Determination of the backbone torsion angle ɛ in nucleic acids

Summary The multiplet structure of cross peaks in double-quantum-filtered COSY NMR spectra is analysed for those resonances that include passive heteronuclear couplings. Interestingly, the cross peak involving the sugar-ring protons H2 and H3 in nucleic acids display an E.COSY-type appearance exclusively when the backbone torsion angle (C4-C3-O3-P) adopts a gauche(-) conformation. This observation allows an unambiguous analysis of the conformation around , without the knowledge of ³J_cp coupling constants.     

Determination of very-long-chain fatty acids in serum by gas chromatography–nitrogen–phosphorus detection following cyanomethylation

A sensitive method for very-long-chain fatty acid analysis was developed by gas chromatography–nitrogen–phosphorus detection by using cyanomethyl derivatization. Bromoacetonitrile as alkylating reagent was used to improve nitrogen phosphorus detection detectability of compounds containing non-nitrogen. The carboxyl group of very-long-chain fatty acid was alkylated to cyanomethyl esters. Reaction conditions were 40 min at 60°C under potassium carbonate base. Heptacosanoic acid was used as an internal standard and hexane was used as a solvent of extraction. The extraction yield was 82.8% or more, relative standard deviation of the precision test was 8.3 % or more and the result of linearity test showed a good correlation coefficient of r²=0.999 in the range of 0.1–50 μg/ml. The quantification limits were 10 ng/ml when 0.5 ml of serum was used. The present method proved simple, rapid, inexpensive and resistant to contaminants. When it was applied to serum samples taken from patients with X-linked adrenoleukodystrophy which is a hereditary X-linked disorder characterized by progressive demyelination and adrenal insufficiency during childhood, relative increase of the concentration of hexacosanoic acid and the concentration ratios of hexacosanoic, lignoceric to behenic acid was observed in comparison with control samples.     

Structure–function correlations in tyrosinases

Tyrosinases are metalloenzymes belonging to the type-3 copper protein family which contain two copper ions in the active site. They are found in various prokaryotes as well as in plants, fungi, arthropods, and mammals and are responsible for pigmentation, wound healing, radiation protection, and primary immune response. Tyrosinases perform two sequential enzymatic reactions: hydroxylation of monophenols and oxidation of diphenols to form quinones which polymerize spontaneously to melanin. Two other members of this family are catechol oxidases, which are prevalent mainly in plants and perform only the second oxidation step, and hemocyanins, which lack enzymatic activity and are oxygen carriers. In the last decade, several structures of plant and bacterial tyrosinases were determined, some with substrates or inhibitors, highlighting features and residues which are important for copper uptake and catalysis. This review summarizes the updated information on structure–function correlations in tyrosinases along with comparison to other type-3 copper proteins.     

Do all backbone polar groups in proteins form hydrogen bonds?

Evidence from proteins and peptides supports the conclusion that intrapeptide hydrogen bonds stabilize the folded form of proteins. Paradoxically, evidence from small molecules supports the opposite conclusion, that intrapeptide hydrogen bonds are less favorable than peptide-water hydrogen bonds. A related issue-often lost in this debate about comparing peptide-peptide to peptide- water hydrogen bonds-involves the energetic cost of an unsatisfied hydrogen bond. Here, experiment and theory agree that breaking a hydrogen bond costs between 5 and 6 kcal/mol. Accordingly, the likelihood of finding an unsatisfied hydrogen bond in a protein is insignificant. This realization establishes a powerful rule for evaluating protein conformations.     

