Comparison of VP4 and VP7 of five murine rotavirus strains.

The nucleotide and deduced amino acid sequences of outer capsid proteins VP4 and VP7 of five murine rotavirus strains (EW, EB, EC, EL, EHP) were determined. Comparisons of the VP7 amino acid sequences of the five murine rotavirus strains with rotavirus strains representative of G serotypes 1-14 showed that the murine strains were highly homologous to one another and more closely related to strains representing G3 than to any other G type. Analysis of the VP4 amino acid sequences of the murine strains revealed the presence of at least two murine P types.

Polypeptide conformational space. Dynamics by solution NMR disorder by X-ray crystallography.

A symmetric dimer of a polypeptide in an organic solvent is shown to be a useful molecular system for observing the effects of crystal packing and solution dynamics on structure determination by X-ray crystallography and solution NMR methods. Organic solvents are not ideal models of a lipid environment, but they represent a better model environment than does water, in fact, for modeling some properties of the lipid environment, organic solvents work very well. In particular, it is a good model for assessing the influence of lipids on local polypeptide dynamics and conformational rearrangements.

Comparison of the rotavirus nonstructural protein NSP1 (NS53) from different species by sequence analysis and northern blot hybridization.

The nucleotide sequence of gene 5 encoding the rotavirus nonstructural protein NSP1 (NS53) of 6 strains (EW, EHP, RRV, I321, OSU, and Gottfried) was determined and compared to 6 previously reported strains (SA11, UK, RF, Hu803, DS-1, and Wa). The 12 rotavirus strains were derived from a total of five separate species (murine, bovine, simian, porcine, and human). Gene sizes ranged from 1564 to 1611 nucleotides in length and the deduced protein sequences were found to be 486 to 495 amino acids in length.

Prosecution of child sexual abuse: which cases are accepted?

This study examined the relationship of case characteristics, maternal support, and child psychopathology to acceptance of child sexual abuse cases for prosecution. Cases referred to prosecutors' offices over a 1-year period in four urban jurisdictions (N = 431) were examined, and a smaller sample of mothers and children (N = 289) were interviewed as well. Background characteristics of the perpetrator and victim, severity of abuse, and nature of available evidence were all significantly related to acceptance for prosecution.

Structural analysis of highly oriented poly(p-phenylene-terephthalamide) by 15N solid-state nuclear magnetic resonance.

The structure of an aromatic polyamide, poly(p-phenylene-terephthalamide) (PPTA), was studied in the solid state using 15N nuclear magnetic resonance (NMR) spectroscopy. Spectra of uniaxially aligned molecules placed with the axis of alignment both parallel with and perpendicular to the applied magnetic field were analyzed to yield the orientations of specific molecular bonds with respect to the fiber axis. The 15N chemical shift tensor was characterized by simulating powder pattern spectra of both PPTA and a model compound, benzanilide.

Side-chain structure and dynamics at the lipid-protein interface: Val1 of the gramicidin A channel.

High resolution dynamics and structural information has been resolved from 2H solid-state NMR spectra of the Val-1 side-chain of the gramicidin channel in a lipid bilayer. Both powder pattern lineshapes and spectra from uniformly aligned samples of gramicidin in lipid bilayers have been analyzed to achieve a fully consistant interpretation of the data. Torsional motions about the C alpha C beta axis (chi 1) are shown to be three-state jumps in which the occupancy of the states is given by the ratio, 75:15:10 for the chi 1 angles of 184 degrees:304 degrees:64 degrees.

Rapidly-frozen polypeptide samples for characterization of high definition dynamics by solid-state NMR spectroscopy.

A method is described for defining anisotropic local dynamics in polypeptides by solid-state NMR. To avoid conformational heterogeneity introduced by large hexagonal ice crystals in low temperature hydrated samples, a fast-freezing technique is used for sample preparation. For a demonstration of this approach, the backbone librational motions of the gramicidin A channel conformation are studied in hydrated DMPC bilayers.

Identification of a new neutralization epitope on VP7 of human serotype 2 rotavirus and evidence for electropherotype differences caused by single nucleotide substitutions.

Neutralizing monoclonal antibodies 2F1 and 1C10, which are specific for VP7 of serotype 2 rotaviruses (G2), were used to select neutralization escape variants of the human serotype 2 rotavirus, DS-1. Neutralization survival patterns generated by monoclonal antibodies 2F1, 1C10, and RV5:3 indicated that 2F1 and 1C10 did not recognize identical epitopes. Direct sequencing of PCR products of gene 8, encoding VP7, revealed that each escape variant possessed only a single nucleotide mutation which resulted in a single amino acid substitution.

High-resolution conformation of gramicidin A in a lipid bilayer by solid-state NMR.

Solid-state nuclear magnetic resonance spectroscopy of uniformly aligned preparations of gramicidin A in lipid bilayers has been used to elucidate a high-resolution dimeric structure of the cation channel conformation solely on the basis of the amino acid sequence and 144 orientational constraints. This initial structure defines the helical pitch as single-stranded, fixes the number of residues per turn at six to seven, specifies the helix sense as right-handed, and identifies the hydrogen bonds. Refinement of this initial structure yields reasonable hydrogen-bonding distances with only minimal changes in the torsion angles.

Studies on the mechanism of 4-aminophenol-induced toxicity to renal proximal tubules.

4-Aminophenol (PAP) is known to cause nephrotoxicity in the rat where it produces selective necrosis to renal proximal tubules. The aim of this work was to investigate the toxicity of PAP and its known nephrotoxic metabolite 4-amino-3-S-glutathionylphenol using a well defined suspension of rabbit renal proximal tubules. PAP at a concentration of 0.

