of photoactive cocaine analogs.

Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility strategies to identify the specific adduction site and binding pose for the crosslinkable, photoactive cocaine analog, RTI 82, which contains a photoactive azide attached at the 2ß position of the tropane pharmacophore. Here, we utilize similar methodology with a different cocaine analog N-[4-(4-azido-3-I-iodophenyl)-butyl]-2-carbomethoxy-3-(4-chlorophenyl)tropane, MFZ 2-24, where the photoactive azide is attached to the tropane nitrogen. In contrast to RTI 82, which crosslinked into residue Phe319 of transmembrane domain (TM) 6, our findings show that MFZ 2-24 adducts to Leu80 in TM1 with modeling and biochemical data indicating that MFZ 2-24, like RTI 82, occupies the central S1 binding pocket with the (+)-charged tropane ring nitrogen coordinating with the (-)-charged carboxyl side chain of Asp79. The superimposition of the tropane ring in the three-dimensional binding poses of these two distinct ligands provides strong experimental evidence for cocaine binding to DAT in the S1 site and the importance of the tropane moiety in competitive mechanisms of DA uptake inhibition. These findings set a structure-function baseline for comparison of typical and atypical DAT inhibitors and how their interactions with DAT could lead to the loss of cocaine-like behaviors.

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.

Functional neuroimaging and clinical features of drug naive patients with de novo Parkinson's disease and probable RBD.

INTRODUCTION: The association between Parkinson Disease (PD) and REM sleep behavior disorder (RBD) has been related to a specific, malignant clinical phenotype. Definite RBD diagnosis requires video-polysomnography that is often unfeasible. A malignant clinical PD-RBD phenotype could be expected also in PD patients with probable RBD. Aim of this cross-sectional study was to evaluate whether a more severe neuropsychological and functional neuroimaging phenotype can be identified in PD patients with probable RBD. METHODS: Thirty-eight de novo, drug naïve PD patients underwent a first-line clinical assessment and a second-line multimodal assessment, including neuropsychological evaluation, (123)I-FP-CIT-SPECT and (18)F-FDG-PET, which were compared between PD patients with (PD + RBD+) and without (PD + RBD-) probable RBD. RESULTS: On first-line assessment, PD + RBD + patients had significantly more constipation (p = 0.02) and showed worse olfaction (p = 0.01) compared with PD + RBD-while the two groups were similar as for age, presence of orthostatic hypotension, UPDRS-III and MMSE scores. On second-line assessment, PD + RBD + patients showed a worse neuropsychological test profile, more severe nigro-striatal dopaminergic impairment, mainly at caudate level in the less affected hemisphere (p = 0.004) and impaired brain glucose metabolism, with relative hypometabolism in posterior cortical regions and relative hypermetabolism mainly in anterior regions of the more affected hemisphere (p = 0.015). CONCLUSIONS: PD patients with probable RBD are likely to have a more severe neuropsychological and functional brain-imaging phenotype already at the time of diagnosis.

Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids.

Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8-8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65mM, 88.3nkatmg(-1) and 2.93S(-1), respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18mM, 81.20nkatmg(-1) and 2.40S(-1) at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56mM, 171.62nkat.mg(-1) and 5.69S(-1), respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34mM, 111.90nkatmg(-1) and 3.30S(-1), respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea.

Reduced Dopamine Transporter Availability and Neurocognitive Deficits in Male Patients with Alcohol Dependence.

Dopamine plays an important role in the development of alcohol dependence, cognitive dysfunction, and is regulated via dopamine transporter activity. Although dopamine transporter activity is critically involved in alcohol dependence, studies observing this relationship are limited. Thus the current study examined whether dopamine transporter availability is associated with developing of alcohol dependence and cognitive dysfunction. Brain imaging with 99mTc-TRODAT-1 as a ligand was used to measure dopamine transporter availability among 26 male patients with pure alcohol dependence and 22 age- and sex- matched healthy volunteers. The Wisconsin Card Sorting Test (WCST) and Tridimensional Personality Questionnaire (TPQ) were administered to assess neurocognitive functioning and personality traits, respectively. Compared to healthy controls, patients with alcohol dependence showed a significant reduction in dopamine transporter availability (p < 0.001), as well as diminished performance on the WCST (p < 0.001). Dopamine transporter availability was negatively correlated with both total and perseverative WCST errors among healthy controls, but only patients with alcohol dependence showed a positive correlation between dopamine transporter availability and a harm avoidance personality profile. Thus, reductions in dopamine transporter availability may play a pathophysiological role in the development of pure alcohol dependence, given its association with neurocognitive deficits. Moreover, personality may influence the development of pure alcohol dependence; however, additional clinical subgroups should be examined to confirm this possibility.

