Activate the endogenous Cu2+ switch for Zn(DDC)2 liposomes conversion: Providing a safer and less toxic alternative in cancer therapy.

The ancient anti-alcohol drug disulfiram (DSF) has gained widespread attention for its highly effective anti-tumor effects in cancer treatment. Our previous studies have developed liposome of Cu (DDC)2 to overcome the limitations, like the poor water solubility. However, Cu (DDC)2 liposomes still have shown difficulties in severe hemolytic reactions at high doses and systemic toxicity, which have limited their clinical use. Therefore, this study aims to exploratively investigate the feasibility of using DSF or DDC in combination also can chelate Zn2+ to form zinc diethyldithiocarbamate (Zn (DDC)2). Furthermore, this study prepared stable and homogeneous Zn (DDC)2 liposomes, which were able to be released in the tumor microenvironment (TME). The released Zn (DDC)2 was converted to Cu (DDC)2 with the help of endogenous Cu2+-switch enriched in the TME, which has a higher stability constant compared with Zn (DDC)2. In other words, the Cu2+-switch is activated at the tumor site, completing the conversion of the less cytotoxic Zn (DDC)2 to the more cytotoxic Cu (DDC)2 for effective tumor therapy so that the Zn (DDC)2 liposomes in vivo achieved the comparable therapeutic efficacy and provided a safer alternative to Cu (DDC)2 liposomes in cancer therapy.

A position statement on the post gene-therapy rehabilitation of aromatic I-amino acid decarboxylase deficiency patients.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare genetic disorder of monoamine neurotransmitter synthesis that presents with a range of symptoms, including motor dysfunction and limited attainment of developmental motor milestones. The approval of eladocagene exuparvovec, a gene therapy for AADC deficiency with demonstrated efficacy for motor improvements, now expands the range of motor outcomes possible for patients with this disorder. However, recommendations and guidelines for therapy following treatment with gene therapy are lacking. To ensure patients can reach their full potential following treatment with gene therapy, it is essential they receive rehabilitation therapies designed specifically with their impairments and goals in mind. Therefore, we highlight specific rehabilitative needs of patients following gene therapy and propose a set of recommendations for the post-treatment period based on collective experiences of therapists, physicians, and caregivers treating and caring for patients with AADC deficiency who have been treated with gene therapy. These recommendations include a focus on periods of intensive therapy, facilitating active movements, training for functional abilities, cognitive and communication training, parent/caregiver empowerment, collaboration between therapists and caregivers to develop in-home programs, and the incorporation of supplemental forms of therapy that patients and their families may find more enjoyable and engaging. Many of these rehabilitative strategies may be employed prior to gene therapy. However, these recommendations will be valuable for therapists, caregivers, and wider treatment teams as they prepare for the post-treatment journey with these patients. Furthermore, the considerations and recommendations presented here may prove beneficial outside the AADC deficiency community as gene therapies and other treatments are developed and approved for other rare diseases.

Melatonin enhances stem strength by increasing lignin content and secondary cell wall thickness in herbaceous peony.

Cut flower quality is severely restrained by stem bending due to low stem strength. Melatonin has been shown to function in many aspects of plant growth and development, yet whether it can enhance stem strength, and the corresponding underlying mechanisms remain unclear. We investigated the role of melatonin in enhancement of stem strength in herbaceous peony (Paeonia lactiflora Pall.) by applying exogenous melatonin and changing endogenous melatonin biosynthesis. Endogenous melatonin content positively correlated with lignin content and stem strength in various P. lactiflora cultivars. Supplementation with exogenous melatonin significantly enhanced stem strength by increasing lignin content and the S/G lignin compositional ratio, up-regulating lignin biosynthetic gene expression. Moreover, overexpression of TRYPTOPHAN DECARBOXYLASE GENE (TDC) responsible for the first committed step of melatonin biosynthesis in tobacco, significantly increased endogenous melatonin, which further increased the S/G ratio and stem strength. In contrast, silencing PlTDC in P. lactiflora decreased endogenous melatonin, the S/G ratio and stem strength. Finally, manipulating the expression of CAFFEIC ACID O-METHYLTRANSFERASE GENE (COMT1), which is involved in both melatonin and lignin biosynthesis, showed even greater effects on melatonin, the S/G ratio and stem strength. Our results suggest that melatonin has a positive regulatory effect on P. lactiflora stem strength.

