Structural analysis of the intracellular domain of (pro)renin receptor fused to maltose-binding protein

In Synechocystis sp. PCC 6803, the loop domain (aa 1–70) of the phycobilisome core-membrane linker, L_CM, was found to interact with the glycosyl transferase homolog, Sll1466. Growth of a Sll1466 knock-out mutant was slightly faster in low light, but strongly inhibited in high light; the phenotype is discussed in relation to the regulation of light energy transfer to photosystem II. At the molecular level, the mutant shows the following changes compared to the wild type: (1) a smaller size and higher mobility of phycobilisomes on the thylakoid membrane, and (2) a changed lipid composition of the thylakoid membrane, especially decreased amounts of digalactosyl diacylglycerol. These results indicate a profound regulatory role for Sll1466 in regulating photosynthetic energy transfer.     

A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor

Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1–EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor’s amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer.     

Application of an improved continuous parallel shaken bioreactor system for three microbial model systems

A continuous parallel shaken bioreactor system, combining the advantages of shaken bioreactors with the advantages of continuous fermentation, was specifically manufactured from quartz glass and provides a geometric accuracy of <1 mm. Two different model systems (facultative anaerobic bacterium C. glutamicum, and Crabtree-negative yeast P. stipitis), whose growth behaviour and metabolite formation are affected by dilution rate and oxygen availability, were studied. The transition from non-oxygen to limited conditions as function of the dilution rate could precisely be predicted applying the approach described by Maier et al. (Biochem Eng J 17:155–167, 2004). In addition, the Crabtree-positive yeast S. cerevisiae was simultaneously studied in the continuous parallel shaken bioreactor system and in a conventional 1-L bioreactor, for comparison. Essentially the same results were obtained in both types of bioreactors. However, many more reading points were obtained with the parallel shaken bioreactor system in the same time at much lower consumption of culture media.     

Metal-induced point defects in DNA: model and mechanisms

The aim of this work was to study the role of H3O+ and transition-metal (TM) ions in keto-enol and amino-imino tautomeric transitions in DNA base pairs and depurination. In this regard, we discuss the thermodynamic model of ion-DNA interactions and UV display of double-proton transfer (DPT) in GC. The probabilities and energies of rare tautomeric forms of GC pairs in DNA induced by H3O+ and TMwere determined being in the range from0.02 (forMg2+) to 1 ( forCu2+), and from 0 kcal/m (for Cu2+) to 2.3 kcal/m (for Mg2+), respectively. It was shown that 3'ACC5'/5'TGG3' site of DNA double helix, which corresponds to the only triplet 5'UGG3' of RNA that codes the most valuable amino acid tryptophan, is a good target for TM ions to attack. It was also shown that the only way to obtain the tryptophan-coding 5'UGG3' triplet in RNA via transition-type G --> A point mutation caused by TM ions is their interaction with the site of a DNA double helix, which corresponds to 5'CGG3' triplet of RNA that codes arginine.     

Preliminary dielectric measurement and analysis protocol for determining the melting temperature and binding energy of short sequences of DNA in solution

Measurement of the real dielectric constant of bulk buffer solutions containing short sequences of DNA as a function of temperature through the DNA melting or denaturiztion transition can be used to determine melting temperatures, T(m), and to estimate the binding energy of the complimentary strands. We describe a preliminary dielectric measurement and analysis protocol to determine these parameters and its application to two known short sequences. The relative real dielectric constant for the bulk solutions was determined over the frequency range of 50 Hz-20 kHz and temperature range of <40-65 degrees C. The measurements were performed on dilute solutions and utilized low electric field strengths. Based on fits to the data by modified sigmoid functions, the melting temperatures, width of transition, and binding energy for the two sequences in solution were estimated. It was observed that the order of the transition appeared to be second order. The results were then compared against predictions of a number of models from the literature that provide theoretical estimates for the melting temperatures of known short sequences of DNA.     

