Cloning and Overexpression of Strictosidine ß-D-Glucosidase Gene Short Sequence from Catharanthus roseus in Escherichia coli.

Purpose: Strictosidine-ß-D-glucosidase (SGD) is considered as a key enzyme in the production of bisindole alkaloids in Catharanthus roseus. The present study illustrated the production of a short sequence of this enzyme in Escherichia coli without codon optimization. Methods: Strictosidine-ß-D-glucosidase (sgd) gene short sequence (1434 bp), which lacks the conserved sequence KGFFVWS and the localization peptide sequence at the C-terminal, was amplified from cDNA of C. roseus leaves, cloned and expressed in Escherichia coli. The activity of the produced protein in cell free lysate was tested using total alkaloid extract of C. roseus leaves. Results: HPLC and LC-MS analysis of the assay mixture revealed the disappearance of the strictosidine peak. Conclusion: SGD short sequence can be produced in Escherichia coli in active form without codon optimization.

Sulfadoxine-pyrimethamine plus artesunate compared with chloroquine for the treatment of vivax malaria in areas co-endemic for Plasmodium falciparum and P. vivax: a randomised non-inferiority trial in eastern Afghanistan.

Chloroquine (CQ) is an effective treatment of choice for vivax malaria in most settings, but with the spread of CQ-resistant Plasmodium falciparum, many countries now use artemisinin-based combination therapy for treatment of falciparum malaria. In areas co-endemic for falciparum and vivax malaria incorrect differential diagnosis is always a risk. In Afghanistan the adoption of sulfadoxine-pyrimethamine plus artesunate (SP+AS) as first-line falciparum treatment raises the prospect of a significant proportion of vivax malaria being misdiagnosed and treated with the combination. SP is considered to have limited efficacy against vivax malaria, and the efficacy of SP+AS against Plasmodium vivax has not been established in areas that are using SP+AS. A randomised, non-inferiority trial comparing SP+AS with CQ monotherapy was undertaken on 190 vivax malaria patients in eastern Afghanistan. Standard WHO procedures for in vivo evaluation of antimalarial drugs were followed. A total of 180 individuals completed the trial to day 42. Using a per protocol analysis, both regimens resulted in > or =96% treatment success at 28 d, but significantly more cases failed in the CQ arm (46%) than in the SP+AS arm (24%) by day 42. In areas where vivax infections might be misdiagnosed as falciparum infections and treated with SP+AS, patient management would be as good, or better than, with the standard CQ treatment.

Biotransformation of chrysin and apigenin by Cunninghamella elegans.

Biotransformation of chrysin by Cunninghamella elegans NRRL 1392 produced apigenin, apigenin 7-sulfate, apigenin 7,4'-disulfate, and a new metabolite identified as chrysin 7-sulfate. On the other hand, fermentation of apigenin, using the same microorganism, yielded apigenin 7-sulfate and apigenin 7,4'-disulfate. The structures of the metabolites were established by spectral analysis, and acid and enzyme hydrolyses in addition to comparison with reference samples.

Efficacy of combination therapy with artesunate plus amodiaquine compared to monotherapy with chloroquine, amodiaquine or sulfadoxine-pyrimethamine for treatment of uncomplicated Plasmodium falciparum in Afghanistan.

INTRODUCTION: In South and Central Asia resistance to chloroquine (CQ) has reached unmanageable levels, and resistance to sulfadoxine-pyrimethamine (SP) is emerging. Amodiaquine (AQ) is widely used in the region, and elsewhere shows only partial resistance to CQ. In Afghanistan, one option for slowing the spread of resistance and improving treatment outcomes is the use of artemisinin combination therapy (ACT). METHODS: The efficacy of CQ, AQ, SP and amodiaquine plus artesunate (AQ/AS) in the treatment of uncomplicated falciparum malaria was investigated using standard World Health Organization (WHO) procedures. Malaria patients were randomized to four treatment groups: 268 were enrolled and 240 completed the trial. RESULTS: There was a high level of cross-resistance between CQ and AQ resistance: adequate clinical and parasitological response by day 42 was 11% after CQ treatment and 9% after AQ treatment. The trend of treatment failure between AQ and CQ was almost identical. Cure rates were considerably improved by the addition of artesunate to AQ or by use of SP; adequate clinical and parasitological response being 72% for AQ/AS and 92% for SP. The combination of AS/AQ substantially reduced the odds of treatment failure relative to AQ monotherapy by day 42 [odds ratio (OR) = 0.03, 95% confidence interval (CI) 0.01-0.1] in addition to reducing the proportion of patients with gametocytes throughout the 42-day period. Gametocyte carriage rate was only marginally higher in the SP than in the CQ- and AQ-treated groups. CONCLUSION: The therapeutic and parasitological cure rates with AS/AQ were inadequate, and the criteria for deploying ACT - namely to prevent further selection of drug resistance from a position of low frequency - was not met in the region. An alternative drug combination to AQ/AS is required for Afghanistan.

O-demethylation and sulfation of 7-methoxylated flavanones by Cunninghamella elegans.

Metabolism of 7-O-methylnaringenin (sakuranetin) by Cunninghamella elegans NRRL 1392 yielded naringenin and naringenin-4'-sulfate. C. elegans also converted 5, 3', 4'-trihydroxy-7-methoxyflavanone into eriodictyol-4'-sulfate. Furthermore, incubation of 5, 4'-dihydroxy-7, 3'-dimethoxyflavanone with the same fungus gave homoeriodictyol (5, 7, 4'-trihydroxy-3'-methoxyflavanone) and homoeriodicytol-7-sulfate. The structures of the new metabolites were established by spectral analysis including 2D-NMR, HR-ESI-FT-MS beside hydrolysis by acid.