Improved rig for dynamically calibrating skinfold calipers: comparison between Harpenden and Slim Guide instruments.

This article describes an improved rig for the dynamic calibration of skinfold calipers. The new unit is 5% lighter and almost 60% smaller than its predecessor (Carlyon et al., 1996, 1998) with a 9.5 mm solid aluminium base and a quick release caliper mount providing stability to both the rig and caliper. Automation of the gap controller with an electric motor standardizes the jaw opening and closing velocity, thereby enabling hands-free operation. Frictional losses in the moving components of the rig have been reduced by replacing the main bush of the swing arm with a bearing, reducing the mass of the swing arm, adding a support wheel to the end of the swing arm, and replacing fishing swivels with a universal joint to allow for changes in the opening screw angle as the caliper's arm moves through its arc. This rig can also be adapted to different types of calipers by changing the position of the load cell, microswitches, and the caliper mount. A universal mounting bracket that can be secured to almost any table supports the rig in a vertical plane when calibrating the load cell. To demonstrate the versatility of the calibration rig, preliminary data are presented for the upscale and downscale jaw pressures of seven Harpenden and seven Slim Guide calipers.

Raman optical activity of filamentous bacteriophages: hydration of alpha-helices.

We report the first observations of vibrational Raman optical activity (ROA) on intact viruses. Specifically, ROA spectra of the filamentous bacteriophages Pf1, M13 and IKe in aqueous solution were measured in the range approximately 600-1800 cm-1. On account of its ability to probe directly the chiral elements of biomolecular structure, ROA has provided a new perspective on the solution structures of these well-studied systems. The ROA spectra of all three are dominated by signatures of helical elements in the major coat proteins, as expected from pre-existing data. The helical elements generate strong sharp positive ROA bands at approximately 1300 and 1342 cm-1in H2O solution, but in2H2O solution the approximately 1342 cm-1bands disappear completely. The spectra are similar to those of polypeptides under conditions that produce alpha-helical conformations. Our present results, together with results from other studies, suggest that the positive approximately 1342 cm-1ROA bands are generated by a highly hydrated form of alpha-helix, and that the positive approximately 1300 cm-1bands originate in alpha-helix in a more hydrophobic environment. The presence of significant amounts of highly hydrated helical sequences accords with the known flexibility of these viruses. Differences of spectral detail for Pf1, M13 and IKe demonstrate that ROA is sensitive to subtle variations of conformation and hydration within the major coat proteins, with M13 and IKe possibly containing more non-helical structure than Pf1. The ROA spectra of Pf1 at temperatures above and below that at which a structural transition is known to occur (approximately 10 degrees C) reveal little difference in the protein conformation between the two forms, but there are indications of changes in DNA structure.

Separation and size determination of circular and linear single-stranded DNAs by alkaline agarose gel electrophoresis.

The electrophoretic mobility of single-stranded DNA (ssDNA) through agarose gels depends on size (number of bases; molecular weight), topology (linear or circular), and conformation (secondary and tertiary folding through base pairing and stacking), as well as on gel concentration and field strength. Under alkaline conditions that disrupt secondary and tertiary structures, the linear and circular forms of a given single-stranded DNA can be easily resolved. The mobility ratios, mulinear/mucircular, depend strongly on gel concentration and less strongly on DNA size. Protocols have been developed to determine conveniently the size within 2% and the fractions of circular and linear forms for ssDNA sizes on the order of 10 kb.

C2, and unusual filamentous bacterial virus: protein sequence and conformation, DNA size and conformation, and nucleotide/subunit ratio.

