Polyphenol oxidase (PPO) arm of catecholamine pathway catalyzes the conversion of L-tyrosine to L-DOPA in Mucuna pruriens (L.) DC var. pruriens: An integrated pathway analysis using field grown and in vitro callus cultures.

Mucuna pruriens (L.) DC var. pruriens is the natural source for L-DOPA, precursor of the neurotransmitter dopamine, used widely in the treatment of Parkinson's disease. However, L-DOPA synthesis in plants is mediated either by Catecholamine (CA) pathway or alternate pathway catalyzed by Cytochrome P450 (CYP450) class of enzymes. Interestingly, the CA pathway itself can be initiated either by tyrosine hydroxylase (TH) or polyphenol oxidase (PPO). The CA pathway mediated synthesis of L-DOPA has not yet been proved in M. pruriens albeit strong indications. Therefore, the present investigation is focused on metabolite analysis of major intermediates of CA pathway up to the formation of dopamine and expression analysis of the selected genes, in different tissues and callus cultures. The four major intermediates, L-tyrosine, tyramine, L-DOPA and dopamine, were detected using NMR spectroscopy and quantified by HPLC in the callus cultures and in different tissues of the field plant, respectively. The various stages of leaf tissue were also analyzed for metabolite profiling. The relative amount of intermediates detected during the ontogeny of leaf indicates that PPO mediated conversion of L-tyrosine to dopamine through L-DOPA is relatively higher compared to dopamine production from tyramine. Among the two possible enzymes, activity of PPO was 6.5-fold more than TH in metabolically active young leaves compared to intermediate leaves. The gene expression profiles comprising upstream genes of L-tyrosine synthesis and downstream up to dopamine synthesis shows strong correlation with L-DOPA synthesis. The study validates CA pathway mediated synthesis of L-DOPA with PPO as candidate enzyme, in M. pruriens.

L-DOPA synthesis in Mucuna pruriens (L.) DC. is regulated by polyphenol oxidase and not CYP 450/tyrosine hydroxylase: An analysis of metabolic pathway using biochemical and molecular markers.

Mucuna pruriens L., commonly known as velvetbean or cow-itch, is a self-pollinated tropical legume of the family Fabaceae, known for its medicinal properties. The active principle L-DOPA extracted from the plant is a potent drug used in the treatment of Parkinson's disease. Although, it is hypothesized that a single step reaction can produce L-DOPA, the presence of optional routes makes the pathway more intricate. For instance, the catecholamine biosynthetic pathway, which leads to L-DOPA production, could occur by hydroxylation of tyrosine to L-DOPA either by polyphenol oxidase (PPO) or tyrosine hydroxylase (TH). Furthermore, Cytochrome P450 (CYP) enzymes can also cause hydroxylation of tyrosine, resulting in L-DOPA synthesis. Therefore, the present investigation was focused on validating the step, which catalyzes the synthesis of L-DOPA, at the biochemical and molecular levels. Enzyme inhibitor studies showed significant inhibition of PPO enzyme with corresponding decrease in L-DOPA synthesis while TH and CYP inhibition had no effect on L-DOPA synthesis. Activity staining of non-denaturing PAGE gel for PPO and TH showed activity only to PPO enzyme. Following in-gel assay and tryptic digestion of the excised stained gel portion, peptide recovery and LC-MS/MS analysis were performed. Degenerate primers based on peptide sequence resulted in an 800bp amplicon. The subsequent sub-cloning, RACE analysis and BLAST search resulted in the isolation of full-length PPO coding sequence of 1800 bp. Structure prediction and phylogenetic analysis of the obtained sequence revealed strong similarity to other plant PPO's like Glycine max, Vigna radiata and Vicia faba of the same family.

Corosolic acid content and SSR markers in Lagerstroemia speciosa (L.) Pers.: a comparative analysis among populations across the Southern Western Ghats of India.

Lagerstroemia speciosa commonly known as 'Banaba' is native of south-east Asia which exhibits both horticultural and therapeutic value. The anti-diabetic and anti-obese property of the tree is attributed to corosolic acid (CRA)-a pentacyclic triterpene seen predominantly in the mature leaves. Although there are studies on either chemical or genetic variation in L. speciosa from different regions, none have dealt with their association to discuss the formation of chemical diversity. For the first time, we have analyzed CRA content in 12 natural populations corresponding to 42 samples seen in the Southern Western Ghats (SWG) using chromatography techniques and genetic variation estimated using SSR markers. Significant variation in percentage distribution of CRA ranging from 0.005% to 0.868% dr.wt. was recorded wherein populations from the north SWG contain relatively more active principle (mean=0.321%) than their counterparts in the south (mean=0.064%). Similarly, SSR data showing relatively high rate of gene flow (Nm=2.72) and low genetic differentiation (FST=0.14) is indicative that populations from north are genetically more diverse than those in the south (Nm=0.48; FST=0.38). The scatter plot derived by Principle Component Analysis (PCA) of chemical and genetic data shows similar pattern of clustering that reveals strong association between the two sets of data. It is concluded that the observed variation in CRA content in natural populations of the species depends more on the genetic background and less on edaphic factors.

Rheumatoid arthritis patients have elevated antibodies to cross-reactive and non cross-reactive antigens from Proteus microbes.

BACKGROUND AND OBJECTIVE: Although a large number of independent studies have shown a paramount role for Proteus mirabilis in the aetiopathogenesis of rheumatoid arthritis (RA), this hypothesis is still controversial among rheumatologists. The main obstacle to its acceptance is the impression that increased Proteus antibodies in RA patients is a secondary phenomenon, occurring as the result of cross-reactivity between bacterial and self-antigens. To shed light on this problem, we examined the link between antibodies to various cross-reactive and non cross-reactive antigenic peptides from P. mirabilis and analysed the relationship between these antibodies and disease severity in patients with RA. METHODS: Using the ELISA method, serum samples from 70 RA patients and 20 healthy controls were screened for total and class-specific antibodies against three human cross-reactive and non-crossreactive synthetic peptides from P. mirabilis haemolysin, urease C and urease F enzymes. An antibody index, which comprised the total concentration of antibodies against these peptides in each sample, was correlated with the biochemical parameters of disease activity and/or severity, such as the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and rheumatoid factors (RF). Furthermore, anti-peptide antibody indices were evaluated among RA patients with different levels of disease activity as defined by ESR and CRP. RESULTS: Significantly elevated levels of total and class-specific IgG antibodies against the 3 Proteus peptides were observed among RA patients compared to healthy controls (p < 0.001). Active RA patients had elevated IgM antibodies against all peptides compared to healthy subjects (p < 0.001). However, no such elevation was observed in IgA anti-peptide antibodies in RA patients. A positive correlation was observed between the antibody indices and ESR (p < 0.001) and CRP (p < 0.01) concentrations, but not the RF status or disease duration. Furthermore, more than 90% of active RA patients showed positive values for the Proteus anti-peptide indices. CONCLUSION: The elevated levels of antibodies against Proteus antigenic epitopes (which are cross-reactive or non cross-reactive with human tissue antigens) observed indicates that this enhanced bacterial immune response in RA patients is specifically triggered by Proteus microbes. Furthermore, the correlation of anti-peptide antibody indices with the biochemical markers of disease activity indicates that these antibodies exert damaging cytotoxic effects on joint tissues during the course of the disease.