WRKY1-mediated regulation of tryptophan decarboxylase in tryptamine generation for withanamide production in Withania somnifera (Ashwagandha).

KEY MESSAGE: WsWRKY1-mediated transcriptional modulation of Withania somnifera tryptophan decarboxylase gene (WsTDC) helps to regulate fruit-specific tryptamine generation for production of withanamides. Withania somnifera is a highly valued medicinal plant. Recent demonstration of novel indolyl metabolites called withanamides in its fruits (berries) prompted us to investigate its tryptophan decarboxylase (TDC), as tryptophan is invariably a precursor for indole moiety. TDC catalyzes conversion of tryptophan into tryptamine, and the catalytic reaction constitutes a committed metabolic step for synthesis of an array of indolyl metabolites. The TDC gene (WsTDC) was cloned from berries of the plant and expressed in E. coli. The recombinant enzyme was purified and characterized for its catalytic attributes. Catalytic and structural aspects of the enzyme indicated its regulatory/rate-limiting significance in generation of the indolyl metabolites. Novel tissue-wise and developmentally differential abundance of WsTDC transcripts reflected its preeminent role in withanamide biogenesis in the fruits. Transgenic lines overexpressing WsTDC gene showed accumulation of tryptamine at significantly higher levels, while lines silenced for WsTDC exhibited considerably depleted levels of tryptamine. Cloning and sequence analysis of promoter of WsTDC revealed the presence of W-box in it. Follow-up studies on isolation of WsWRKY1 transcription factor and its overexpression in W. somnifera revealed that WsTDC expression was substantially induced by WsWRKY1 resulting in overproduction of tryptamine. The study invokes a key role of TDC in regulating the indolyl secondary metabolites through enabling elevated flux/supply of tryptamine at multiple levels from gene expression to catalytic attributes overall coordinated by WsWRKY1. This is the first biochemical, molecular, structural, physiological and regulatory description of a fruit-functional TDC.

Microbial Synthesis of Human-Hormone Melatonin at Gram Scales.

Melatonin is a commercially attractive tryptophan-derived hormone. Here we describe a bioprocess for the production of melatonin using Escherichia coli to high titers. The first engineered strain produced 0.13 g/L of melatonin from tryptophan under fed-batch fermentation conditions. A 4-fold improvement on melatonin titer was further achieved by (1) protein engineering of rate-limiting tryptophan hydroxylase to improve 5-hydroxytryptophan biosynthesis and (2) chromosomal integration of aromatic-amino-acid decarboxylase to limit byproduct formation and to minimize gene toxicity to the host cell. Fermentation optimization improved melatonin titer by an additional 2-fold. Deletion of yddG, a tryptophan exporter, exhibited an additive beneficial effect. The final engineered strain produced ∼2.0 g/L of melatonin with tryptophan supplemented externally and ∼1.0 g/L with glucose as the sole carbon source for tryptophan supply. This study lays the foundation for further developing a commercial melatonin-producing E. coli strain.

Bioavailability and Neuroprotectivity of 3-(3, 4-dimethoxy phenyl)-1-4 (methoxy phenyl) prop-2-en-1-one against Schizophrenia: an in silico approach.

