Observation of a distinct transition in the mode of interconversion of ring pucker conformers in non-crystalline d-ribose-2'-d from 2H NMR spin-alignment

Internal motions of d-ribose selectively ²H-labeled at the 2 position were measured using solid state ²H NMR experiments. A sample of d-ribose-2 -d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of –74 and –60°C the C2–H2 bond was observed to undergo two kinds of motions which were similar to those of C2–H2/H2 found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609–8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6 ± 0.7 kcal mol^–1, and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy 4 kcal mol^–1. At –74°C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at –60°C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2–H2 bond are most likely from interconversions between C2-endo and C3-endo by way of the O4-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2–H2 bond within the C2-endo and C3-endo potential energy minima.     

The dynamic impact of CpG methylation in DNA

Solid-state deuterium NMR is used to investigate perturbations of the local, internal dynamics in the EcoRI restriction binding site, -GAATTC- induced by cytidine methylation. Methylation of the cytidine base in this sequence is known to suppress hydrolysis by the EcoRI restriction enzyme. Previous solid-state deuterium NMR studies have detected large amplitude motions of the phosphate-sugar backbone at the AT-CG junction of the unmethylated DNA sequence. This study shows that methylation of the cytidine base in a CpG dinucleotide reduces the amplitudes of motions of the phosphate-sugar backbone. These observations suggest a direct link between suppression of the amplitudes of localized, internal motions of the sugar-phosphate backbone of the DNA and inhibition of restriction enzyme cleavage.     

Biochemical and Clinical Improvement of Cytotoxic State by Amantadine Sulphate

  1. The main idea of the open clinical trial was to compare the income and outcome clinical picture and the evolution of the biochemical markers in the defined intervals in closed head injury group patients.2. In the group of 32 patients, mean age 40.78±15.36 years suffering from closed traumatic brain injury the following markers were measured: glycaemia, malondialdehyde (MDA) as marker of lipid peroxidation, beta-caroten, total SH groups as marker of protein oxidation in the following intervals: between the 1st and the 3rd, between the 3rd and the 7th, between the 1st and the 7th day respectively.3. Glycaemia significantly decreased since the 1st day till the 3rd day (p < 0.05) and since the 1st day till the 7th day (p < 0.05) but it was not significantly changed since the 3rd day till the 7th day (p > 0.05).4. MDA 1st × MDA 3rd p > 0.05 insignificant change, MDA 1st × MDA 7th p < 0.001—high significant decrease, MDA 3rd × MDA 7th—p < 0.0001—very high significant decrease.5. Beta-caroten the 1st × beta-caroten the 3rd day was insignificantly changed—p > 0.05, the 3rd × the 7th day beta-caroten increased significantly—p < 0.0002, the 1st day × 7th day beta-caroten significantly increased—p < 0.0001.6. We examined the SH groups only in nine patients, due to technical problems and SH groups decrease on the 3rd day (p < 0.005).7. In 18 amantadine sulphate subgroups (randomly selected), there was 5.5% lethality and mean outcome GCS (outGCS) 9.83±3.8, while lethality of the control subgroup (n=14) was 42.9%, mean outGCS 6.28±3.5.     

Hydration dependent dynamics in RNA

The essential role played by local and collective motions in RNA function has led to a growing interest in the characterization of RNA dynamics. Recent investigations have revealed that even relatively simple RNAs experience complex motions over multiple time scales covering the entire ms–ps motional range. In this work, we use deuterium solid-state NMR to systematically investigate motions in HIV-1 TAR RNA as a function of hydration. We probe dynamics at three uridine residues in different structural environments ranging from helical to completely unrestrained. We observe distinct and substantial changes in ²H solid-state relaxation times and lineshapes at each site as hydration levels increase. By comparing solid-state and solution state ¹³C relaxation measurements, we establish that ns–μs motions that may be indicative of collective dynamics suddenly arise in the RNA as hydration reaches a critical point coincident with the onset of bulk hydration. Beyond that point, we observe smaller changes in relaxation rates and lineshapes in these highly hydrated solid samples, compared to the dramatic activation of motion occurring at moderate hydration.     

Genetic dissection of Al tolerance QTLs in the maize genome by high density SNP scan

Background

Aluminum (Al) toxicity is an important limitation to food security in tropical and subtropical regions. High Al saturation on acid soils limits root development, reducing water and nutrient uptake. In addition to naturally occurring acid soils, agricultural practices may decrease soil pH, leading to yield losses due to Al toxicity. Elucidating the genetic and molecular mechanisms underlying maize Al tolerance is expected to accelerate the development of Al-tolerant cultivars.

Results

Five genomic regions were significantly associated with Al tolerance, using 54,455 SNP markers in a recombinant inbred line population derived from Cateto Al237. Candidate genes co-localized with Al tolerance QTLs were further investigated. Near-isogenic lines (NILs) developed for ZmMATE2 were as Al-sensitive as the recurrent line, indicating that this candidate gene was not responsible for the Al tolerance QTL on chromosome 5, qALT5. However, ZmNrat1, a maize homolog to OsNrat1, which encodes an Al³⁺ specific transporter previously implicated in rice Al tolerance, was mapped at ~40 Mbp from qALT5. We demonstrate for the first time that ZmNrat1 is preferentially expressed in maize root tips and is up-regulated by Al, similarly to OsNrat1 in rice, suggesting a role of this gene in maize Al tolerance. The strongest-effect QTL was mapped on chromosome 6 (qALT6), within a 0.5 Mbp region where three copies of the Al tolerance gene, ZmMATE1, were found in tandem configuration. qALT6 was shown to increase Al tolerance in maize; the qALT6-NILs carrying three copies of ZmMATE1 exhibited a two-fold increase in Al tolerance, and higher expression of ZmMATE1 compared to the Al sensitive recurrent parent. Interestingly, a new source of Al tolerance via ZmMATE1 was identified in a Brazilian elite line that showed high expression of ZmMATE1 but carries a single copy of ZmMATE1.