Spectral diagnostics of the interaction between pyridoxine hydrochloride and bovine serum albumin in vitro

The interaction between pyridoxine hydrochloride (VB6) and bovine serum albumin (BSA) were studied by spectroscopic methods including fluorescence spectroscopy and UV-visible absorption spectra. The quenching mechanism of fluorescence of BSA by VB6 was discussed. The number of binding sites n and observed binding constant K(b) was measured by fluorescence quenching method. The thermodynamic parameters DeltaH(theta), DeltaG(theta), DeltaS(theta) at different temperatures were calculated and the results indicate the binding reaction is mainly entropy-driven and hydrophobic interaction played major role in the reaction. The distance r between donor (BSA) and acceptor (VB6) was obtained according to FOrster theory of non-radiation energy transfer. Synchronous fluorescence and three-dimensional fluorescence spectra were used to investigate the structural change of BSA molecules with addition of VB6, the result indicates that the secondary structure of BSA molecules is changed in the presence of VB6.     

Rapid ab initio prediction of RNA pseudoknots via graph tree decomposition

The prediction of RNA secondary structure including pseudoknots remains a challenge due to the intractable computation of the sequence conformation from nucleotide interactions under free energy models. Optimal algorithms often assume a restricted class for the predicted RNA structures and yet still require a high-degree polynomial time complexity, which is too expensive to use. Heuristic methods may yield time-efficient algorithms but they do not guarantee optimality of the predicted structure. This paper introduces a new and efficient algorithm for the prediction of RNA structure with pseudoknots for which the structure is not restricted. Novel prediction techniques are developed based on graph tree decomposition. In particular, based on a simplified energy model, stem overlapping relationships are defined with a graph, in which a specialized maximum independent set corresponds to the desired optimal structure. Such a graph is tree decomposable; dynamic programming over a tree decomposition of the graph leads to an efficient optimal algorithm. The final structure predictions are then based on re-ranking a list of suboptimal structures under a more comprehensive free energy model. The new algorithm is evaluated on a large number of RNA sequence sets taken from diverse resources. It demonstrates overall sensitivity and specificity that outperforms or is comparable with those of previous optimal and heuristic algorithms yet it requires significantly less time than the compared optimal algorithms. The preliminary version of this paper appeared in the proceedings of the 6th Workshop on Algorithms for Bioinformatics (WABI 2006).     

Changes in MicroRNA expression patterns in human fibroblasts after low-LET radiation

Exposure to radiation provokes cellular responses controlled in part by gene expression networks. MicroRNAs (miRNAs) are small non-coding RNAs which mostly regulate gene expression by degrading the messages or inhibiting translation. Here, we investigated changes in miRNA expression patterns after low (0.1 Gy) and high (2.0 Gy) doses of X-ray in human fibroblasts. At early (0.5 h) and late (6 and 24 h) time points, irradiation caused qualitative and quantitative differences in the down-regulation of miRNA levels, including miR-92b, 137, 660, and 656. A transient up-regulation of miRNAs was observed after 2 h post-irradiation following high doses of radiation, including miR-558 and 662. MicroRNA levels were inversely correlated with targets from mRNA and proteomic profiling after 2.0 Gy of radiation. MicroRNAs miR-579, 608, 548-3p, and 585 are noted for targeting genes involved in radioresponsive mechanisms, such as cell cycle checkpoint and apoptosis. We suggest here a model in which miRNAs may act as "hub" regulators of specific cellular responses, immediately down-regulated so as to stimulate DNA repair mechanisms, followed by up-regulation involved in suppressing apoptosis for cell survival. Taken together, miRNAs may mediate signaling pathways in sequential fashion in response to radiation, and may serve as biodosimetric markers of radiation exposure.     

UBE1DC1, an ubiquitin-activating enzyme, activates two different ubiquitin-like proteins