Schizophrenia is a major debilitating disorder worldwide. Schizophrenia is a result of multi-gene mutation and psycho-social factors. Mutated amino acid sequences in genes of DOPA such as TH, DDC, DBH, VMAT2, and NMDA (SET-1) have been implicated as major factors causing schizophrenia. In addition mutations in genes other than the DOPA genes such as RGS4, NRG1, COMT, AKT1 and DTNBP1 (SET 2) have also been implicated in the pathogenesis of schizophrenia. Several medicinal herbs and their bioactive constituents have been reported to be involved in ameliorating different neurological disorders including schizophrenia. The present study is mainly focused to study the effect of bioactive compound isolated from the celastrus panuculatus on DOPA and other related genes of schizophrenia using in silico approach. Moledular docking study was carriedout aginast all the selected targets with the lingds i.e. compound and clozapine using the autodock vina 4.0 module implemented in Pyrx 2010.12. The 3 D structures of genes of intrest were retrieved from the protein data bank (PDB). The bioavailability and pharmacological properties of the ligands were determined using OSIRIS server. The novelty of the compound was determined based on fitness, docking and bioavailability score. From the results it is observed that, the compoud has exhibited best dock score against all the selected targets than the clozapie except DBH and VMAT2 in SET-1 targets of DOPA genes. Where as the compound has shown best pharmacokinetic and biologicl property score than the clozapine. Hence, the compound can be considered for further in vitro and in vivo studies to determine the therapeutic efficacy and drug candidacy of the compound in future.

Rumen-Protected 5-Hydroxytryptophan Improves Sheep Melatonin Synthesis in the Pineal Gland and Intestinal Tract.

BACKGROUND Based on the extensive biological effects of melatonin (MLT), it is beneficial to increase the MLT content in the bodies of animals at a specific physiological stage. This study was conducted to investigate the effect of a diet supplemented with rumen-protected (RP) 5-hydroxytryptophan (5-HTP) on the pineal gland and intestinal tract MLT synthesis of sheep. MATERIAL AND METHODS Eighteen Kazakh sheep were assigned randomly to 3 diet groups: control group (CT, corn-soybean meal basal diet), CT+111 group (111 mg/kg BW RP 5-HTP), and CT+222 group (222 mg/kg BW RP 5-HTP). The gene expressions of aromatic amino acid decarboxylase (AADC), arylalkylamine N-acetyltransferase (AA-NAT), hydroxyindole-O-methyltransferase (HIOMT), monoamine oxidase A (MAOA), and the intermediates of MLT synthesis were observed from the pineal gland and intestinal tract by the reverse transcription (RT)-PCR method. The 5-HTP, 5-HT, N-acetylserotonin (NAS), MLT, and 5-hydroxyindole acetic acid (5-HIAA) contents in the pineal gland and intestinal tract were analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry. RESULTS The study showed that the pineal gland HIOMT expression (P<0.05), MLT (P<0.05) and 5-HIAA (P<0.05) levels in the 222 mg/kg group significantly increased compared to those in the CT and CT+111 mg/kg groups. In addition, the AADC (P<0.01) and AA-NAT (P<0.05) gene expression levels in the duodenum and jejunum were increased by the supplementation of RP 5-HTP. CONCLUSIONS Rumen-protected 5-hydroxytryptophan promoted melatonin synthesis in the pineal gland and intestinal tract during the natural light period.

Clinical Metabolomics to Segregate Aromatic Amino Acid Decarboxylase Deficiency From Drug-Induced Metabolite Elevations.

