DNA mutagenesis in 2- and 20-yr-old Panax ginseng cell cultures

Previously, Panax ginseng var. Mimaki C.A. Meyer had been shown to accumulate genetic mutations during long-term propagation of a callus culture over a period of 20 yr. In this study, we analyzed the mutation types and frequency in a 2-yr-old P. ginseng callus culture and compared it with the 20-yr-old callus culture, and leaves of cultivated plants. We analyzed the sequence variability between the Actin genes, which are a family of housekeeping genes; phenylalanine ammonia-lyase (PAL) and dammarenediol synthase (DDS), which actively participate in the biosynthesis of ginsenosides; and the somatic embryogenesis receptor kinases (SERK), which control plant development. The frequency of point mutations in the Actin, PAL, DDS, and SERK genes in the 2-yr-old P. ginseng callus culture was markedly higher than in cultivated plants, but lower than in the 20-yr-old callus culture. Most of the mutations in the 2-yr-old P. ginseng calli were A?G and T?C transitions, as in the 20-yr-old calli and intact P. ginseng plants. The number of nonsynonymous mutations was higher in the 2- and 20-yr-old callus cultures than the number of nonsynonymous mutations in cultivated P. ginseng. Interestingly, the total number of N→G or N→C substitutions in the analyzed genes was 1.6 times higher than the total number of N→A or N→T substitutions. Using a methylation-sensitive DNA fragmentation assay, we showed the level of methylcytosine to be higher in the DNA of the 20-yr-old P. ginseng calli that than in the DNA of the 2-yr-old cultures.     

Phylogenetic analyses of endoparasitic Acanthocephala based on mitochondrial genomes suggest secondary loss of sensory organs

The metazoan taxon Syndermata (Monogononta, Bdelloidea, Seisonidea, Acanthocephala) comprises species with vastly different lifestyles. The focus of this study is on the phylogeny within the syndermatan subtaxon Acanthocephala (thorny-headed worms, obligate endoparasites). In order to investigate the controversially discussed phylogenetic relationships of acanthocephalan subtaxa we have sequenced the mitochondrial (mt) genomes of Echinorhynchus truttae (Palaeacanthocephala), Paratenuisentis ambiguus (Eoacanthocephala), Macracanthorhynchus hirudinaceus (Archiacanthocephala), and Philodina citrina (Bdelloidea). In doing so, we present the largest molecular phylogenetic dataset so far for this question comprising all major subgroups of Acanthocephala. Alongside with publicly available mt genome data of four additional syndermatans as well as 18 other lophotrochozoan (spiralian) taxa and one outgroup representative, the derived protein-coding sequences were used for Maximum Likelihood as well as Bayesian phylogenetic analyses. We achieved entirely congruent results, whereupon monophyletic Archiacanthocephala represent the sister taxon of a clade comprising Eoacanthocephala and monophyletic Palaeacanthocephala (Echinorhynchida). This topology suggests the secondary loss of lateral sensory organs (sensory pores) within Palaeacanthocephala and is further in line with the emergence of apical sensory organs in the stem lineage of Archiacanthocephala.     

Evaluation of the mechanisms involved in leucine-induced oxidative damage in cerebral cortex of young rats

Maple syrup urine disease (MSUD) is a metabolic disorder caused by the deficiency of the activity of the mitochondrial enzyme complex branched-chain l-2-keto acid dehydrogenase. The metabolic block results in tissue and body fluid accumulation of the branched-chain amino acids leucine (Leu), isoleucine and valine, as well as of their respective α-keto acids. Neurological sequelae are usually present in MSUD, but the pathophysiologic mechanisms of neurotoxicity are still poorly known. It was previously demonstrated that Leu elicits oxidative stress in rat brain. In the present study we investigated the possible mechanisms involved in Leu-induced oxidative damage. We observed a significant attenuation of Leu-elicited increase of thiobarbituric acid-reactive substances (TBA-RS) measurement when cortical homogenates were incubated in the presence of the free radical scavengers ascorbic acid plus trolox, dithiothreitol, glutathione, and superoxide dismutase, suggesting a probable involvement of superoxide and hydroxyl radicals in this effect. In contrast, the use of N^ω-nitro-l-arginine methyl ester or catalase (CAT) did not affect TBA-RS values. We also demonstrated an inhibitory effect of Leu on the activities of the antioxidant enzymes CAT and gluthathione peroxidase, as well as a significant reduction in the membrane-protein thiol content from mitochondrial enriched preparations. Furthermore, dichlorofluorescein levels were increased although not significantly by Leu. Taken together, our present data indicate that an unbalance between free radical formation and inhibition of critical enzyme activities may explain the mechanisms involved in the Leu-induced oxidative damage.     

