TSAR: a program for automatic resonance assignment using 2D cross-sections of high dimensionality, high-resolution spectra

While NMR studies of proteins typically aim at structure, dynamics or interactions, resonance assignments represent in almost all cases the initial step of the analysis. With increasing complexity of the NMR spectra, for example due to decreasing extent of ordered structure, this task often becomes both difficult and time-consuming, and the recording of high-dimensional data with high-resolution may be essential. Random sampling of the evolution time space, combined with sparse multidimensional Fourier transform (SMFT), allows for efficient recording of very high dimensional spectra (≥4 dimensions) while maintaining high resolution. However, the nature of this data demands for automation of the assignment process. Here we present the program TSAR (Tool for SMFT-based Assignment of Resonances), which exploits all advantages of SMFT input. Moreover, its flexibility allows to process data from any type of experiments that provide sequential connectivities. The algorithm was tested on several protein samples, including a disordered 81-residue fragment of the δ?subunit of RNA polymerase from Bacillus subtilis containing various repetitive sequences. For our test examples, TSAR achieves a high percentage of assigned residues without any erroneous assignments.     

LIMITED EFFECT OF REACTIVE OXYGEN SPECIES ON THE COMPOSITION OF SUSCEPTIBLE ESSENTIAL AMINO ACIDS IN THE MIDGUTS OF Lymantria Dispar CATERPILLARS

The essential amino acids (EAAs) arginine, histidine, lysine, and methionine, as well as cysteine (semiessential), are believed to be susceptible to reactions with reactive oxygen species (ROS) in biological systems. The decreased availability of these EAAs could harm insect nutrition, since several of them can also be limiting for protein synthesis. However, no in vivo studies have quantified the effect of ROS in the midguts of insect herbivores on EAA composition. This study examined the association between elevated levels of ROS in the midgut fluid of Lymantria dispar caterpillars and the compositions of EAAs (protein‐bound + protein‐free) in their midgut fluid and frass. Contrary to expectation, the compositions of EAAs were not significantly decreased by ROS in midgut fluid ex vivo when incubated with phenolic compounds. Two in vivo comparisons of low‐ and high‐ROS‐producing leaves also showed similar results: there were no significant decreases in the compositions of EAAs in the midgut fluids and/or frass of larvae with elevated levels of ROS in their midguts. In addition, waste nitrogen excretion was not significantly increased from larvae on high‐ROS treatments, as would be expected if ROS produced unbalanced EAA compositions. These results suggest that L. dispar larvae are able to tolerate elevated levels of ROS in their midguts without nutritionally significant changes in the compositions of susceptible EAAs in their food.     

A SALL4 zinc finger missense mutation predicted to result in increased DNA binding affinity is associated with cranial midline defects and mild features of Okihiro syndrome

Truncating mutations of the gene SALL4 on chromosome 20q13.13–13.2 cause Okihiro and acro-renal-ocular syndromes. Pathogenic missense mutations within the SALL4 or SALL1 genes have not yet been reported, raising the question which phenotypic features would be associated with them. Here we describe the first missense mutation within the SALL4 gene. The mutation results in an exchange of a highly conserved zinc-coordinating Histidine crucial for zinc finger (ZF) structure within a C2H2 double ZF domain to an Arginine. Molecular modeling predicts that this exchange does not result in a loss of zinc ion binding but leads to an increased DNA-binding affinity of the domain. The index patient shows mild features of Okihiro syndrome, but in addition cranial midline defects (pituitary hypoplasia and single central incisor). This finding illustrates that the phenotypic and functional effects of SALL4 missense mutations are difficult to predict, and that other SALL4 missense mutations might lead to phenotypes not overlapping with Okihiro syndrome. This study has received approval by the institutional review board (Ethics committee) of the Medical Faculty, University of Freiburg, Germany. Jan Miertus and Wiktor Borozdin have equally contributed to this work.     