Inovirus C2 is 1295 nm long and 6.8 nm in diameter, and its mass is 24 million Da. Its genome is a topologically circular, single-stranded DNA molecule of 8100 nucleotides. The DNA is packed in the virion as two antiparallel strands, with a rise per nucleotide in each strand of 3.2 A; it can be assigned spectroscopic properties like those of base-stacked, right-handed, double-stranded DNA. The stoichiometric ratio (n/s) of nucleotides to subunits of the major coat protein is close to 2. The protein subunit contains 52 amino acids, and the DNA sequence of its gene does not encode a signal peptide. The protein conformation in the virion is helical, mostly alpha-helix with perhaps some 3(10)-helix. The amino acid sequence of the DNA interaction domain of the subunit is unique among Inovirus species. On the basis of its coat protein sequence and available theories of helical symmetry in such structures, C2 appears to be either an unusual member of filamentous virus symmetry class II or the defining member of a new symmetry class.

An electrostatic spatial resonance model for coaxial helical structures with applications to the filamentous bacteriophages.

A model is presented that treats the symmetry matching problem in structures made of two interacting coaxial helices of point charges. The charges are sources of a potential field that mediates a non-specific attractive interaction between the helices. The problem is represented in Fourier space, which affords the most generality. It is found that coaxial helices with optimally mated symmetries can lock into spatial resonance configurations that maximize their interaction. The resonances are represented as vectors in a discrete three-dimensional space. Two algebraic relations are given for the four symmetry parameters of two helices in resonance. One-start inner helices interacting with coaxial one-start or NR-start outer helices are considered. Applications are made to the filamentous bacteriophages Ff, Pf1, Xf, and Pf3. The interaction given by the linearized Poisson-Boltzmann equation is calculated in this formalism to allow comparison of the electrostatic free energy of interaction of different resonance structures. Experimental nucleotide/subunit ratios are accounted for, and models for the DNA-protein interfaces are presented, with particular emphasis on Pf1.

Pf1 virus structure: helical coat protein and DNA with paraxial phosphates.

The helical path of the DNA in filamentous bacteriophage Pf1 was deduced from different kinds of existing structural information, including results from x-ray fiber diffraction. The DNA has the same pitch, 16 angstroms, as the surrounding helix of protein subunits; the rise and rotation per nucleotides are 6.1 angstroms and 132 degrees, respectively; and the phosphates are 2.5 angstroms from the axis. The DNA in Pf1 is, therefore, the most extended and twisted DNA structure known. On the basis of the DNA structure and extensive additional information about the protein, a model of the virion is proposed. In the model, the DNA bases reach out, into the protein, and the lysine and arginine side chains reach in, between the DNA bases, to stabilize the paraxial phosphate charges; the conformation of the protein subunit is a combination of alpha and 3(10) helices.

Ultraviolet absorbance and circular dichroism of Pf1 virus: nucleotide/subunit ratio of unity, hyperchromic tyrosines and DNA bases, and high helicity in the subunits.

Data have been obtained for the Pf1 virion that establish its stoichiometry and conformational features of its DNA and its protein. The absorbance spectrum of the dissociated virus under alkaline denaturing conditions is fit exactly by spectra for DNA and protein at a mole ratio of one nucleotide per protein subunit. This result, together with three previous values by independent methods, establishes that the nucleotide/subunit ratio (n/s) of Pf1 is unity. The absorbance spectrum of DNA in the intact native virus is assigned as the spectrum for heat denatured Pf1 DNA, with epsilon (P) = 8400 M-1 cm-1 at 259 nm. The absorbance spectrum assigned to protein (two tyrosines) in the intact virus has = 2500 M-1 cm-1 per tyrosine at lambda max of 281.5 nm; this is the most red-shifted and hyperchromic tyrosine spectrum known. The CD spectrum of the intact virus from 250 to 320 nm has no apparent DNA contribution, but has a strong contribution from the red-shifted tyrosine(s). The CD spectrum from 185 to 250 nm has the shape of alpha-helical CD reference spectra, but is perceptibly blue-shifted, with a crossover from negative to positive ellipticity at 199.7 nm, and it has very high amplitudes (e.g. [theta 207.5nm] = -44,000 deg cm2 dmol-1). This spectrum indicates completely helical protein in the virus, with a predominance of alpha-helix and perhaps some 3(10)-helix. The unit n/s ratio, the high absorbance and negligible near-UV CD for the DNA bases, and the high amplitudes for the helical protein are critical input data for the determination of Pf1 virus structure.

