Monthon Lertcanawanichakul

Expression of Chitinase-Encoding Genes in Bacillus thuringiensis and Toxicity of Engineered B. thuringiensis subsp. aizawai Toward Lymantria dispar Larvae

Chitinase genes from Aeromonas hydrophila and Bacillus circulans No.4.1 were cloned into the plasmid pHY300PLK and designated as pHYA2 and pHYB43, respectively. Both plasmids were introduced into various strains of B. thuringiensis by electroporation. Plasmid pHYB43 was generally structurally stable, but showed lower segregrational stability than pHYA2 in B. thuringiensis subsp. aizawai when grown under nonselective conditions. The production of chitinase from B. thuringiensis subsp. aizawai harboring pHYB43 or pHYA2 could be detected after native polyacrylamide gel electrophoresis by using 4-methylumbelliferyl β-D-N,N′- diacetylchitobioside as the substrate. Moreover, B. thuringiensis subsp. aizawai harboring pHYB43 gave 15 times higher chitinase activity than when harboring pHYA2, as determined by means of a colorimetric method using glycol chitin as the substrate. In addition, B. thuringiensis subsp. aizawai harboring pHYB43 was more toxic to gypsy moth larvae (Lymantria dispar) than parental B. thuringiensis subsp. aizawai or its clone harboring pHYA2.

Demethoxycurcumin from Curcuma longa Rhizome Suppresses iNOS Induction in an in vitro Inflamed Human Intestinal Mucosa Model

BACKGROUND It is known that inducible nitric oxide synthase (iNOS)/nitric oxide (NO) plays an integral role during intestinal inflammation, an important factor for colon cancer development. Natural compounds from Curcuma longa L. (Zingiberaceae) have long been a potential source of bioactive materials with various beneficial biological functions. Among them, a major active curcuminoid, demethoxycurcumin (DMC) has been shown to possess anti-inflammatory properties in lipopolysaccharide (LPS)-activated macrophages or microglia cells. However, the role of DMC on iNOS expression and NO production in an in vitro inflamed human intestinal mucosa model has not yet been elucidated. This study concerned inhibitory effects on iNOS expression and NO production of DMC in inflamed human intestinal Caco-2 cells. An in vitro model was generated and inhibitory effects on NO production of DMC at 65 μM for 24-96 h were assessed by monitoring nitrite levels. Expression of iNOS mRNA and protein was also investigated. DMC significantly decreased NO secretion by 35-41% in our inflamed cell model. Decrease in NO production by DMC was concomitant with down-regulation of iNOS at mRNA and protein levels compared to proinflammatory cytokine cocktail and LPS-treated controls. Mechanism of action of DMC may be partly due to its potent inhibition of the iNOS pathway. Our findings suggest that DMC may have potential as a therapeutic agent against inflammation-related diseases, especially in the gut.

In vitro antimicrobial and antioxidant activities of bioactive compounds (secondary metabolites) extracted from Streptomyces lydicus A2

In the present study, we report antibacterial and antioxidant activities of bioactive compounds extracts (ethyl acetate extract, methanolic extract, n-hexane extract) of Streptomyces lydicus A2 isolated from air in Sciencetific and Technological Equipments Building, Walailak University, Thailand. The S. lydicus A2 is potential to produce bioactive compounds showed various biological activities. In previous research, its culture broth or extract shows antimicrobial activitie. In this study, extract prepared from S. lydicus A2 stored at -80 oC was re-evaluated the antimicrobial activity when grew in various medium and incubation time to find out storage effect toward the antimicrobial activity. The extract (fractions) from S. lydicus A2 still inhibit test bacteria eventhough it decreases 50% than the extract prepared from S. lydicus A2 without -80 oC storage. Antimicrobial activity against Staphylococcus aureus and Bacillus cereus was only exhibited by the methanolic fraction S. lydicus A2. Only, the ethyl acetate extract was found to possess dose dependent DPPH free radical scavenging. The isolate A2 could be a potential candidate for the development of novel therapeutic agents active against pathogens and free radicals. Further studies on genomic characterization of isolate and structure determination of the bioactive compounds are under progress.

Construction of Plasmid Vector for Expression of Bacteriocin N15-Encoding Gene and Effect of Engineered Bacteria on Enterococcus faecalis

A 6.09-kb plasmids vector pOri253 was constructed from the plasmid pIL253 (5.2 kb) and a 0.89-kb fragment of oriColE1 from pBluescript II KS. The bifunctional plasmid pOri253 conferred erythromycin resistance in both Escherichia coli and Enterococcus faecalis. It has unique sites for EcoRI, BamHI, SalI, and PstI derived from pIL253 and was lost at a low rate in E. faecalis JCM8726 when cultured in Man, Rogosa, & Sharpe broth without antibiotic. The lactococcal promoter P23 was inserted at one end of the pOri253 multicloning site. Gene expression was assessed by an entAI gene, which produced bacteriocin N15. The E. faecalis harboring constructed plasmid carrying P23 (pOrient23) had more antibacterial activity than parental E. faecalis JCM8726 and its clone harboring non-P23-containing plasmid (pOrient), as determined by means of an overlay method.