Rapid immobilization of applied nitrogen in saline–alkaline soils

The dynamics of inorganic N are important in soil, and this applies particularly to the saline–alkaline soils of the former lake Texcoco in Mexico with high pH and salinity where a forestation program was started in the 1970s. In soils of lake Texcoco, in Mexico, more than 50% of applied N could not be accounted for one day after application of 200 mg kg^–1 soil along with glucose amendment. It was not clear whether this was due to abiotic or biotic processes, the form of inorganic N applied or the result of applying an easily decomposable substrate. We investigated this by adding glucose and 200 mg kg^–1 soil as (NH₄)₂SO₄-N or KNO₃-N to sterilized and unsterilized soil. The changes in inorganic and ninhydrin N, microbial biomass C and production of CO₂ were then monitored. Between the time of applying N and extraction with 0.5 M K₂SO₄, i.e., after ca 2 h, approximately 110 mg NH₄ ⁺-N kg^–1 dry soil could not be accounted for in the unsterilized and sterilized soil and that remained so for the entire incubation in the sterilized soil. After 1 day this increased to 140 mg NH₄ ⁺-N kg^–1 dry soil in the unsterilized control and 170 mg NH₄ ⁺-N kg^–1 dry soil in C amended soil. Volatilization of NH₃ accounted for 56 mg NH₄ ⁺-N kg^–1 so the rest appeared to be adsorbed on the soil matrix. The NH₃ volatilization and NH₄ ⁺ fixed in the soil matrix remained constant over time and no oxidation to NO₂ ^– or NO₃ ^– had occurred, so unaccounted N in unsterilized soil was probably incorporated into the microbial biomass in excess of what was required for metabolic activity. The unaccounted N was ca 70 mg NO₃ ^––N in nitrate amended soil after 3 days and 138 NO₃ ^––N when glucose was additionally added. Losses through abiotic processes were absent as inferred from changes in sterilized soil and the aerobic incubation inhibited possible losses through denitrification. It was inferred that NO₃ ^– that could not be accounted for was taken up by micro-organisms in excess of what was required for metabolic activity.     

Shift in soil–plant nitrogen dynamics of an alpine–nival ecotone

We investigated the nitrogen (N) dynamics of an alpine–nival ecotone on Mt. Schrankogel, Tyrol, Austria, in relation to temperature. Natural abundance of ¹⁵N was used as a tool to elucidate differences in N cycling along an altitudinal transect ranging from 2,906 to 3,079 m, corresponding to a gradient in mean annual temperature of 2.4 °C. The amount of total soil N, of plant available N and soil C/N ratio decreased significantly with increasing altitude, whereas soil pH increased. Soil δ ¹⁵N decreased with increasing altitude from +2.2 to −2.1‰ and δ ¹⁵N of plant tissues (roots and leaves) decreased from −3.7 to −5.5‰. The large shift in soil δ ¹⁵N of 4.3‰ from the lowest to the highest site suggested substantial differences in N cycling in alpine and nival ecosystems in the alpine nival ecotone investigated. We concluded that N cycling at the alpine–nival ecotone is likely to be controlled by various factors: temperature, soil age and development, atmospheric N deposition and plant competition. Our results furthermore demonstrate that the alpine–nival ecotone may serve as a sensitive indicator of global change.     

Simultaneous acquisition of 13Cα–15N and 1H–15N–15N sequential correlations in proteins: application of dual receivers in 3D HNN

We describe here, adaptation of the HNN pulse sequence for multiple nuclei detection using two independent receivers by utilizing the detectable ¹³C^α transverse magnetization which was otherwise dephased out in the conventional HNN experiment. It enables acquisition of 2D ¹³C^α–¹⁵N sequential correlations along with the standard 3D ¹⁵N–¹⁵N–¹H correlations, which provides directionality to sequential walk in HNN, on one hand, and enhances the speed of backbone assignment, on the other. We foresee that the implementation of dual direct detection opens up new avenues for a wide variety of modifications that would further enhance the value and applications of the experiment, and enable derivation of hitherto impossible information.     

The evolution of signal–reward correlations in bee- and hummingbird-pollinated species of Salvia

Within-individual variation in floral advertising and reward traits is a feature experienced by pollinators that visit different flowers of the same plant. Pollinators can use advertising traits to gather information about the quality and amount of rewards, leading to the evolution of signal–reward correlations. As long as plants differ in the reliability of their signals and pollinators base their foraging decisions on this information, natural selection should act on within-individual correlations between signals and rewards. Because birds and bees differ in their cognitive capabilities, and use different floral traits as signals, we tested the occurrence of adaptive divergence of the within-individual signal–reward correlations among Salvia species that are pollinated either by bees or by hummingbirds. They are expected to use different floral advertising traits: frontal traits in the case of bees and side traits in the case of hummingbirds. We confirmed this expectation as bee- and hummingbird-pollinated species differed in which specific traits are predominantly associated with nectar reward at the within-individual level. Our findings highlight the adaptive value of within-individual variation and covariation patterns, commonly disregarded as ‘environmental noise’, and are consistent with the hypothesis that pollinator-mediated selection affects the correlation pattern among floral traits.     