Pattern formation in icosahedral virus capsids: the papova viruses and Nudaurelia capensis beta virus.

The capsids of the spherical viruses all show underlying icosahedral symmetry, yet they differ markedly in capsomere shape and in capsomere position and orientation. The capsid patterns presented by the capsomere shapes, positions, and orientations of three viruses (papilloma, SV40, and N beta V) have been generated dynamically through a bottom-up procedure which provides a basis for understanding the patterns. A capsomere shape is represented in two-dimensional cross-section by a mass or charge density on the surface of a sphere, given by an expansion in spherical harmonics, and referred to herein as a morphological unit (MU). A capsid pattern is represented by an icosahedrally symmetrical superposition of such densities, determined by the positions and orientations of its MUs on the spherical surface. The fitness of an arrangement of MUs is measured by an interaction integral through which all capsid elements interact with each other via an arbitrary function of distance. A capsid pattern is generated by allowing the correct number of approximately shaped MUs to move dynamically on the sphere, positioning themselves until an extremum of the fitness function is attained. The resulting patterns are largely independent of the details of both the capsomere representation and the interaction function; thus the patterns produced are generic. The simplest useful fitness function is sigma 2, the average square of the mass (or charge) density, a minimum of which corresponds to a "uniformly spaced" MU distribution; to good approximation, the electrostatic free energy of charged capsomeres, calculated from the linearized Poisson-Boltzmann equation, is proportional to sigma 2. With disks as MUs, the model generates the coordinated lattices familiar from the quasi-equivalence theory, indexed by triangulation numbers. Using fivefold MUs, the model generates the patterns observed at different radii within the T = 7 capsid of papilloma and at the surface of SV40; threefold MUs give the T = 4 pattern of Nudaurelia capensis beta virus. In all cases examined so far, the MU orientations are correctly found.

An exact description of five-membered ring configurations. II. Applications to furanose rings in DNA and RNA, analysis of errors, and bond angle bending energy.

The method developed in Paper I (the preceding paper in this issue) for the exact parameterization of the five-atom furanose ring has been applied to rings in macromolecular structures. The data include coordinates for 322 rings in A- and B-form DNA; 60 rings in Z-form DNA; 29 rings in RNA molecules; and 53 rings in two specific DNA-protein complexes. Characteristic distributions of internal coordinates [q, P, S, gamma; (bj)] for the macromolecular data sets are found to be closely similar to the corresponding distributions for the small molecule data sets, even though the uncertainties in the atomic coordinates are larger for the larger molecules. To aid in the interpretation of such data rules of thumb are given. It is shown, analytically, that the distribution of the direction angle gamma is largely determined by the bond angle bending potential. This analytical result is fully confirmed by the data, which show that the average gamma value is roughly near 90 degrees. The experimentally observed gamma for each ring tends to fall near the theoretical gamma min calculated for it by minimizing the bond angle bending potential while holding the other internal parameters constant. Such unmistakable trends are discernible by considering large data sets using the full parameterization. The effects of uncertainties in the input Cartesian coordinates are treated, and it is shown that the new parameters S and gamma are more sensitive to error than are q and P, but that the uncertainties in S and gamma decrease when S is large. The Jacobian J of the transformation from Cartesian coordinates to the coordinates [q, P, S, gamma; (bj)] is calculated. The Jacobian, which can be used in statistical studies, aids in understanding the sampling of configuration space, the distribution of S values, and the propagation of error.

An exact description of five-membered ring configurations. I. Parameterization via an amplitude S, an angle gamma, the pseudorotation amplitude q and phase angle P, and the bond lengths.