BACKGROUND: Phenotyping technologies featured in the diagnosis of inborn errors of metabolism, such as organic acid, amino acid, and acylcarnitine analyses, recently have been supplemented by broad-scale untargeted metabolomic phenotyping. We investigated the analyte changes associated with aromatic amino acid decarboxylase (AADC) deficiency and dopamine medication treatment. METHODS: Using an untargeted metabolomics platform, we analyzed ethylenediaminetetraacetic acid plasma specimens, and biomarkers were identified by comparing the biochemical profile of individual patient samples to a pediatric-centric population cohort. RESULTS: Elevated 3-methoxytyrosine (average z score 5.88) accompanied by significant decreases of dopamine 3-O-sulfate (-2.77), vanillylmandelate (-2.87), and 3-methoxytyramine sulfate (-1.44) were associated with AADC deficiency in three samples from two patients. In five non-AADC patients treated with carbidopa-levodopa, levels of 3-methoxytyrosine were elevated (7.65); however, the samples from non-AADC patients treated with DOPA-elevating drugs had normal or elevated levels of metabolites downstream of aromatic l-amino acid decarboxylase, including dopamine 3-O-sulfate (2.92), vanillylmandelate (0.33), and 3-methoxytyramine sulfate (5.07). In one example, a plasma metabolomic phenotype pointed to a probable AADC deficiency and prompted the evaluation of whole exome sequencing data, identifying homozygosity for a known pathogenic variant, whereas whole exome analysis in a second patient revealed compound heterozygosity for two variants of unknown significance. CONCLUSIONS: These data demonstrate the power of combining broad-scale genotyping and phenotyping technologies to diagnose inherited neurometabolic disorders and suggest that metabolic phenotyping of plasma can be used to identify AADC deficiency and to distinguish it from non-AADC patients with elevated 3-methoxytyrosine caused by DOPA-raising medications.

The central anorexigenic mechanism of amylin in Japanese quail (Coturnix japonica) involves pro-opiomelanocortin, calcitonin receptor, and the arcuate nucleus of the hypothalamus.

Amylin is a 37-amino acid peptide hormone that exerts anorexigenic effects in humans and animals. We demonstrated that central injection of amylin into chicks affected feeding and related behaviors via the hypothalamus and brainstem, although the molecular mechanisms remained elusive. Thus, the objective of this study was to investigate the molecular mechanisms underlying anorexigenic effects of amylin in 7 day-old Japanese quail. Food but not water intake was reduced after intracerebroventricular amylin injection, and the behavior analysis indicated that this was associated with decreased food pecks and preening. Whole hypothalamus and hypothalamic nuclei including the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), dorsomedial nucleus (DMN) and lateral hypothalamic area (LH) were extracted from quail at 1h post-injection for total RNA isolation. Real time PCR was performed to quantify mRNA abundance of amylin receptors, appetite-associated neuropeptides and monoamine-synthesis-related enzymes. Central amylin injection increased the mRNA abundance of calcitonin receptor (CALCR), receptor activity modifying protein 1 (RAMP1), pro-opiomelanocortin (POMC), and aromatic l-amino acid decarboxylase (AADC) in the hypothalamus and individual hypothalamic nuclei. Relative quantities of CALCR and POMC mRNA were greater in the ARC of the amylin- than vehicle-treated group. Thus, amylin-mediated effects on food intake may involve POMC, monoamine synthesis, and amylin receptor 1 (a complex of CALCR and RAMP1) in the ARC. Together, these data provide novel insights on the hypothalamic-specific molecular mechanisms of amylin-induced food intake.

Structural and functional analogies and differences between histidine decarboxylase and aromatic l-amino acid decarboxylase molecular networks: Biomedical implications.

Human histidine decarboxylase (HDC) and dopa decarboxilase (DDC) are highly homologous enzymes responsible for the synthesis of biogenic amines (BA) like histamine, and serotonin and dopamine, respectively. The enzymes share many structural and functional analogies, while their product metabolisms also follow similar patterns that are confluent in some metabolic steps. They are involved in common physiological functions, such as neurotransmission, gastrointestinal track function, immunity, cell growth and cell differentiation. As a consequence, metabolic elements of both BA subfamilies are also co-participants in a long list of human diseases. This review summarizes the analogies and differences in their origin (HDC and DDC) as well as their common pathophysiological scenarios. The major gaps of information are also underlined, as they delay the possibility of holistic approaches that would help personalized medicine and pharmacological initiatives for prevalent and rare diseases.

Missing proof of cooperative synthesis of dopamine by non-dopaminergic neurons.

L-DOPA accumulation in the extracellular medium was detected when the transfer of L-DOPA from the neurons containing tyrosine hydroxylase to the neurons containing aromatic L-amino acid decarboxylase was blocked, under conditions of inhibition of the L-DOPA degradation enzyme. Thus, the missing proof confirming the existence of cooperative synthesis of dopamine by neurons non-dopaminergic was obtained.