Determination of 13Cα relaxation times in uniformly 13C/15N-enriched proteins

Summary Relaxation times of ¹³C carbons of uniformly ¹³C/¹⁵N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T₁, T₂ and the heteronuclear NOE of ¹³C in uniformly ¹³C/¹⁵N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T₁ and the heteronuclear NOE were similar to those of the corresponding ¹⁵N experiments published previously. The determination of T₂ for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T₂ evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T₂ evolution period were simulated in order to optimize the bandwidth for which reliable T₂ relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual ¹H–¹³C cross peaks, in a series of (¹H, ¹³C)-ct-HSQC spectra with a modified CPMG sequence as well as a T_1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.     

Computer simulation of the influence of hydrogen on stress–order correlations in amorphous silicon

This paper reports the computational simulation of the correlations between atomic-level stress and local structure fluctuations in a computational model of amorphous silicon. A single parameter has been identified, which uniquely characterises the structural order in these structures. This parameter is the linear combination of the SDs of the first and second nearest neighbour separations. The stress fluctuations, under progressive hydrogen incorporation, show two clear dependences on this parameter, and therefore on the structural order. This dual dependency clearly alludes to structural network configurations that contain low and high hydrogen concentrations. The implications of the results on the local geometry of tetrahedrally bonded amorphous solids are discussed.     

Determination of Pneumococcal Serotypes in Meningitis Cases in Niger, 2003–2011

Background

The epidemiology of pneumococcal meningitis in the African ‘meningitis belt’ is poorly studied. In order to ensure an effective vaccination strategy and post-vaccination surveillance, we examined the serotype distribution patterns of pneumococcal meningitis in Niger over the period 2003–2011.

Methods

Cerebrospinal fluid (CSF) samples were collected from different health facilities throughout Niger in the frame of the national microbiological surveillance of meningitis. Determination of the serotype of CSF positive for pneumococci was performed using a sequential multiplex PCR method (SM-PCR) adapted with a national algorithm in which 32 different serotypes were covered and grouped into eight consecutive PCR.

Results

The SM-PCR assay could predict the Sp serotype for 779 CSF (88.7%), 98 CSF (11.3%) were not-typeable in our national-adapted algorithm. In total, 26 different serotypes were identified. Serotype 1 (n = 393) was the most prevalent and accounted for 45.3% of infections, followed by serogroups/serotypes 12F/(12A)/(44)/(46) (7.3%), 6/(6A/6B/6C/6D) (5.4%), 14 (5.2%), 5 (4.6%), 23F (4.2%), 45 (3.6%), 2 (3.1%), 18/(18A/18B/18C/18F) (2.9%) and 17 others serotypes with a prevalence of less than 2%. The proportion of serotype 1 in infants(<2 years old) represented only 4.3% of the cases affected by this serotype. In contrast, serotypes 5, 6, 14, 19A and 23F were only detected in very young children.

Conclusions

The proportion of serotype 1 in the pneumococcal meningitis cases and the theoretical vaccine coverage across all age groups advocates for the introduction of a conjugate vaccine (PCV10 or 13) into the Expanded Programme on Immunization (EPI) in Niger. Post-vaccine introduction surveillance supported by molecular approaches will be essential to provide a comprehensive picture of the impact of the vaccine on the burden reduction of pneumococcal meningitis and on pneumococcal serotype distribution.     

[15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N–1H, aromatic 13C–1H and side-chain 15N–1H2 correlations in large proteins

This paper describes a [¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide ¹⁵N–¹H groups, side chain ¹⁵N–¹H₂ groups and aromatic ¹³C–¹H groups in otherwise highly deuterated proteins. The ¹⁵N–¹H and ¹³C–¹H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the ¹H dimension. The side-chain ¹⁵N–¹H₂ groups, which are suppressed in conventional [¹⁵N,¹H)-TROSY, are observed with high sensitivity in the ¹⁵N–¹H subspectrum. [¹⁵N,¹H]/[¹³C,¹H]-TROSY was used as the heteronuclear correlation block in a 3D [¹H,¹H]-NOESY-[¹⁵N,¹H]/[¹³C,¹H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain ¹⁵N–¹H₂ groups.     

