Minor diterpenoid glycosides from the leaves of Stevia rebaudiana

From the commercial extract of the leaves of Stevia rebaudiana, three new diterpenoid glycosides were isolated besides eight known steviol glycosides including stevioside, rebaudiosides A–F and dulcoside A. The structures of the three compounds were identified as 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-kaur-16-en-18-oic acid-(6-O-β-d-xylopyranosyl-β-d-glucopyranosyl) ester (1), 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-17-hydroxy-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (2), and 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-17-oxo-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (3) on the basis of extensive NMR and MS spectral studies. Another known diterpenoid glycoside, 13-[(2-O-β-d-glucopyranosyl-β-d-glucopyranosyl) oxy]-kaur-15-en-18-oic acid β-d-glucopyranosyl ester (4) was also isolated and its complete NMR spectral assignments were made on the basis of COSY, HSQC and HMBC spectral data.     

2-deoxy-D-glucose combined with ferulic acid enhances radiation response in non-small cell lung carcinoma cells

The present study was undertaken to investigate the radiosensitizing effects of 2-deoxy-D-glucose (2DG), a glycolytic inhibitor, and ferulic acid (FA), a phenolic prooxidant, in relatively radioresistant human non-small cell lung carcinoma cells (NCI-H460). NCI-H460 cells were treated with 4 mM 2DG and/or 53.8 μM FA for 24 h and then exposed to 2 Gy irradiation. Compared to cells that were 2 Gy-irradiated alone (50%), FA and 2DG with radiation (FA+2DG+IR) showed additional decrease in cell viability (15%). This has been further validated by decreased (86%) colony formation in 2DG+FA+IR group compared to 2DG (29%), FA (24%) and IR (37%) group alone. Increased apoptotic cells (84%) in 2DG+FA+IR group further confirm the radiosensitizing property of 2DG or FA. In NCI-H460 cells 2DG decreased NADPH levels (10%) and FA increased ROS levels leading to enhanced oxidative damage in the 2DG+FA+IR group. This was reflected as altered mitochondrial membrane potential, increased lipid peroxidative markers (TBARS), DNA damage and decreased intracellular glutathione (GSH) levels in combined treatment groups when compared to radiation or 2DG or FA treatment alone. The present study suggests that FA and 2DG act by increasing oxidative damage in NCI-H460 cells.     

Organization of the CTX prophage in environmental isolates of Vibrio mimicus

The organization of the CTX prophage in environmental strains of Vibrio mimicus was investigated. Sixteen hundred non-sucrose fermenting vibrios were examined for ctx gene by hybridization. Out of 1,600 isolates, 6 V. mimicus isolates contained ctxA gene. The organization of CTX prophage was determined by RFLP using ctxA probe. The CTX prophage integrated at a single site in V. mimicus genome which was present as a single copy flanked by at least a single RS element. Ribotype pattern revealed that a particular clone of V. mimicus acquired the CTXPhi in the aquatic environment. This study demonstrated that V. mimicus could act as a reservoir of CTXPhi in the aquatic environment.     

Rapid identification of female Culexmosquito species using Expert System in the South East Asian region

Rapid identification of mosquito (vector) species is critical for vector control and disease management. Pictorial keys of mosquito species are currently used for the identification of new mosquito species. However, this approach is not very effective. Here, we describe the use of an ID3 algorithm (part of artificial intelligence) for the rapid identification of the South East Asian female Culex mosquito species.

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Purification of lipase from Cunninghamella verticillata by stepwise precipitation and optimized conditions for crystallization

Lipase from the oil-mill waste isolate Cunninghamella verticillata was purified by stepwise precipitation using acetone, as a sequel to our earlier conventional column chromatographic method [Gopinath et al. (2002)World Journal of Microbiology and Biotechnology 18, 449–458]. The yield of purified lipase was approx. 4-fold higher than by the previous method and the purified lipase was obtained with 70–80% acetone saturations. The enzyme was resolved as a single band with homogeneity both by native and by SDS–PAGE. The optimum condition for the lipase to crystallize was 5 g of enzyme in 0.05 M sodium phosphate buffer (pH 6.5) with 5 mM FeCl₂ and 10% 2-methyl 2,4-pentanediol (MPD).These authors equally contributed to this work     

Antitubercular agents. Part 1: synthesis of phthalimido- and naphthalimido-linked phenazines as new prototype antitubercular agents

The preparation and antitubercular properties of a series of phthalimido- and naphthalimido-linked phenazines are described. Some of these new compounds inhibited the growth of Mycobacterium tuberculosis ATCC 27294, Mycobacterium avium ATCC 49601, Mycobacterium intracellulare ATCC 13950 and some clinical isolates.     

Exoribonuclease R interacts with endoribonuclease E and an RNA helicase in the psychrotrophic bacterium Pseudomonas syringae Lz4W

Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni(2+)-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes.     

The structure of antimicrobial pexiganan peptide in solution probed by Fourier transform infrared absorption, vibrational circular dichroism, and electronic circular dichroism spectroscopy

Pexiganan (Gly-Ile-Gly-Lys-Phe-Leu-Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys-Ile-Leu-Lys-Lys), a 22 amino acid peptide, is an analogue of the magainin family of antimicrobial peptides present in the skin of the African clawed frog. Conformational analysis of pexiganan was carried out in different solvent environments for the first time. Organic solvents, trifluoroethanol (TFE) and methanol, were used to study the secondary structural preferences of this peptide in the membrane-mimicking environments. In addition, aqueous (D2O) and dimethyl sulfoxide (DMSO) solutions were also investigated to study the role of hydrogen bonding involved in the secondary structure formation. Fourier transform infrared absorption, vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) measurements were carried out under the same conditions to ascertain the conformational assignments in different solvents. All these spectroscopic measurements suggest that the pexiganan peptide has the tendency to adopt different structures in different environments. Pexiganan appears to adopt an alpha-helical conformation in TFE, a sheet-stabilized beta-turn structure in methanol, a random coil with beta-turn structure in D2O, and a solvated beta-turn structure in DMSO.     

Distinction of sperm-binding site and reactive site for trypsin inhibition on p12 secreted from the accessory sex glands of male mice

Six variants of P12, a Kazal-type trypsin inhibitor in the secretion of male mouse accessory sexual glands, were made using single-site mutations including R19L, Y21V, D22G, R43G, K44S, and R45T, based on one-letter-code mutation of amino acids. The other two variants, Nd10 and Cd8, were made using the deletion of 10 and 8 residues from the N- and C-terminals, respectively. Their CD profiles revealed maintenance of the P12 conformation in the seven variants, excluding Cd8, which became unfolded. Only R19L entirely lost the ability while the other variants were as strong as P12 in inhibiting the trypsin digestion of N-benzoyl-Phe-Val-Arg 7-amido-4-methylcoumarin. The immunocytochemical results demonstrated that D22G and Cd8 failed to bind to sperm, Y21V very weakly did so, and the other variants retained their sperm-binding abilities. Concomitantly, the immunocytochemical stainability of each ligand was parallel to its inhibitory effect on 125I-P12-sperm binding, and a synthetic oligopeptide corresponding to residues 18-24 of P12 was able to inhibit P12-sperm binding. The data together concluded that R19 was essential for protease inhibition and D22 and/or Y21 mainly being responsible for the binding of P12 to sperm. The steric arrangement of R19, Y21, and D22 on the tertiary structure of P12 is discussed.