Accelerated laboratory test to study fungal biodeterioration of cementitious matrix

The present study aimed to develop an accelerated laboratory test to study the biodeteriorative effect of different fungal strains to a cementitious matrix. The test developed in this study permits to obtain a rapid fungal development on cement specimens. Three months of experiments only are needed to obtain first results, which is rather shorter than other test developed to date to study fungal biodeterioration. Results are mainly related to aesthetical biodeterioration. Results show that in these experimental conditions, fungal growth occurs since the first week of incubation. Stereomicroscopy observations showed that microbial growth was noticed only on the surface of specimens, while PAS staining revealed the real extent of microbial growth on and within the matrix as later confirmed by SEM observations of cross section showing the penetration of hyphae inside the matrix. Test can be used with short time of incubation, to test and to compare bioreceptivity of cement-based materials; and several months of incubation should allow the study of mechanisms involved in biodeterioration.     

Multiple quadrature detection in reduced dimensionality experiments

A new, simple procedure is proposed which enables acquisition of two or more chemical shifts encoded in a common dimension simultaneously in quadrature. For n chemical shifts projected in a single dimension, the expected effect is obtained by interleaved acquisition and appropriate combination of 2ⁿ data sets per increment of respective evolution time. The particular chemical shifts can be calculated from sums and differences of signal frequencies obtained by different combination of the acquired data sets. In comparison to the established reduced dimensionality (RD) techniques, the proposed method enhances resolution due to reduction of the number of signals and requires less evolution time increments owing to narrower spectral width in the RD-domain. We show examples of the application of the new approach to the 2D HNCA and HN(CO)CA techniques with two, and 2D HACANH with three frequencies simultaneously encoded in the t ₁ evolution period, for ¹³C,¹⁵N-labeled ubiquitin.     

Antigenic structure of rabies virus glycoprotein: ordering and immunological characterization of the large CNBr cleavage fragments.

After rabies virus glycoprotein was treated with CNBr, the peptide mixture was fractionated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CNBr-cleaved peptide fragments were resolved into seven peptide bands under reducing conditions and six peptide bands under nonreducing conditions. The isolated nonreduced polypeptides were further analyzed by electrophoresis under reducing conditions. The N-terminal amino acid sequences were determined for the peptides in each of the isolated bands. The sequence data identified eight CNBr peptides and allowed the peptide fragments to be ordered within the deduced amino acid sequence of the glycoprotein. Analysis of the nonreduced CNBr peptides revealed two conformations of the glycoprotein. Two CNBr peptide fragments were specifically immunoprecipitated with a hyperimmune anti-rabies glycoprotein serum. These two and one other CNBr peptide induced the production of rabies virus-neutralizing antibodies, indicating the existence of at least three distinct antigenic sites on the rabies virus glycoprotein.     

S100A6 mediates nuclear translocation of Sgt1: a heat shock-regulated protein

Sgt1 was originally identified in yeast as a suppressor of the Skp1 protein. Later, it was found that Sgt1 is present in plant and mammalian organisms and that it binds other ligands such as S100A6, a calcium-binding protein. In this work we show that in HEp-2 cells Sgt1 translocates to the nucleus due to heat shock. We also found that in HEp-2 cells with diminished level of S100A6, due to stable transfection with siRNA against S100A6, such translocation occurred at a much smaller scale in comparison with cells expressing a normal level of S100A6. Moreover, translocation of Sgt1 was observed in HEp-2 cells treated with thapsigargin instead of heat shock. In contrast thapsigargin was ineffective in cells with diminished level of S100A6. Thus, our results suggest that increase in intracellular concentration of Ca²⁺, transduced by S100A6, is necessary for nuclear translocation of the Sgt1 protein.     

Reconstruction of non-uniformly sampled five-dimensional NMR spectra by signal separation algorithm

A method for five-dimensional spectral reconstruction of non-uniformly sampled NMR data sets is proposed. It is derived from the previously published signal separation algorithm, with major alterations to avoid unfeasible processing of an entire five-dimensional spectrum. The proposed method allows credible reconstruction of spectra from as little as a few hundred data points and enables sensitive resonance detection in experiments with a high dynamic range of peak intensities. The efficiency of the method is demonstrated on two high-resolution spectra for rapid sequential assignment of intrinsically disordered proteins, namely 5D HN(CA)CONH and 5D (HACA)CON(CO)CONH.