2H NMR determination of the global correlation time of the gramicidin channel in a lipid bilayer.

A detailed experimental description of molecular dynamics requires an accurate description of the global correlation time. For the gramicidin cation-selective channel the local dynamics of the polypeptide backbone are thought to play a very significant role in the functional process of this channel which occurs on the 10-100-ns time scale. By solid-state NMR spectroscopy an experimental description of the local dynamics including a description of the axis about which the motions occur, the amplitude of the motions, whether they are diffusional or discontinuous and the frequency of the motions is possible.

High-resolution structure and dynamic implications for a double-helical gramicidin A conformer.

The high-resolution structure of a dimeric conformer of gramicidin A, a 15-residue polypeptide, has been determined in the mixed-solvent system of benzene and ethanol by 2D NMR techniques. NOEs, coupling constants and hydrogen-bond information were used to generate 744 experimental constraints for the dimer. Stereoassignment of most beta-methylene groups was achieved by analysis of 3J alpha beta, d alpha beta(i,i), dN beta(i,i) and dN beta(i + 1,i) distances, and consideration of the initial backbone structure determinations.

Tryptophans in membrane proteins: indole ring orientations and functional implications in the gramicidin channel.

Orientational constraints generated from solid-state NMR of uniformly aligned gramicidin A in hydrated lipid bilayers have been used to determine the indole ring orientations for the four tryptophans of the gramicidin A monomer with respect to the bilayer normal and the channel axis. 15N epsilon 1 labeled tryptophan has been incorporated into gramicidin at positions 9, 11, 13, and 15. The chemical shift tensor orientation has been oriented with respect to the N-H bond via doubly labeled sample in which the 15N epsilon 1-1H has been exchanged for 2H.

A method for studying the structure of uniaxially aligned biopolymers using solid state 15N-nmr: application to Bombyx mori silk fibroin fibers.

Recent advances in the application of solid state nmr spectroscopy to uniformly aligned biopolymers have opened a window through which to view the detailed structure of biological macromolecules that are unable to be seen with standard techniques for structure determination such as x-ray diffraction. Atomic resolution structural details are obtained from solid state nmr data in the form of bond orientations, which yield the relative positions of specific atoms within the molecule. For static aligned systems such as fibers, in which rapid reorientation about the axis of alignment does not occur, it has generally been necessary to perform trial and error line-shape simulations to extract structural details from nmr spectra arising from a single type of nuclear spin interaction.

Orientational constraints as three-dimensional structural constraints from chemical shift anisotropy: the polypeptide backbone of gramicidin A in a lipid bilayer.

Chemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high-resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization.

A conformational rearrangement in gramicidin A: from a double-stranded left-handed to a single-stranded right-handed helix.

A conformational transition is described for the polypeptide, gramicidin A, in which a dimer that forms a left-handed intertwined antiparallel helix is converted to a single-stranded amino terminus to amino terminus right-handed helix. The starting structure is determined here by solution NMR methods while reference is made to the well-established folding motif of gramicidin in a lipid bilayer for the ultimate conformation of this transition. Furthermore, an organic solvent system of benzene and ethanol in which gramicidin has a unique conformation is identified.

Structure of an isolated gramicidin A double helical species by high-resolution nuclear magnetic resonance.

A conformational species of gramicidin A has been isolated in dioxane by high pressure liquid chromatography and characterized by circular dichroism and two-dimensional proton nuclear magnetic resonance. Double-quantum filtered two-dimensional correlation spectroscopy, two-dimensional homonuclear Hartman Hahn spectroscopy and two-dimensional nuclear Overhauser effect spectra at 500 MHz were used to obtain virtually complete proton assignments and produce 192 distance constraints. Protocols to determine the state of aggregation, monomer-specific assignment of nuclear Overhauser enhancement values, hydrogen bonding pattern and helix handedness are described.

Solid-state C NMR spectroscopy of a C carbonyl-labeled polypeptide.

High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For (13)C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled (14)N from the peptide bond.

Molecular dynamics computations and solid state nuclear magnetic resonance of the gramicidin cation channel.

This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other.

Solid-state nuclear magnetic resonance derived model for dynamics in the polypeptide backbone of the gramicidin A channel.

The dynamics of the backbone of the gramicidin A transmembrane cation channel in dimyristoylphosphatidylcholine bilayers have been investigated using solid state 15N nuclear magnetic resonance (n.m.r.

Experimental determination of torsion angles in the polypeptide backbone of the gramicidin A channel by solid state nuclear magnetic resonance.

An analytical method for the determination of torsion angles from solid state 15N nuclear magnetic resonance (n.m.r.

Toxicity of N-(3,5-dichlorophenyl)succinimide and metabolites to rat renal proximal tubules and mitochondria.

The acute nephrotoxicity caused by N-(3,5-dichlorophenyl) succinimide (NDPS) has been shown to be due to a metabolite(s) of the parent compound. This study examined the toxicity of NDPS, its known metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (NDHSA), N-(3,5-dichlorophenyl)malonamic acid (DMA), N-(3,5-dichlorophenyl)succinamic acid (NDPSA), and two postulated metabolites N-(3,5-dichlorophenyl)maleamic acid (NDPMA) and N-(3,5-dichlorophenyl)maleimide (NDPM) to suspensions of renal proximal tubules (RPT) prepared from male Fischer 344 rats. Tubule viability and mitochondrial function were not adversely affected by exposure of RPT to either 1 mM NDPS, NDHS, NDHSA, DMA, NDPSA, or NDPMA for 4 h.