Changes in concentraions of free radicals and amino acids in developing maize inflorescences and fruits

The changes in the concentration of free radicals and bound amino acids in developing maize inflorescences and fruits were ascertained. The concentration rises markedly when tissues disintegrate and desiccate. A slight increase in the concentration may be ascribed to the rise of metabolic processes connected with kernel formation.     

Conformational variability of recombination R-triplex formed by the mammalian telomeric sequence

Alignment of three nucleic acids strands, in which the third strand is identical to one of the DNA duplex strands, occurs in various cellular systems. In the case of telomeric t-loops, recognition between the DNA duplex and the homologous single strand is likely to be mediated by proteins through formation of the transient recombination-type R-triplex. Earlier, using 2-aminopurine as a fluorescent reporting base, we evaluated the thermodynamic characteristics of intramolecular R-triplex formed by a mixed nucleotide sequence. Here, we used this approach to explore a propensity of the telomeric TTAGGG repeat to form the R-triplex. The circular dichroism spectral changes detected upon formation of the R-triplex suggest that this process is accompanied by specific conformational changes in DNA, including a local destabilization of the target duplex next to a GGG run revealed by the fluorescence of the reporting 2-aminopurine base. Surprisingly, stability of the R-triplex formed by telomeric sequence depends strikingly on the counter ion, being higher for Na⁺ than for Li⁺. Taken together these findings indicate a significant conformational variability of telomeric DNA in the context of recombination-type R-triplex, a phenomenon of possible biological relevance.     

Topology of the disulfide bonds in the antiviral lectin scytovirin

The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re‐examined the disulfide topology. N‐terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7–Cys55, Cys20–Cys32, Cys26–Cys38, Cys68–Cys80, and Cys74–Cys86. We also analyzed the N‐terminal domain of SVN (SD1, residues 1–48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20–Cys26, Cys32–Cys38) or SD1B (Cys20–Cys32, Cys26–Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the “cystine clusters” [Cys20–Cys32/Cys26–Cys38; SD1] and [Cys68–Cys80/Cys74–C‐86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578–2584). Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require considerable precautions when being fragmented for the purpose of disulfide assignment.     

Developmental and metabolic changes induced by anoxia in diapausing and non-diapausing flesh fly pupae

Summary Anaerobic metabolism was compared in nondiapausing (ND) and diapausing (D) pupae of the flesh fly,Sarcophage crassipalpis using in vivo¹³C NMR spectroscopy. Anoxia-induced changes in the development of ND and D pupae were correlated with oxidative metabolism and mitochondrial integrity. ND pupae tolerated 1 day of anoxia without any obvious developmental effect, while D pupae tolerated up to 6 days of anoxia. Longer exposure to anoxia (3 days in ND pupae and up to 14 days in D pupae) allowed development to the pharate adult stage but precluded eclosion. Four-day anoxia applied to ND pupae during 4–6 days post-pupariation arrested development in a stage indistinguishable from diapause. This morphological stasis was accompanied by 80% suppression of oxidative metabolism and a 100% increase in glycerol concentration. However, unlike a true diapause, this arrest could not be terminated with 20-hydroxyecdysone or hexane. Four-day anoxia treatment applied to D pupae stimulated development and raised their oxygen consumption. The anoxia-induced changes in oxidative metabolism were not accompanied by mitochondrial changes. Exposure to 95%PO₂ atmosphere had no apparent developmental or metabolic effects on ND or D pupae. Major metabolites (lipids, trehalose, glycogen, glycerol, glutamine, and alanine) were detected in the ND and D pupae but their rates of turnover differed. Anoxia induced synthesis of glycerol and alanine in both D and ND pupae. Injected labeled glucose was incorporated primarily into trehalose and glycogen by both D and ND pupae. The rate of incorporation in ND pupae was approximately twice that observed in D pupae. Anoxia resulted in glycerol and alanine synthesis in both groups of pupae, but more glycerol was labeled in ND pupae and more alanine in D pupae. Glycogen and trehalose were depleted in the D pupae under anoxia. Cold acclimation had no effect on the steady-state or rate of synthesis of metabolites.     

The Thermodynamic Basis for Viral RNA Detection by the RIG-I Innate Immune Sensor

RIG-I is a cytoplasmic surveillance protein that contributes to the earliest stages of the vertebrate innate immune response. The protein specifically recognizes 5′-triphosphorylated RNA structures that are released into the cell by viruses, such as influenza and hepatitis C. To understand the energetic basis for viral RNA recognition by RIG-I, we studied the binding of RIG-I domain variants to a family of dsRNA ligands. Thermodynamic analysis revealed that the isolated RIG-I domains each make important contributions to affinity and that they interact using different strategies. Covalent linkage between the domains enhances RNA ligand specificity while reducing overall binding affinity, thereby providing a mechanism for discriminating virus from host RNA.     