Melatonin mediates selenium-induced tolerance to cadmium stress in tomato plants.

Both selenium (Se) and melatonin reduce cadmium (Cd) uptake and mitigate Cd toxicity in plants. However, the relationship between Se and melatonin in Cd detoxification remains unclear. In this study, we investigated the influence of three forms of Se (selenocysteine, sodium selenite, and sodium selenate) on the biosynthesis of melatonin and the tolerance against Cd in tomato plants. Pretreatment with different forms of Se significantly induced the biosynthesis of melatonin and its precursors (tryptophan, tryptamine, and serotonin); selenocysteine had the most marked effect on melatonin biosynthesis. Furthermore, Se and melatonin supplements significantly increased plant Cd tolerance as evidenced by decreased growth inhibition, photoinhibition, and electrolyte leakage (EL). Se-induced Cd tolerance was compromised in melatonin-deficient plants following tryptophan decarboxylase (TDC) gene silencing. Se treatment increased the levels of glutathione (GSH) and phytochelatins (PCs), as well as the expression of GSH and PC biosynthetic genes in nonsilenced plants, but the effects of Se were compromised in TDC-silenced plants under Cd stress. In addition, Se and melatonin supplements reduced Cd content in leaves of nonsilenced plants, but Se-induced reduction in Cd content was compromised in leaves of TDC-silenced plants. Taken together, our results indicate that melatonin is involved in Se-induced Cd tolerance via the regulation of Cd detoxification.

Isolation of Catharanthus roseus (L.) G. Don Nuclei and Measurement of Rate of Tryptophan decarboxylase Gene Transcription Using Nuclear Run-On Transcription Assay.

BACKGROUND: An accurate assessment of transcription 'rate' is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription 'rate'. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and therefore serves as a 'molecular hub' to understand gene expression profiles. RESULTS: The protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA). However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli. CONCLUSIONS: Our study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear 'run-on' transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run-on transcript could be further subjected to RNA-Seq for global nuclear run-on assay (GNRO-Seq) for genome wide in-situ measurement of transcription rate of plant genes.

Characterization of A11 neurons projecting to the spinal cord of mice.

The hypothalamic A11 region has been identified in several species including rats, mice, cats, monkeys, zebrafish, and humans as the primary source of descending dopamine (DA) to the spinal cord. It has been implicated in the control of pain, modulation of the spinal locomotor network, restless leg syndrome, and cataplexy, yet the A11 cell group remains an understudied dopaminergic (DAergic) nucleus within the brain. It is unclear whether A11 neurons in the mouse contain the full complement of enzymes consistent with traditional DA neuronal phenotypes. Given the abundance of mouse genetic models and tools available to interrogate specific neural circuits and behavior, it is critical first to fully understand the phenotype of A11 cells. We provide evidence that, in addition to tyrosine hydroxylase (TH) that synthesizes L-DOPA, neurons within the A11 region of the mouse contain aromatic L-amino acid decarboxylase (AADC), the enzyme that converts L-DOPA to dopamine. Furthermore, we show that the A11 neurons contain vesicular monoamine transporter 2 (VMAT2), which is necessary for packaging DA into vesicles. On the contrary, A11 neurons in the mouse lack the dopamine transporter (DAT). In conclusion, our data suggest that A11 neurons are DAergic. The lack of DAT, and therefore the lack of a DA reuptake mechanism, points to a longer time of action compared to typical DA neurons.

Aromatic L-amino acid decarboxylase deficiency is a cause of long-fasting hypoglycemia.

