Biosynthesis of pleuromutilin congeners using an Aspergillus oryzae expression platform.

Pleuromutilin is an antibiotic diterpenoid made by Clitopilus passeckerianus and related fungi, and it is the progenitor of a growing class of semi-synthetic antibiotics used in veterinary and human medicine. To harness the biotechnological potential of this natural product class, a full understanding of its biosynthetic pathway is essential. Previously, a linear pathway for pleuromutilin biosynthesis was established. Here we report two shunt pathways involving Pl-sdr and Pl-atf that were identified through the rational heterologous expression of combinations of pleuromutilin biosynthetic genes in Aspergillus oryzae. Three novel pleuromutilin congeners were isolated, and their antimicrobial activity was investigated, alongside that of an additional derivative produced through a semi-synthetic approach. It was observed that the absence of various functional groups - 3 ketone, 11 hydroxyl group or 21 ketone - from the pleuromutilin framework affected the antibacterial activity of pleuromutilin congeners. This study expands our knowledge on the biosynthesis of pleuromutilin and provides avenues for the development of novel pleuromutilin analogues by combining synthetic biology and synthetic chemistry.

Bioassay-Guided Isolation of Two Flavonoids from Derris scandens with Topoisomerase II Poison Activity.

Derris scandens (ROXB.) BENTH. (Fabaceae) is used as an alternative treatment for cancer in Thai traditional medicine. Investigation of the topoisomerase II (Top2) poison of compounds isolated from this plant may reveal new drug leads for the treatment of cancer. Bioassay-guided isolation was performed on an extract of D. scandens stems using a yeast cell-based assay. A yeast strain expressing the top2-1 temperature-sensitive mutant was used to assay Top2 activity. At the permissive temperature of 25°C, yeast cells were highly sensitive to Top2 poison agents. At the semi-permissive temperature of 30°C, where enzyme activity was present but greatly diminished, cells displayed only marginal sensitivity. The bioassay-guided fractionation of the extract led to the isolation of two known isoflavones: 5,7,4'-trihydroxy-6,8-diprenylisoflavone (1) and lupalbigenin (2). These two compounds also displayed cytotoxicity against three different cancer cell lines, KB, MCF-7 and NCI-H187. In conclusion, Top2 poison agents from D. scandens are reported for the first time, substantiating the use of D. scandens in Thai traditional medicine for cancer treatment.

Complete Genome Sequences of Two Genetically Distinct Variants of Porcine Epidemic Diarrhea Virus in the Eastern Region of Thailand.

Porcine epidemic diarrhea virus (PEDV) has continued to cause sporadic outbreaks in Thailand since 2007. Previously, PEDV in Thailand was a new variant containing an insertion and deletion in the spike gene. Herein, full-length genome sequences are reported for two variants of PEDV isolates from pigs displaying diarrhea in Thailand.

Detection of paeoniflorin and albiflorin by immunostaining technique using anti-paeoniflorin monoclonal antibody.

INTRODUCTION: Paeoniae radix is one of the most important crude drugs used in Kampo medicines (KMs). A part of its pharmaceutical properties is due to the presence of paeoniflorin (PF) and albiflorin (AF). OBJECTIVE: For the specific and easy identification of PF and AF, an immunostaining technique was developed using anti-PF monoclonal antibody (MAb). METHODOLOGY: PF and AF were treated with a NaIO4 solution and reacted with bovine serum albumin (BSA) preparing PF- and AF-BSA conjugates on the polyethersulphone (PES) membrane. Anti-PF MAb was bound and then antibody labelled with peroxidase directed against anti-PF MAb. Finally, a substrate was added and then PF and AF were detected. RESULTS: Anti-PF MAb recognised not only PF but also AF when 10 µg was present on the PES membrane. As little as 0.5 µg of PF and AF were still detected under immunostaining. Various Paeoniae radix samples and KMs were qualitatively analysed, and total amounts of PF and AF were visually detected by immunostaining technique. This method was applied to investigate the distribution of PF and AF in fresh peony root using immunoblotting by transferred from peony root to the PES membrane. CONCLUSION: The technique permitted the visualisation of PF and AF on PES membrane using immunostaining. The immunostaining technique established would be a powerful tool for probing the sources of PF and AF in plant extracts.

A topoisomerase II poison screen of ethnomedicinal Thai plants using a yeast cell-based assay.

ETHNOPHARMACOLOGICAL RELEVANCE: The plants used in this study had previously been identified and used by Thai folk practitioners, who had knowledge of Thai traditional medicine, as alternative treatments for cancer. Investigation into the mechanism of the Topoisomerase II (Top2) poison of these plants may give rise to new drug leads for cancer treatment. AIM OF THE STUDY: This study aimed to screen ethnomedicinal plants used in Thai traditional medicine for Top2 poison activity using a yeast cell-based assay and also to validate the traditional uses of these plants by examining the Top2 poison activity. MATERIALS AND METHODS: Thirty Thai medicinal plants were harvested and identified. Plant methanol extracts were prepared and screened in vitro using a yeast cell-based assay. Mutant yeast strains carrying the top2-1 allele, which encodes a temperature-sensitive topoisomerase, were used to establish the yeast spot test. Strains carrying this mutation grow normally at 25°C and generally have a wild-type drug sensitivity. These yeast strains are able to grow at 30°C, but the Top2 activity is greatly reduced at this temperature, which causes the strains to be highly drug resistant to anti-Top2 agents. Cell growth was measured by colony survival after exposure to various concentrations of extracts at different temperatures. RESULTS: The extracts of six out of thirty ethnomedicinal plants, Curcuma longa, C. zedoaria, Derris scandens, Grangea maderaspatana, Stephania pierrei and S. suberosa, were found to have Top2 poison activity against the yeast cells. CONCLUSIONS: The yeast screening system confirmed the proposed anti-cancer mechanisms of plants used in Thai traditional medicine by traditional doctors.