Growth and morphological response of old-forest lichens transplanted into a young and an old Picea abies forest

This experimental study focuses on why old-forest lichens such as Lobaria scrobiculata and Platismatia norvegica are scarce in younger spruce stands. Understanding the factors limiting the distribution of species is important for developing appropriate methods for forest management aiming to maintain biodiversity. A successful growth of L. scrobiculata and P. norvegica was found in the young planted stand as the rate of growth did not differ between the young stand and the old spruce forest where they naturally occurred, during 14 months of transplantation. In the young forest environment, L. scrobiculata showed a significantly higher specific thallus weight, and a slightly higher water-holding capacity. This morphological response is probably due to a higher light exposure in the young stand and consequently a higher rate of desiccation. The ubiquitous species Platismatia glauca showed a significantly higher rate of growth in the young forest than in the old forest, and a positive relationship between growth rate and light exposure was found. This transplant study has shown that the environmental conditions in younger planted forests are not necessarily unfavourable for growth of old-forest lichens. Other factors, such as limited dispersal ability and poor diaspore production, are probably important for explaining the species scarcity in younger stands.     

Interaction of reactive oxygen and nitrogen species with albumin- and methemoglobin-bound dinitrosyl-iron complexes.

Destructive effect of superoxide anions O2- derived from KO(2) or xanthine-xanthine oxidase system on dinitrosyl-iron complexes bound with bovine albumin or methemoglobin (DNIC-BSA or DNIC-MetHb) was demonstrated. The sensitivity of DNIC-BSA synthesized by the addition of DNIC with cysteine, thiosulfate or phosphate (DNIC-BSA-1, DNIC-BSA-2 or DNIC-BSA-3, respectively) to destructive action of O2- decreased in row: DNIC-BSA-1>DNIC-BSA-3>DNIC-BSA-2. The estimated rate constant for the reaction between O2- and DNIC-BSA-3 was equal to approximately 10(7)M(-1)s(-1). However, hydrogen peroxide and tert-butyl hydrogenperoxide (t-BOOH) did not induce any noticeable degradation of DNIC-BSA-3 even when used at concentrations exceeding by one order of magnitude those of the complex. As to their action on DNIC-MetHb both hydrogen peroxide and t-BOOH-induced rapid degradation of the complex. Both agents could induce the process due to the effect of alkylperoxyl or protein-derived free radicals formed at the interaction of the agents with ferri-heme groups of MetHb. Peroxynitrite (ONOO(-)) could also initiate protein-bound DNIC degradation more efficiently in the reaction with DNIC-BSA-3. Higher resistance of DNIC-MetHb to peroxynitrite was most probably due to the protective action of heme groups on ONOO(-). However, the analysis allows to suggest that the interaction of protein-bound DNICs with O2- is the only factor responsible for the degradation of the complexes in cells and tissues.     

Analysis of bacterial communities in a municipal duck pond during a phytoplankton bloom and isolation of Anatilimnocola aggregata gen. nov., sp. nov., Lacipirellula limnantheis sp. nov. and Urbifossiella limnaea gen. nov., sp. nov. belonging to the phylum Planctomycetes

Waterbodies such as lakes and ponds are fragile environments affected by human influences. Suitable conditions can result in massive growth of phototrophs, commonly referred to as phytoplankton blooms. Such events benefit heterotrophic bacteria able to use compounds secreted by phototrophs or their biomass as major nutrient source. One example of such bacteria are Planctomycetes, which are abundant on the surfaces of marine macroscopic phototrophs; however, less data are available on their ecological roles in limnic environments. In this study, we followed a cultivation-independent deep sequencing approach to study the bacterial community composition during a cyanobacterial bloom event in a municipal duck pond. In addition to cyanobacteria, which caused the bloom event, members of the phylum Planctomycetes were significantly enriched in the cyanobacteria-attached fraction compared to the free-living fraction. Separate datasets based on isolated DNA and RNA point towards considerable differences in the abundance and activity of planctomycetal families, indicating different activity peaks of these families during the cyanobacterial bloom. Motivated by the finding that the sampling location harbours untapped bacterial diversity, we included a complementary cultivation-dependent approach and isolated and characterized three novel limnic strains belonging to the phylum Planctomycetes.