A method is developed which exactly parameterizes the configurations of five membered rings. In addition to the five bond lengths, (bj), four other parameters are needed to describe completely all configurations of a three-dimensional figure with five sides. Two of these parameters are taken as the phase angle, P, and amplitude, q (A), introduced by Cremer and Pople (J. Am. Chem. Soc. 97, 1354 (1975)) which give exactly the displacements of the atoms in any five-membered ring from a special plane (the CP plane). For the two other parameters, a second amplitude, S (A), and an orientation angle, gamma (the upper case Greek letter "gamma"), are introduced. These two new parameters describe the "distortion" of the projection of the ring in the CP plane. gamma is the angle in the CP plane at which the projected ring is maximally "spread" or "stretched," where the stretch is a least squares measure of position along a line through the ring center. S measures the difference between maximum stretch (in direction gamma) and minimum stretch (in direction gamma +/- 90 degrees) such that S = 0 denotes maximum symmetry. Transformations from Cartesian coordinates to the new internal coordinates, and from the new internal coordinates to the Cartesian set, are exact inverse transformations. Fortran source codes MAKERING and BREAKRING for these transformations are available. The set of four parameters aids in direct comparisons of ring structures and in detailed numerical analyses of data. They also aid the systematic generation of all possible rings, useful for theoretical studies of ring conformation. To demonstrate the descriptive method, we have generated distributions of q, P, S, and gamma for furanose rings in the structures of 665 nucleosides determined by X-ray crystallography.

Tryptophan contributions to the unusual circular dichroism of fd bacteriophage.

The circular dichroism (CD) spectrum of fd bacteriophage has a deep minimum at 222 nm characteristic of highly alpha-helical protein, but there is a shoulder at 208 nm rather than a minimum, with a 222/208-nm amplitude ratio near 1.5 rather than near 1. Oxidation of fd phage with the tryptophan reagent N-bromosuccinimide (NBS) changes the ratio. In this report, the NBS titration of fd is followed by CD and three other spectroscopies, the results of which yield an explanation of the unusual CD spectrum. Absorbance, fluorescence, and Raman data show the oxidation to have two phases, the first of which involves the destruction of tryptophan and the second, tryptophan and tyrosine. Raman spectra reveal the invariance of an environmentally-sensitive tyrosine Fermi resonance doublet during the first oxidative phase. Raman spectra also show that little or no change of alpha-helicity occurs in the first or second oxidation phase, although very slight changes in the helix parameters might be occurring. Concurrent with the destruction of tryptophan during the first phase is the appearance in CD difference spectra ([theta]NBS-treated fd - [theta]native fd) of positive maxima at 208-210 nm and negative maxima at 224 nm, with crossovers at 217 nm. Enormous difference ellipticities, per oxidized subunit of 50 amino acids, of +490,000 +/- 80,000 deg cm2 dmol-1 at 208 nm and -520,000 +/- 110,000 deg cm2 dmol-1 at 224 nm have been derived from the data.(ABSTRACT TRUNCATED AT 250 WORDS)

Export of infectious particles by Escherichia coli transfected with the RF DNA of Pf1, a virus of Pseudomonas aeruginosa strain K.

Pf1 is a filamentous, single-stranded DNA virus that has Pseudomonas aeruginosa (strain K) as host. It is the longest of the filamentous bacterial viruses, and the DNA within it has the most extended conformation known. Pf1 virus cannot infect Escherichia coli (strain MM294) cells, but when these cells are transfected with the double-stranded replicative form of Pf1 DNA (RF DNA, 7.35 kb), they export low levels of infectious particles that create plaques on lawns of P. aeruginosa. Several different structural species, at least two of which are infectious, are exported. One of them, called Epf1, has virtually the same structure as Pf1, but the amount of Epf1 exported by E. coli is 10(4) lower than the amount of Pf1 exported by P. aeruginosa. The results imply that host factors affect not only the efficiency of virus assembly and export, but also the actual structures of the species exported.

Development and testing of the Stressor Scale for Pediatric Oncology Nurses.