Heteronuclear relayed E.COSY revisited: Determination of 3J(Hα,Cγ) couplings in Asx and aromatic residues in proteins

Constant-time 3D heteronuclear relayed E.COSY [Schmidt et al. (1996) J. Biomol. NMR, 7, 142–152], as based on generic 2D small-flip-angle HMQC-COSY [Schmidt et al. (1995) J. Biomol. NMR, 6, 95–105], has been modified to allow for quantitative determination of heteronuclear three-bond ³ J(H,C) couplings. The method is applicable to amino acid spin topologies with carbons in the position which lack attached protons, i.e. to asparagine, aspartate, and aromatic residues in uniformly ¹³C-enriched proteins. The pulse sequence critically exploits heteronuclear triple-quantum coherence (HTQC) of CH₂ moieties involving geminal H proton pairs, taking advantage of improved multiple-quantum relaxation properties, at the same time avoiding scalar couplings between those spins involved in multiple-quantum coherence, thus yielding E.COSY-type multiplets with a splitting structure that is simpler than with the original scheme. Numerical least-squares 2D line-shape simulation is used to extract ³ J(H,C) coupling constants which are of relevance to side-chain ₁ dihedral-angle conformations in polypeptides. Methods are demonstrated with recombinant ¹⁵N,¹³C-enriched ribonuclease T1 and Desulfovibrio vulgaris flavodoxin with bound oxidized FMN.     

Supramolecular organization of fucoxanthin–chlorophyll proteins in centric and pennate diatoms

Fucoxanthin–chlorophyll proteins (FCP) are the major light-harvesting proteins of diatom algae, a major contributor to marine carbon fixation. FCP complexes from representatives of centric (Cyclotella meneghiniana) and pennate (Phaeodactylum tricornutum) diatoms were prepared by sucrose gradient centrifugation and studied by means of electron microscopy followed by single particle analysis. The oligomeric FCP from a centric diatom were observed to take the form of unusual chain-like or circular shapes, a very unique supramolecular assembly for such antennas. The existence of the often disputed oligomeric form of FCP in pennate diatoms has been confirmed. Contrary to the centric diatom FCP, pennate diatom FCP oligomers are very similar to oligomeric antennas from related heterokont (Stramenopila) algae. Evolutionary aspects of the presence of novel light-harvesting protein arrangement in centric diatoms are discussed.     

Vibrational distribution of nitrogen molecules in the C3Πu state in a near-surface microwave plasma in nitrogen at pressures of 1–5 Torr

The radiation of the second positive nitrogen system has been used to study the spatial dependence of the vibrational distribution of nitrogen molecules in the C³Π_u state in the near-surface plasma layer of an electrode microwave discharge in nitrogen at pressures of 1–5 Torr. It has been shown that the vibrational distribution changes at a scale of 100 μm. It has been concluded that this state is populated owing to the electron impact from the ground state. The possibility of using the local approximation for the electron energy distribution function to explain the experimental results has been analyzed.     

Genotype–phenotype correlations in Leber hereditary optic neuropathy

Leber hereditary optic neuropathy (LHON), acute or subacute vision loss due to retinal ganglion cell death which in the long run leads to optic nerve atrophy is one of the most widely studied maternally inherited diseases caused by mutations in mitochondrial DNA. Although three common mutations, 11778G>A, 14484T>C or 3460G>A are responsible for over 90% of cases and affect genes encoding complex I subunits of the respiratory chain, their influence on bioenergetic properties of the cell is marginal and cannot fully explain the pathology of the disease. The following chain of events was proposed, based on biochemical and anatomical properties of retinal ganglion cells whose axons form the optic nerve: mitochondrial DNA mutations increase reactive oxygen species production in these sensitive cells, leading to caspase-independent apoptosis. As LHON is characterized by low penetrance and sex bias (men are affected about 5 times more frequently than women) the participation of the other factors—genetic and environmental—beside mtDNA mutations was studied. Mitochondrial haplogroups and smoking are some of the factors involved in the complex etiology of this disease.     

On the calculation of 3 J αβ-coupling constants for side chains in proteins

Structural knowledge about proteins is mainly derived from values of observables, measurable in NMR spectroscopic or X-ray diffraction experiments, i.e. absorbed or scattered intensities, through theoretically derived relationships between structural quantities such as atom positions or torsional angles on the one hand and observable quantities such as squared structure factor amplitudes, NOE intensities or (3) J-coupling constants on the other. The standardly used relation connecting (3) J-couplings to torsional angles is the Karplus relation, which is used in protein structure refinement as well as in the evaluation of simulated properties of proteins. The accuracy of the simple and generalised Karplus relations is investigated using side-chain structural and (3) J (αβ)-coupling data for three different proteins, Plastocyanin, Lysozyme, and FKBP, for which such data are available. The results show that the widely used Karplus relations are only a rough estimate for the relation between (3) J (αβ)-couplings and the corresponding χ(1)-angle in proteins.