Flavone c-glycosides from Trollius ledebouri reichb.

A new acylated C-glycosylflavone (1) was isolated from Trollius ledebouri Reichb together with two known C-glycosyflavones (2, 3). The structures were elucidated by spectroscopic methods, including HRMS, IR, 1H and 13C NMR and 2D experiments (COSY, HMQC and HMBC). The anti-inflammatory activities of 1-3 were tested on TPA-induced mice ear edema (in vivo).

Japonicins A and B from the flowers of Inula japonica.

Two new flavonols japonicins, A (1) and B (2), along with nine known flavonoids were isolated from an antidepressant fraction, which was separated from the 70% alcohol extract of the flowers of Inula japonica Thumb. The structures of compounds 1 and 2 were determined as 3,3',4',5,9,10-hexahydroxy-12-methylchroman[2,3-h]flavone and 8-(1-(3,4-dihydroxyphenyl)ethyl)-3,3',4',5,7-pentahydroxyflavone by the analyses of physical constants and spectral data. The special flavonoid structure having the substituent 1-phenylethyl at C-8 position was discovered for the first time.

[Secondary metabolites of Gliocladium sp., a growth accelerating fungus for Anoectochilus roxburghii (Wall.) Lindl].

OBJECTIVE: To study the secondary metabolites of fungus Gliocladium sp. that helps accelerate the growth of A. roxburghii. METHOD: Compoud isolation by chromatography and structure elucidation by chemical and spectral analyses. RESULTS: Five compounds were obtained and elucidated as: 8(E)-N-(2'-hydroxypalmityl)-1-O-beta-gly-copyranosyl-3-hydroxyl-9-methyl-2- octodecanine-4, 8-diene (I), N-(2'-hydroxytetracosanoyl)-1,3,4-trihydroxy-2-octodecanine(II), 7, 22-diene-3-hydroxy-6,9-epidioxyergosta(III), ergostol(IV) and alpha-palmitin(V). CONCLUSION: I, II, III were obtained from Gliocladium sp. for the first time.

Mutational analysis of the roles of residues in Escherichia coli elongation factor Ts in the interaction with elongation factor Tu.

The crystal structure of the Escherichia coli elongation factor (EF)-Tu.Ts complex indicates that there are extensive contacts between EF-Tu and EF-Ts. To determine the importance of these contacts in the interaction between E. coli EF-Tu and EF-Ts, residues in EF-Ts at the interface of these two proteins were mutated. The binding constants governing the interaction of the resulting EF-Ts variants with E. coli EF-Tu were determined. The effects of these mutations on the ability of EF-Ts to stimulate GDP exchange with EF-Tu.GDP and on its ability to stimulate the activity of EF-Tu in polymerization were tested. The results indicate that Arg-12, Met-19, and Met-20 in the N-terminal domain of EF-Ts and His-147 and Lys-166 and/or His-167 in subdomain C of EF-Ts are crucial in the interaction between EF-Tu and EF-Ts. Lys-23, Val-234, Met-235, and the C-terminal helix h13 are less important. The binding constants of the EF-Ts variants governing their interactions with EF-Tu correlate well with their activities in stimulating GDP exchange with EF-Tu. Mutations prepared in EF-Tu indicate that His-19 and Gln-114 but not Glu-348 in EF-Tu are moderately important for its interaction with EF-Ts.

Regulation of the activity of chloroplast translational initiation factor 3 by NH2- and COOH-terminal extensions.

The mature form of the chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis consists of an internal region homologous to prokaryotic IF3 flanked by long NH2- and COOH-terminal extensions. Sequences in these extensions reduce the activity of the homology domain in promoting initiation complex formation with chloroplast mRNAs and 30 S ribosomal subunits. A series of deletions of the NH2- and COOH-terminal extensions of IF3chl were constructed and tested for their effects on the activity of the homology domain. About half of the inhibitory effect arises from sequences within 9 residues of the junction between the NH2-terminal extension and the homology domain. The remaining inhibitory effect is the result of sequences in the COOH-terminal extension. The equilibrium constant governing the binding of the homology domain of IF3chl to 30 S subunits is estimated to be 1.3 x 10(7) M-1. Sequences close to the junction of the NH2-terminal extension and the homology domain reduce this binding constant about 10-fold. Sequences in the COOH-terminal extension have a similar negative effect. The negative effects of these two regions are cumulative, resulting in a 100-fold reduction of the binding constant. The 9 residues at the NH2-terminal extension effectively prevent the proofreading activity of IF3chl. The entire COOH-terminal extension reduces the proofreading ability by about half. These results are discussed in terms of the proposed three-dimensional structure of the homology domain of IF3chl.

Structural and mechanistic studies on chloroplast translational initiation factor 3 from Euglena gracilis.

Chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis contains a central region (homology domain) that is homologous to prokaryotic IF3. The homology domain is preceded by a long NH2-terminal extension (head), and followed by a 64 amino acid COOH-terminal extension (tail). Sequences in these extensions reduce the activity of the homology domain. To gain insight into these effects, a possible three-dimensional structure for the homology region of IF3chl has been modeled using the X-ray coordinates from the N- and C-domains of Bacillus stearothermophilus IF3. In B. stearothermophilus IF3, these two compact domains are thought to fold independently and are separated by a helical lysine-rich linker. The modeled structure suggests that IF3chl has a similar overall fold although some subtle differences are predicted to occur. Both the head and tail regions of IF3chl are oriented toward the linker region in the homology domain where they may potentially interfere with its function. Circular dichroism spectropolarimetry (CD) indicates that the lysine-rich linker region in IF3chl is not in a helical conformation and is probably a random coil. CD analysis indicates that a portion of the tail region of IF3chl is helical and that the tail has a direct interaction with the linker region in the homology domain. Site-directed mutagenesis of the linker indicates that two conserved lysine residues are important for the function of IF3chl and play a role in the binding of IF3chl to the 30S ribosomal subunit. Mutation of these residues affects the interaction of the homology domain with the tail.

Expression and functional analysis of Euglena Gracilis chloroplast initiation factor 3.

A portion of a cDNA predicted to encode the mature form of Euglena gracilis chloroplast translational initiation factor 3 (IF-3chlM, molecular mass, 46 402) and the portion of this factor homologous to bacterial IF-3 (IF-3chlH, molecular mass 22 829) have been cloned and expressed in Escherichia coli as histidine-tagged proteins. The homology domain can be expressed in reasonable levels in E. coli. However, IF-3chlM is quite toxic and can only be produced in small amounts. Both forms of the chloroplast factor are associated with E. coli ribosomes. Purification procedures have been developed for both IF-3chlM and IF-3chlH using Ni-NTA affinity chromatography followed by ion exchange chromatography. IF-3chlM and IF-3chlH are active in promoting ribosome dissociation and in promoting the binding of fMet-tRNA to E. coli ribosomes. However, IF-3chlH has at least 5-fold more activity than either native IF-3chl or IF-3chlM in promoting initiation complex formation on chloroplast 30S ribosomal subunits in the presence of a mRNA carrying a natural translational initiation signal. This observation suggests that regions of IF-3chl lying outside of the homology domain may down-regulate the activity of this factor.