Aromatic interactions with naphthylalanine in a β‐hairpin peptide

Stable peptides have been explored as epitope mimics for protein–protein and protein–nucleic acid interactions; however, presentation of a regular structure is critical. Aromatic interactions are ubiquitous and are competent at stabilizing a β‐hairpin fold. The greatest stabilization has been reported from pairs of tryptophan side chains. Naphthylalanine residues are often used as tryptophan replacements, but it is not clear if 1‐naphthylalanine or 2‐naphthylalanine is adequate at replicating the geometry and stability observed with tryptophan aromatic interactions. Herein, a 12‐residue peptide has been constructed with laterally disposed aromatic amino acids. A direct comparison is made between tryptophan and other bicyclic, unnatural amino acids. Significant stabilization is gained from all bicyclic amino acids; however, geometric analysis shows that only 1‐naphthylalanine adopts a similar edge to face geometry as tryptophan, whereas the 2‐naphthylalanine appears most similar to a substituted phenylalanine. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Discovery and development of exome‐based,co‐dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.)

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)‐based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP‐based wheat markers have been made available via the use of next‐generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial‐scale breeding programmes. To identify exome‐based co‐dominant SNP‐based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re‐sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co‐dominant. Validation of a subset of these putative co‐dominant markers confirmed that 96% were true polymorphisms and 65% were co‐dominant SNP assays. The new co‐dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web‐based database to facilitate their use on genotyping programmes worldwide.     

Microwave-assisted Synthesis of Novel Thiazolocarbazoles and Evaluation as Potential Anticancer Agents. Part III

Novel 6-substituted thiazolocarbazole derivatives have been synthesized under microwave irradiation via the corresponding imino-1,2,3-dithiazoles. In vitro antitumor potential of these polyheterocyclic compounds was evaluated. Among all the tested thiazolocarbazoles, compound 10 is the most effective in inhibiting cell growth.     

Synthesis of Biotin-Containing Phosphoramidite Linker with Polyether Spacer Arm

A phosphoramidite linker unit, based on glycerol backbone and containing a biotin residue attached through a tetraethylene glycol spacer arm, was synthesized. DMTr-Glycidol and tetraethylene glycol were used as starting materials. After conversion of one of hydroxy groups in tetraethylene glycol into an amino group, the epoxy cycle in DMTr-glycidol was opened by this amino alcohol, resulting in the corresponding ether and some quantity of secondary amine. After attaching of biotin residue to the ether followed by phosphitylation, the desirable linker was obtained. The structure of the linker was confirmed by ¹H-¹H COSY, ¹H-¹³C HSQC, ¹H-¹³C HMBC, ¹H-¹⁵N HSQC, and ¹H-¹⁵N HMBC spectra. The resulted phosphoramidite linker unit is suitable for use in common DNA synthesizers. This approach can be used for preparation of various modifiers containing reporter groups attached to the primary amino function using conventional procedures.     

Conjugated Antisense Oligonucleotides for Inhibition of Human Cytomegalovirus In Vitro

Abstract

Several thiono triester containing oligonucleotide phosphorothioates linked with a lipophilic group have been synthesized. Some of these modified antisense oligonucleotides show potent anti-HCMV activity as well as improved cellular association and nuclease resistance.     

Plant diversity does not buffer drought effects on early‐stage litter mass loss rates and microbial properties

Human activities are decreasing biodiversity and changing the climate worldwide. Both global change drivers have been shown to affect ecosystem functioning, but they may also act in concert in a non‐additive way. We studied early‐stage litter mass loss rates and soil microbial properties (basal respiration and microbial biomass) during the summer season in response to plant species richness and summer drought in a large grassland biodiversity experiment, the Jena Experiment, Germany. In line with our expectations, decreasing plant diversity and summer drought decreased litter mass loss rates and soil microbial properties. In contrast to our hypotheses, however, this was only true for mass loss of standard litter (wheat straw) used in all plots, and not for plant community‐specific litter mass loss. We found no interactive effects between global change drivers, that is, drought reduced litter mass loss rates and soil microbial properties irrespective of plant diversity. High mass loss rates of plant community‐specific litter and low responsiveness to drought relative to the standard litter indicate that soil microbial communities were adapted to decomposing community‐specific plant litter material including lower susceptibility to dry conditions during summer months. Moreover, higher microbial enzymatic diversity at high plant diversity may have caused elevated mass loss of standard litter. Our results indicate that plant diversity loss and summer drought independently impede soil processes. However, soil decomposer communities may be highly adapted to decomposing plant community‐specific litter material, even in situations of environmental stress. Results of standard litter mass loss moreover suggest that decomposer communities under diverse plant communities are able to cope with a greater variety of plant inputs possibly making them less responsive to biotic changes.