Exonuclease V from Saccharomyces cerevisiae. A 5'----3'-deoxyribonuclease that produces dinucleotides in a sequential fashion

A novel deoxyribonuclease, exonuclease V, has been purified approximately 30,000-fold from Saccharomyces cerevisiae. Exonuclease V is localized in the nucleus. The nuclease degrades single-stranded, but not double-stranded, DNA from the 5'-end. The products of exonuclease action are dinucleotides, except the 3'-terminal tri- and tetranucleotides which are not degraded. Studies with synthetic oligo- and polynucleotides with specified sequence elements showed that exonuclease V cleaves off dinucleotides as primary digestion products. Thus, the polymers (pT)9pA(pT)n and (pT)10pA(pT)n yielded pTpA and pApT as digestion products, respectively. Removal of the 5'-terminal phosphate from the DNA substrate results in reduced binding of the enzyme to the substrate. In addition, the initial hydrolytic cut by exonuclease V on the dephosphorylated substrate produces a mixture of dinucleoside monophosphates and trinucleoside diphosphates. The enzyme is processive in action.     

Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera

Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.     

Brain Transcriptional and Epigenetic Associations with Autism

BackgroundAutism is a common neurodevelopmental syndrome. Numerous rare genetic etiologies are reported; most cases are idiopathic.Conclusions/SignificanceThis work highlights two largely unrecognized molecular pathophysiological themes in autism and suggests differing molecular bases for autism behavioral endophenotypes.     

Mapping and characterization of mangrove plant communities in Hong Kong

Ecological surveys were carried out to investigate the distribution and characterization of remaining mangrove stands in Hong Kong. The field studies indicate that 43 mangrove stands, excluding Mai Po Nature Reserve, still remained along the coastline of Hong Kong despite tremendous reclamation and development which occurred in the past 40 years. Most mangrove stands were found in Deep Bay (western part)and Sai Kung District (eastern coasts). The total areas occupied by these mangrove stands were 178 ha,varying from a very small stand (with 1–2 mangrove shrubs) to fairly extensive mangroves in Deep Bay (> 10 ha). It appeared that mangrove stands located in Deep Bay area were larger than those in the eastern coasts. Twenty plant species were identified from these stands, with 13 being exclusive or associate mangrove species. The major constituent species were Kandelia candel, Aegiceras corniculatum, Excoecaria agallocha and Avicennia marina. Rare species such as Heritiera littoralis were only found in a few mangrove stands. Out of the 43remaining mangrove stands, 23 were more worthwhile for conservation and their plant community structures were further investigated by transect and quadrat analyses. The importance values (sum of relative abundance,frequency and dominance) show that K. candel was the most dominant species. Species richness and Simpson's indices together with tree height, tree density and canopy area fluctuated significantly between mangrove stands. These values were used to prioritize the conservation potential of the remaining mangrove stands in Hong Kong. This revised version was published online in August 2006 with corrections to the Cover Date.     

1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

Gene characterization, analysis of expression and in vitro synthesis of dihydroflavonol 4-reductase from [Citrus sinensis (L.) Osbeck

Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) catalyzes the reduction of dihydroflavonols to leucoanthocyanins, a key "late" step in the biosynthesis of anthocyanins. In this study we showed that a strong reduction in DFR expression occurs in the non-red orange cultivar (Navel and Ovale) compared to that of the red orange (Tarocco) suggesting that the enzyme could be involved in the lack of production of anthocyanins. Therefore, we isolated and compared the cDNAs, the genomic clones, as well as the promoter regions of blood and blond orange dfrs. Our data revealed that the cDNA sequences of pigmented and non-pigmented orange DFRs were 100% homologous and contained a 1017 bp open reading frame which encodes a protein of 338 amino acid residues, corresponding to a molecular mass of 38010.76 Da, with a theoretical pI of 5.96. Moreover, we found that there were no significant differences in non-coding regions (introns and 5' upstream region) of dfr sequences. Southern blot analysis of genomic DNA indicated that dfr was present as a single copy gene in both cultivars. From these findings the low expression level of blond orange dfr, which might play a role in the phenotypic change from blood to blond orange, is thought to be the result of a likely mutation in a regulatory gene controlling the expression of dfr. In addition, here we reported the successful expression of orange DFR cDNAs leading to an active DFR enzyme which converts dihydroquercetin to leucoanthocyanidin, thus confirming the involvement of the isolated genes in the biosynthesis of anthocyanins. Moreover, as far as we know, this is the first report concerning the in vitro expression of DFR from fruit flesh whose biochemical properties might be very different from those of other plant organ DFRs.     

Reducing the immunogenicity and improving the in vivo activity of trichosanthin by site-directed pegylation.

PEG modification (PEGylation) has been shown to reduce immunogenicity and prolong circulating half-life of proteins. In the present study, site-directed PEGylation was used to reduce immunogenicity and prolong plasma half-life of trichosanthin (TCS). Four TCS mutants, i.e. S7C, Q219C, K173C and [K173C,Q219C] (KQ), were constructed by site-directed mutagenesis. PEG modifications were done by reacting PEG5k-maleimide or PEG20k-maleimide reagent with the newly introduced cysteine residue of the mutants. The plasma clearance rate of PEGylated TCS mutants decreased up to 100-fold and the decrease was inversely proportional to the effective molecular size. The in vitro activities such as ribosome-inactivating activity and cytotoxicity were also decreased. However, the in vivo abortifacient activity was, slightly decreased, unchanged, or even enhanced in some preparations. PEG5k modification had little effect on immunogenicity. However, PEG20k modification significantly reduced immunogenicity. All PEG20k modified TCS mutants induced lower level IgG and IgE antibodies. In particular, PEG20k-KQ and PEG20k-K173C induced weaker systemic anaphylaxis reaction in guinea pigs. In conclusion, the present results suggest that PEG20k is better than PEG5k for reducing immunogenicity and prolonging plasma half-life. The conjugate can become a better therapeutic agent.     

Phytochemical composition of Potentilla anserina L. analyzed by an integrative GC-MS and LC-MS metabolomics platform

Potentilla anserina L. (Rosaceae) is known for its beneficial effects of prevention of pre-menstrual syndrome (PMS). For this reason P. anserina is processed into many food supplements and pharmaceutical preparations. Here we analyzed hydroalcoholic reference extracts and compared them with various extracts of different pharmacies using an integrative metabolomics platform comprising GC-MS and LC-MS analysis and software toolboxes for data alignment (MetMAX Beta 1.0) and multivariate statistical analysis (COVAIN 1.0). Multivariate statistics of the integrated GC-MS and LC-MS data showed strong differences between the different plant extract formulations. Different groups of compounds such as chlorogenic acid, kaempferol 3-O-rutinoside, acacetin 7-O-rutinoside, and genistein were reported for the first time in this species. The typical fragmentation pathway of the isoflavone genistein confirmed the identification of this active compound that was present with different abundances in all the extracts analyzed. As a result we have revealed that different extraction procedures from different vendors produce different chemical compositions, e.g. different genistein concentrations. Consequently, the treatment may have different effects. The integrative metabolomics platform provides the highest resolution of the phytochemical composition and a mean to define subtle differences in plant extract formulations.