UNLABELLED: OBJECTIVE/CONTEXT: Long-fasting hypoglycemia in children may be induced by neurotransmitter disorders. CASE REPORT: A 5-year-old girl with a medical history of chronic diarrhea presented three episodes of severe hypoglycemia (20 mg/dL) between ages 3 and 5 years. She became pale and sweaty with hypothermia (33.5°C), bradycardia (45 bpm), and acidosis and presented a generalized seizure. During the 17-hour fast test performed to determine the etiology of her hypoglycemia, insulin and C-peptide were appropriately low, and human GH, IGF-I, cortisol, amino acids, and acylcarnitines were in the usual range for fasting duration. However, the presence of vanillactic and vanilpyruvic acids in urine led us to investigate the metabolism of dopamine and serotonin in the cerebrospinal fluid. Indeed, these results indicated an aromatic L-amino acid decarboxylase deficiency that impairs the synthesis of serotonin, dopamine, and catecholamines. The diagnosis was confirmed by the low aromatic L-amino acid decarboxylase (AADC) enzyme activity in plasma (5 pmol/min/mL; reference value, 20-130) and the presence of two heterozygous mutations, c.97G>C (p.V33L, inherited from her father) and c.1385G>C (p.R462P, inherited from her mother) in the DCC gene. She was supplemented with pyridoxine and raw cornstarch (1 g/kg) at evening dinner to reduce the night fast. The episodes of hypoglycemia and the chronic diarrhea were suppressed. CONCLUSION: Here is the first case report of long-fasting hypoglycemia due to a nontypical AADC deficiency. Hypoglycemia was severe, but the other neurological clinical hallmarks present in AADC-deficient patients were mild to moderate. Thus, neurotransmitter disorders should be considered in any patients presenting hypoglycemia with urine excretion of vanillactic acid.

Integration of ¹H NMR and UPLC-Q-TOF/MS for a comprehensive urinary metabonomics study on a rat model of depression induced by chronic unpredictable mild stress.

Depression is a type of complex psychiatric disorder with long-term, recurrent bouts, and its etiology remains largely unknown. Here, an integrated approach utilizing (1)H NMR and UPLC-Q-TOF/MS together was firstly used for a comprehensive urinary metabonomics study on chronic unpredictable mild stress (CUMS) treated rats. More than twenty-nine metabolic pathways were disturbed after CUMS treatment and thirty-six potential biomarkers were identified by using two complementary analytical technologies. Among the identified biomarkers, nineteen (10, 11, 16, 17, 21-25, and 27-36) were firstly reported as potential biomarkers of CUMS-induced depression. Obviously, this paper presented a comprehensive map of the metabolic pathways perturbed by CUMS and expanded on the multitude of potential biomarkers that have been previously reported in the CUMS model. Four metabolic pathways, including valine, leucine and isoleucine biosynthesis; phenylalanine, tyrosine and tryptophan biosynthesis; tryptophan metabolism; synthesis and degradation of ketone bodies had the deepest influence in the pathophysiologic process of depression. Fifteen potential biomarkers (1-2, 4-6, 15, 18, 20-23, 27, 32, 35-36) involved in the above four metabolic pathways might become the screening criteria in clinical diagnosis and predict the development of depression. Moreover, the results of Western blot analysis of aromatic L-amino acid decarboxylase (DDC) and indoleamine 2, 3-dioxygenase (IDO) in the hippocampus of CUMS-treated rats indicated that depletion of 5-HT and tryptophan, production of 5-MT and altered expression of DDC and IDO together played a key role in the initiation and progression of depression. In addition, none of the potential biomarkers were detected by NMR and LC-MS simultaneously which indicated the complementary of the two kinds of detection technologies. Therefore, the integration of (1)H NMR and UPLC-Q-TOF/MS in metabonomics study provided an approach to identify the comprehensive potential depression-related biomarkers and helpful in further understanding the underlying molecular mechanisms of depression through the disturbance of metabolic pathways.

Role of aromatic aldehyde synthase in wounding/herbivory response and flower scent production in different Arabidopsis ecotypes.