Conclusions

High ZmMATE1 expression, controlled either by three copies of the target gene or by an unknown molecular mechanism, is responsible for Al tolerance mediated by qALT6. As Al tolerant alleles at qALT6 are rare in maize, marker-assisted introgression of this QTL is an important strategy to improve maize adaptation to acid soils worldwide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-153) contains supplementary material, which is available to authorized users.     

The orientation of insulin receptors in the red cell membrane

High-affinity binding of insulin to receptors in human erythrocyte membranes occurred at the external surface, but not at the cytoplasmic surface of the plasma membrane, as assessed by insulin binding to right-side-out and inside-out membrane vesicles. Even after prolonged (3 h) incubation at 22°C, binding at the cytoplasmic membrane aspect remained negligible. The data indicate that the insulin receptor displays its hormone-binding site exclusively toward the extracellular space and that transmembrane mobility (“flip-flop”) of the receptor from one to the other membrane leaflet is severely restricted.     

Proton nuclear magnetic resonance studies on the variant-3 neurotoxin from Centruroides sculpturatus Ewing: sequential assignment of resonances

We report the sequential assignment of resonances to specific residues in the proton nuclear magnetic resonance spectrum of the variant-3 neurotoxin from the scorpion Centruroides sculpturatus Ewing (range southwestern U.S.A.). A combination of two-dimensional NMR experiments such as 2D-COSY, 2D-NOESY, and single- and double-RELAY coherence transfer spectroscopy has been employed on samples of the protein dissolved in D2O and in H2O for assignment purposes. These studies provide a basis for the determination of the solution-phase conformation of this protein and for undertaking detailed structure-function studies of these neurotoxins that modulate the flow of sodium current by binding to the sodium channels of excitable membranes.     

Dynamics of bases in hydrated [d(CGCGAATTCGCG)]2

Solid-state 2H NMR spectroscopy has been used to investigate the dynamics of a DNA oligonucleotide with a defined sequence, [d(CGCGAATTCGCG)]2, which contains the EcoRI binding site. Quadrupole echo line shapes and spin-lattice relaxation times were obtained as a function of hydration on two different deuterated samples, both in the form of the Na salt. In one sample, the C8 protons of all purines in the self-complementary dodecamer were exchanged for deuterons. In the other sample, a specifically labeled thymidine (C6 deuterated) was synthetically incorporated at the seventh position (counting 5' to 3') in the sequence. The general trends for both samples were quite similar. At all levels of hydration, the data reveal the presence of a rapid, small-amplitude libration of the bases (tau c less than or equal to 1 ns, 6 degrees-10 degrees amplitude). At the higher hydration levels (80% relative humidity or higher), the results indicate the presence of a much slower motion (tau c approximately 10-100 microseconds), which at 80% relative humidity is of small amplitude (approximately 5 degrees) and at higher hydration levels may be of larger amplitude. There is no evidence for large-amplitude (greater than +/- 10 degrees) motion on a nanosecond or faster time scale under any hydration condition. The 2H NMR results were analyzed with a dynamical model which treats the oligonucleotide as a deformable filament and which can include collective torsional fluctuations. The slow motion observed at high hydration levels is attributed to the uniform twisting mode (of the entire helix).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequential 1H NMR assignments and secondary structure of aponeocarzinostatin in solution

Sequential assignments and secondary structural analysis have been accomplished for the 113-residue apoprotein of the antitumor drug neocarzinostatin (NCS) from Streptomyces carzinostaticus. A total of 98% of the main-chain and 77% of the side-chain resonances have been sequence specifically assigned by use of information from coherence transfer experiments and by sequential and interstrand NOEs. Because of the complexity of the NCS spectrum, several sequential assignment strategies were employed to complete the analysis. Apo-NCS consists of three antiparallel beta-sheeted domains by NMR analysis. There is an extensive four-strand antiparallel beta-sheet, and two two-stranded domains. One of the two-strand domains is contiguous, S72-N87, with chain reversal occurring through the region L77-R82. The other two-stranded domain has the section G16-A24 antiparallel with respect to the region S62-R70. This secondary structure is consistent with the crystal structure of holo-NCS at 2.8-A resolution.     

Phylogenetic relationships of typical antbirds (Thamnophilidae) and test of incongruence based on Bayes factors

Background  

The typical antbirds (Thamnophilidae) form a monophyletic and diverse family of suboscine passerines that inhabit neotropical forests. However, the phylogenetic relationships within this assemblage are poorly understood. Herein, we present a hypothesis of the generic relationships of this group based on Bayesian inference analyses of two nuclear introns and the mitochondrial cytochrome b gene. The level of phylogenetic congruence between the individual genes has been investigated utilizing Bayes factors. We also explore how changes in the substitution models affected the observed incongruence between partitions of our data set.