Ubiquitin and ubiquitin-like proteins are known to be covalently conjugated to a variety of cellular substrates via a three-step enzymatic pathway. These modifications lead to the degradation of substrates or change its functional status. The ubiquitin-activating enzyme (E1) plays a key role in the first step of ubiquitination pathway to activate ubiquitin or ubiquitin-like proteins. Ubiquitin-activating enzyme E1-domain containing 1 (UBE1DC1) had been proved to activate an ubiquitin-like protein, ubiquitin-fold modifier 1 (Ufm1), by forming a high-energy thioester bond. In this report, UBE1DC1 is proved to activate another ubiquitin-like protein, SUMO2, besides Ufm1, both in vitro and in vivo by immunological analysis. It indicated that UBE1DC1 could activate two different ubiquitin-like proteins, SUMO2 and Ufm1, which have no significant similarity with each other. Subcellular localization in AD293 cells revealed that UBE1DC1 was especially distributed in the cytoplasm; whereas UBE1DC1 was mainly distributed in the nucleus when was cotransfected with SUMO2. It presumed that UBE1DC1 greatly activated SUMO2 in the nucleus or transferred activated-SUMO2 to nucleus after it conjugated SUMO2 in the cytoplasm.     

Thermodynamics of Agricultural Sustainability: The Case of US Maize Agriculture

This paper considers the local, field-scale sustainability of a productive industrial maize agrosystem that has replaced a fertile grassland ecosystem.

Using the revised thermodynamic approach of Svirezhev (1998 Svirezhev, Y. M. 1998. “Thermodynamic orientors: How to use Thermodynamic concepts in ecology”. In Eco Targets, Goal Functions, and Orientors, 102–122. Berlin: Springer Verlag. [Crossref] [Google Scholar], 2000 Svirezhev, Y. M. 2000. Thermodynamics and ecology. Ecological Modelling, 132: 11–22. [Crossref], [Web of Science ®] [Google Scholar]) and Steinborn and Svirezhev (2000) Steinborn, W. and Svirezhev, Y. M. 2000. Entropy as an indicator of sustainability in agro-ecosystems: North Germany case study. Ecol. Mode., 133: 247–257. [Crossref], [Web of Science ®] [Google Scholar], it is shown that currently this agrosystem is unsustainable in the U.S., with or without tilling the soil. The calculated average erosion rates of soil necessary to dissipate the entropy produced by U.S. maize agriculture, 23–45 t ha^?1 yr^?1, are bounded from above by an experimental estimate of mean soil erosion by conventional agriculture worldwide, 47 t ha^?1 yr^?1, (Montgomery, 2007 Montgomery, D. R. 2007. Soil erosion and agricultural sustainability. PNAS, 104(33): 13268–13272. [Crossref], [PubMed], [Web of Science ®] [Google Scholar]). Between 1982 and 1997, US agriculture caused an estimated 7–23 t ha^?1 yr^?1 of average erosion with the mean of 15 t ha^?1 yr^?1 (USDA-NRCS Database). The lower mean erosion rate of no till agriculture, 1.5 t ha^?1 yr^?1 (Montgomery, 2007 Montgomery, D. R. 2007. Soil erosion and agricultural sustainability. PNAS, 104(33): 13268–13272. [Crossref], [PubMed], [Web of Science ®] [Google Scholar]), necessitates the elimination of weeds and pests with field chemicals—with the ensuing chemical and biological soil degradation, and chemical runoff—to dissipate the produced entropy. The increased use of field chemicals that replace tillers is equivalent to the killing or injuring of up to 300 kg ha^?1 yr^?1 of soil flora and fauna. Additional soil degradation, not calculated here, occurs by acidification, buildup of insoluble metal compounds, and buildup of toxic residues from field chemicals. The degree of unsustainability of an average U.S. maize field is high, requiring 6–13 times more energy to reverse soil erosion and degradation, etc., than the direct energy inputs to maize agriculture. This additional energy, if spent, would not increase maize yields. The calculated “critical yield” of “organic” maize agriculture that does not use field chemicals and fossil fuels is only 30 percent lower than the average maize yield of 8.7 tons per hectare (～140 bu/acre) assumed here. This critical yield would not likely be achieved and sustained by large monocultures, but might be achieved by more balanced organic polycultures (Baum et al., 2008 Baum, A. W., Patzek, T. W., Bender, M., Renich, S. and Jackson, W. 2008. The Visible, Sustainable Farm: A Comprehensive Energy Analysis of a Midwestern Farm 1–34. Posted at petroleum.berkeley.edu/papers/Biofuels/SSF?Report3-051408.pdf [Google Scholar]).