Aromatic L-amino acid decarboxylases (AADCs) are key enzymes operating at the interface between primary and secondary metabolism. The Arabidopsis thaliana genome contains two genes, At2g20340 and At4g28680, encoding pyridoxal 5'-phosphate-dependent AADCs with high homology to the recently identified Petunia hybrida phenylacetaldehyde synthase involved in floral scent production. The At4g28680 gene product was recently biochemically characterized as an L-tyrosine decarboxylase (AtTYDC), whereas the function of the other gene product remains unknown. The biochemical and functional characterization of the At2g20340 gene product revealed that it is an aromatic aldehyde synthase (AtAAS), which catalyzes the conversion of phenylalanine and 3,4-dihydroxy-L-phenylalanine to phenylacetaldehyde and dopaldehyde, respectively. AtAAS knock-down and transgenic AtAAS RNA interference (RNAi) lines show significant reduction in phenylacetaldehyde levels and an increase in phenylalanine, indicating that AtAAS is responsible for phenylacetaldehyde formation in planta. In A. thaliana ecotype Columbia (Col-0), AtAAS expression was highest in leaves, and was induced by methyl jasmonate treatment and wounding. Pieris rapae larvae feeding on Col-0 leaves resulted in increased phenylacetaldehyde emission, suggesting that the emitted aldehyde has a defensive activity against attacking herbivores. In the ecotypes Sei-0 and Di-G, which emit phenylacetaldehyde as a predominant flower volatile, the highest expression of AtAAS was found in flowers and RNAi AtAAS silencing led to a reduction of phenylacetaldehyde formation in this organ. In contrast to ecotype Col-0, no phenylacetaldehyde accumulation was observed in Sei-0 upon wounding, suggesting that AtAAS and subsequently phenylacetaldehyde contribute to pollinator attraction in this ecotype.

Integrating carbon-halogen bond formation into medicinal plant metabolism.

Halogenation, which was once considered a rare occurrence in nature, has now been observed in many natural product biosynthetic pathways. However, only a small fraction of halogenated compounds have been isolated from terrestrial plants. Given the impact that halogenation can have on the biological activity of natural products, we reasoned that the introduction of halides into medicinal plant metabolism would provide the opportunity to rationally bioengineer a broad variety of novel plant products with altered, and perhaps improved, pharmacological properties. Here we report that chlorination biosynthetic machinery from soil bacteria can be successfully introduced into the medicinal plant Catharanthus roseus (Madagascar periwinkle). These prokaryotic halogenases function within the context of the plant cell to generate chlorinated tryptophan, which is then shuttled into monoterpene indole alkaloid metabolism to yield chlorinated alkaloids. A new functional group-a halide-is thereby introduced into the complex metabolism of C. roseus, and is incorporated in a predictable and regioselective manner onto the plant alkaloid products. Medicinal plants, despite their genetic and developmental complexity, therefore seem to be a viable platform for synthetic biology efforts.

Neuroprotective effect of Rosmarinus officinalis extract on human dopaminergic cell line, SH-SY5Y.

Hydrogen peroxide (H2O2) is a major Reactive Oxygen Species (ROS), which has been implicated in many neurodegenerative conditions including Parkinson's disease (PD). Rosmarinus officinalis (R. officinalis) has been reported to have various pharmacological properties including anti-oxidant activity. In this study, we investigated the neuroprotective effects of R. officinalis extract on H2O2-induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that H2O2-induced cytotoxicity in SH-SY5Y cells was suppressed by treatment with R. officinalis. Moreover, R. officinalis was very effective in attenuating the disruption of mitochondrial membrane potential and apoptotic cell death induced by H2O2. R. officinalis extract effectively suppressed the up-regulation of Bax, Bak, Caspase-3 and -9, and down-regulation of Bcl-2. Pretreatment with R. officinalis significantly attenuated the down-regulation of tyrosine hydroxylase (TH), and aromatic amino acid decarboxylase (AADC) gene in SH-SY5Y cells. These findings indicate that R. officinalis is able to protect the neuronal cells against H2O2-induced injury and suggest that R. officinalis might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.