Pediatric oncology nursing has been identified as a stressful specialty, but the exact sources of the stress have not been comprehensively specified nor put into measurable form. Effective interventions can best be developed when the stressors are known and measured. The purpose of this study was to develop and test an instrument that could accurately and sensitively measure the job-related stressors for pediatric oncology nurses. A 61-item, visual analogue instrument, the Stressor Scale for Pediatric Oncology Nurses (SSPON), was inductively developed from interviews with 30 pediatric oncology nurses. Subsequently, the SSPON was tested in three geographically separated samples of pediatric oncology nurses (n = 78). After an item analysis, 11 items were deleted from the scale. Reliability of the SSPON was estimated using both stability and internal consistency methods. The test-retest correlation coefficient was 0.88, and the total scale alpha coefficient was 0.94. Content validity was estimated using a two-stage process of developmental and judgment quantification with a panel of three specialty nurses. Construct validity was estimated through testing of theoretically derived hypotheses and a cluster analysis. Findings indicated the SSPON is content valid. Six meaningful clusters that represented sources of job-related stress were identified and defined. The revised SSPON appears to have adequate psychometric properties for a new instrument.

Sugar pucker and phosphodiester conformations in viral genomes of filamentous bacteriophages: fd, If1, IKe, Pf1, Xf, and Pf3.

The laser Raman spectra of filamentous viruses contain discrete bands which are assignable to molecular vibrations of the encapsidated, single-stranded DNA genomes and which are informative of their molecular conformations. Discrimination between Raman bands of the DNA and those of the coat proteins is facilitated by analysis of viruses containing deuterium-labeled amino acids. Specific DNA vibrational assignments are based upon previous studies of A-, B-, and Z-DNA oligonucleotide crystals of known structure [Thomas, G.J., Jr., & Wang, A.H.-J. (1988) in Nucleic Acids and Molecular Biology (Eckstein, F., & Lilley, D.M.J., Eds.) Vol. 2, Springer-Verlag, Berlin]. The present results show that canonical DNA structures are absent from six filamentous viruses: fd, If1, IKe, Pfl, Xf, and Pf3. The DNAs in three viruses of symmetry class I (fd, If1, IKe) contain very similar nucleoside sugar puckers and glycosyl torsions, deduced to be C3'-endo/anti. However, nucleoside conformations are not the same among the three class II viruses examined: Pf1 and Xf DNAs contain similar conformers, deduced to be C2'-endo/anti, whereas Pf3 DNA exhibits bands usually associated with C3'-endo/anti conformers. Conformation-sensitive Raman bands of the DNA 3'-C-O-P-O-C-5' groups show that in all class I viruses and in Pf1 the ssDNA backbones do not contain regularly ordered phosphodiester group geometries, like those found in ordered single- and double-stranded nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermal difference circular dichroism of Pf1 filamentous virus and effects of mercury(II), silver(I), and copper(II).

The circular dichroism (CD) of Pfl filamentous virus has been examined over the temperature range 0-40 degrees C, in the absence and presence of Hg(II), Ag(I), and Cu(II). Thermal difference CD spectra were obtained by subtraction of spectra recorded above and below a thermally induced structure transition near 12 degrees C. The thermal difference spectra look like they arise from shifts in two exciton bands, one centered at 230 nm and the other at 290 nm. The amplitudes on either side of a crossover at 230 nm are 10 times those of a crossover at 290 nm. It is proposed that the difference spectra result from thermally induced shifts in coupled oscillator interactions between Tyr40 residues of the coat protein and the guanine and cytosine bases of the DNA. Metal ions can reduce or block these shifts. The changes in ellipticities at 220, 237, and 270 nm induced by changing the temperature have inflections near 12 degrees C. Ag(I) and Hg(II), which are known to bind to the DNA bases in Pfl, reduce or eliminate the inflections in the thermal profiles, depending on the metal ion type and concentration. Cu(II) ions do not affect the profiles. The spectral changes and the effects of the metal ions indicate intimate contact between the DNA bases and the protein subunits in the virion.

A theory of the symmetries of filamentous bacteriophages.