The Securinega alkaloids.

Securinega alkaloids represent a family of plant secondary metabolites known for 50 years. Securinine (1), the most abundant and studied alkaloid of this series was isolated by Russian researchers in 1956. In the following years, French and Japanese scientists reported other Securinega compounds and extensive work was done to elucidate their intriguing structures. The homogeneity of this family relies mainly on its tetracyclic chemical backbone, which features a butenolide moiety (cycle D) and an azabicyclo[3.2.1]octane ring system (rings B and C). Interestingly, after a period of latency of 20 years, the Securinega topic reemerged as a prolific source of new natural structures and to date more than 50 compounds have been identified and characterized. The oligomeric subgroup gathering dimeric, trimeric, and tetrameric units is of particular interest. The unprecedented structure of the Securinega alkaloids was the subject of extensive synthetic efforts culminating in several efficient and elegant total syntheses. The botanical distribution of these alkaloids seems limited to the Securinega, Flueggea, Margaritaria, and Breynia genera (Phyllanthaceae). However, only a limited number of plant species have been considered for their alkaloid contents, and additional phytochemical as well as genetic studies are needed. Concerning the biosynthesis, experiments carried out with radiolabelled aminoacids allowed to identify lysine and tyrosine as the precursors of the piperidine ring A and the CD rings of securinine (1), respectively. Besides, plausible biosynthetic pathways were proposed for virosaine A (38) and B (39), flueggine A (46), and also the different oligomers flueggenine A-D (48-51), fluevirosinine A (56), and flueggedine (20). The case of nirurine (45) and secu'amamine (37) remains elusive and additional studies seem necessary to understand their mode of production. The scope of biological of activities of the Securinega alkaloids was mainly centered on the CNS activity of securinine (1), although the exact mechanism of action remained in part unknown. Nevertheless, for its stimulant and antispasmodic effects securinine nitrate was marketed as a drug in the USSR until the early 1990s. Moreover, securinine (1) and several other Securinega alkaloids recently demonstrated promising anticancer properties. In particular securinine (1) demonstrated markedly benefits in the treatment of acute myeloid leukemia.

Anatoxin-a producing Tychonema (Cyanobacteria) in European waterbodies.

In order to identify the cyanobacterial species responsible of anatoxin-a (ATX) production in Lake Garda (Northern Italy), an intensive isolation and culturing of filamentous cyanobacteria were established since 2014 from environmental samples. In this work, we report a detailed account of the strategy adopted, which led to the discovery of a new unexpected producer of ATX, Tychonema bourrellyi. So far, this species is the first documented example of cultured Oscillatoriales able to produce ATX isolated from pelagic freshwater ecosystems. The isolated filaments were identified adopting a polyphasic approach, which included microscopic species identification, genetic characterisation and phylogenetic analyses based on 16S rRNA genes. The taxonomic identification was further confirmed by the high (>99%) rbcLX sequence similarities of the T. bourrellyi strains of Lake Garda with those deposited in DNA sequence databases. More than half of the isolates were shown to produce a significant amount of ATX, with cell quota ranging between 0.1 and 2.6 µg mm(-3), and 0.01 and 0.35 pg cell(-1). The toxic isolates were tested positive for anaC of the anatoxin-a synthetase (ana) gene cluster. These findings were confirmed with the discovery of one ATX producing T. bourrellyi strain isolated in Norway. This strain and a further non-ATX producing Norwegian Tychonema bornetii strain tested positive for the presence of the anaF gene of the ana gene cluster. Conversely, none of the Italian and Norwegian Tychonema strains were positive for microcystins (MCs), which was also confirmed by the absence of mcyE PCR products in all the samples analysed. This work suggests that the only reliable strategy to identify cyanotoxins producers should be based on the isolation of strains and their identification with a polyphasic approach associated to a concurrent metabolomic profiling.