A mathematical model is presented which explains the symmetries observed for the protein coats of filamentous bacterial viruses. Three viruses (Ff, IKe, and If1) all have five-start helices with rotation angles of 36 degrees and axial translations of 16 A (Type I symmetry), and three other viruses (Pf1, Xf, and Pf3) all have one-start helices with rotation angles of approximately equal to 67 degrees and translations of approximately 3 A (Type II symmetry). The coat protein subunits in each group diverge from each other in amino acid sequence, and Type II viruses differ dramatically in DNA structure. Regardless of the differences, both Type I and Type II symmetry can be understood as direct, natural consequences of the close-packing of alpha-helical protein subunits. In our treatment, an alpha-helical subunit is modeled as consisting of two interconnected, flexible tubular segments that follow helical paths around the DNA, one in an inner layer and the other in an outer layer. The mathematical model is a set of algebraic equations describing the disposition of the flexible segments. Solutions are described by newly introduced symmetry indices and other parameters. An exhaustive survey over the range of indices has produced a library of all structures that are geometrically feasible within our modeling scheme. Solutions which correspond in their rotation angles to Type I and Type II viruses occur over large ranges of the parameter space. A few solutions with other symmetries are also allowed, and viruses with these symmetries may exist in nature. One solution to the set of equations, obtained without any recourse to the x-ray data, yields a calculated x-ray diffraction pattern for Pf1 which compares reasonably with experimental patterns. The close-packing geometry we have used helps explain the near constant linear mass density of known filamentous phages. Helicoid, rigid cylinder, and maximum entropy structure models proposed by others for Pf1 are reconciled with the flexible tube models and with one another.

Raman spectroscopy of mercury (II) binding to two filamentous viruses: Ff (fd, M13, f1) and Pf1.

Ff and Pf1 are filamentous bacteriophages. Each contains, in a central core region surrounded by protein, a circular single-stranded DNA molecule, and it is known that the DNA bases are sites of Hg(II) binding. In the present study, Raman spectra were obtained for the two viruses in the presence of increasing amounts of Hg(II), with ratios (m) of Hg(II) added per nucleotide residue in the range 0 less than m less than 2.0. Hg(II) binding to the viruses induces Raman intensity changes in previously assigned Raman lines of viral DNA, demonstrating metal binding to the DNA bases, but also in many lines assigned to protein. The overall structures of the viruses do not change with Hg(II) binding, and the Raman spectra indicate little, if any, change in protein secondary structure. Changes in certain protein Raman lines induced by Hg(II) binding to the DNA for low values of m are attributed to altered interactions between solvent and protein side chains, aliphatic groups being the most affected. The nature of such changes for both viruses suggests DNA-protein linkage. In Pf1, lines assigned to ring vibrations of all four bases are perturbed upon initial addition of Hg(II) to m = 0.25. In Ff, however, lines assigned to base ring vibrations are not perturbed until m greater than or equal to 0.5. The results provide additional evidence for fundamentally different DNA structures in Ff and Pf1.

Nucleotide sequence of the genome of Pf3, an IncP-1 plasmid-specific filamentous bacteriophage of Pseudomonas aeruginosa.

The circular, single-stranded DNA genome of the Pf3 bacteriophage was sequenced in its entirety by each of two methods, the M13-dideoxy chain termination method and the chemical degradation method. It consists of 5,833 nucleotides. With respect to both the DNA and the protein sequences, there is virtually no homology between Pf3 and the phages Ff (M13, f1, and fd) and IKe. However, similarities between these phages were noted with respect to their overall genome organizations. The gene for the single-stranded DNA-binding protein is followed by the gene for the major coat protein and then by a transcription termination signal. Open reading frames for seven other proteins were predicted, and their sizes and order show a fair correspondence to the sizes and order of the genes of the Ff phages and IKe. In addition, several regions have the potential to form stem and loop structures similar to those in the intergenic region of the Ff phage genome, but in Pf3 some are within open reading frames. Evolutionary relationships between Pf3 and the Ff phages and IKe are thus apparent through the correspondence of overall gene order rather than through primary sequence homologies.