Conformational changes in dopamine transporter intracellular regions upon cocaine binding and dopamine translocation.

The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward- and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward- and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.

Increased 99mTc TRODAT-1 uptake in anaplastic oligodendroglioma.

(99m)Tc TRODAT-1, a selective dopamine transporter SPECT imaging agent, has demonstrated its efficacy in identifying patients with Parkinson disease. Primary or metastatic brain neoplasm uptake of TRODAT-1 is rarely reported in literatures. A 51-year-old female patient underwent TRODAT-1 study for bradykinesia and altered cognitive function; the images showed abnormal extrastriatal uptake in the right frontal lobe subsequent to operation, and pathological examination confirmed anaplastic oligodendroglioma. Care should be taken in interpreting TRODAT-1 image; any focus on abnormal accumulation of radiotracer should not be overlooked because it can be brain neoplasm as demonstrated in this case.

The incidental suggestive meningioma presenting as high 18F FP-CIT uptake on PET/CT study.

A 76-year-old woman with about a 13-month history of bradykinesia, gait disturbance and resting tremor of right upper extremity was referred to Nuclear Medicine Department. A PET-CT with 229 MBq of F FP-CIT was performed to diagnose suspected early Parkinson's disease (PD). The PET-CT showed a mass lesion with highly intense focal dopamine transporter uptake in the right frontal lobe. A subsequent brain magnetic resonance image also showed a mass in the right frontal lobe demonstrating homogeneous enhancement and extensive surrounding edema, highly suggestive of a brain tumor.

Phormidium autumnale growth and anatoxin-a production under iron and copper stress.

Studies on planktonic cyanobacteria have shown variability in cyanotoxin production, in response to changes in growth phase and environmental factors. Few studies have investigated cyanotoxin regulation in benthic mat-forming species, despite increasing reports on poisoning events caused by ingestion of these organisms. In this study, a method was developed to investigate changes in cyanotoxin quota in liquid cultures of benthic mat-forming cyanobacteria. Iron and copper are important in cellular processes and are well known to affect growth and selected metabolite production in cyanobacteria and algae. The effect of iron (40-4000 µg L(-1)) and copper (2.5-250 µg L(-1)) on growth and anatoxin-a quota in Phormidium autumnale was investigated in batch culture. These concentrations were chosen to span those found in freshwater, as well as those previously reported to be toxic to cyanobacteria. Anatoxin-a concentrations varied throughout the growth curve, with a maximum quota of between 0.49 and 0.55 pg cell(-1) measured within the first two weeks of growth. Growth rates were significantly affected by copper and iron concentrations (P < 0.0001); however, no statistically significant difference between anatoxin-a quota maxima was observed. When the iron concentrations were 800 and 4000 µg L(-1), the P. autumnale cultures did not firmly attach to the substratum. At 250 µg L(-1) copper or either 40 or 4000 µg L(-1) iron, growth was suppressed.

Autologous transplantation of GDNF-expressing mesenchymal stem cells protects against MPTP-induced damage in cynomolgus monkeys.

Glial cell-derived neurotrophic factor (GDNF) has shown beneficial effects in models of Parkinson's disease. The mild results observed in the double-blind clinical trial by intraputamenal infusion of recombinant GDNF proteins warrant a search for alternative delivery methods. In this study, we investigated the function of autologous mesenchymal stem cells (MSCs) expressing GDNF (GDNF-MSCs) for protection against MPTP-induced injury in cynomolgus monkeys. MSCs were obtained from the bone marrow of individual monkeys and gene-modified to express GDNF. Following unilateral engraftment of GDNF-MSCs into the striatum and substantia nigra, the animals were challenged with MPTP to induce a stable systemic Parkinsonian state. The motor functions were spared in the contralateral limbs of monkeys receiving GDNF-MSCs, but not in those receiving MSCs alone. In the striatum of the grafted hemisphere, dopamine levels were higher and dopamine uptake was enhanced. The results suggest that autologous MSCs may be a safe vehicle to deliver GDNF for enhancing nigro-striatum functions.