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PubMed1985

Sequence-dependent energetics of the B-Z transition in supercoiled DNA containing nonalternating purine-pyrimidine sequences.

The likelihood that a given DNA sequence will adopt the Z conformation in negatively supercoiled DNA depends on the energy difference between the B form and the Z form for that sequence relative to other sequences in the same molecule. This energy can be viewed simply as a sum of energies for the nearest-neighbor interactions within the sequence plus the energy required to stabilize the B-Z boundaries. Knowledge of these energetic terms would be of value in predicting when sequences become left-handed in response to negative superhelicity. Here we present an approach that can be used to determine the free-energy changes associated with all the nearest-neighbor interactions that can occur in Z-DNA. Synthetic stretches of d(C-G)n containing one or two transversions were cloned into plasmids, and the extent of the B-Z transition as a function of negative superhelicity was determined for each insert by two-dimensional agarose gel electrophoresis. By subjecting the data to statistical mechanical analysis, it was possible to evaluate the energetic penalty resulting from each base-pair (bp) substitution. Guanine to cytosine transversions cost 2.4 kcal (1 cal = 4.18 J)/(mol X bp), whereas guanine to thymine transversions cost 3.4 kcal/(mol X bp), to stabilize in the Z conformation. We have used these numbers, along with energetic values determined by others for the B-Z transition, to predict that certain strictly nonalternating purine and pyrimidine sequences may adopt the Z form readily.

Base Sequence↗
PubMed2001

Genomic stability of murine leukemia viruses containing insertions at the Env-3' untranslated region boundary.

Retroviruses containing inserts of exogenous sequences frequently eliminate the inserted sequences upon spread in susceptible cells. We have constructed replication-competent murine leukemia virus (MLV) vectors containing internal ribosome entry site (IRES)-transgene cassettes at the env-3' untranslated region boundary in order to examine the effects of insert sequence and size on the loss of inserts during viral replication. A virus containing an insertion of 1.6 kb replicated with greatly attenuated kinetics relative to wild-type virus and lost the inserted sequences in a single infection cycle. In contrast, MLVs containing inserts of 1.15 to 1.30 kb replicated with kinetics only slightly attenuated compared to wild-type MLV and exhibited much greater stability, maintaining their genomic integrity over multiple serial infection cycles. Eventually, multiple species of deletion mutants were detected simultaneously in later infection cycles; once detected, these variants rapidly dominated the population and thereafter appeared to be maintained at a relative equilibrium. Sequence analysis of these variants identified preferred sites of recombination in the parental viruses, including both short direct repeats and inverted repeats. One instance of insert deletion through recombination with an endogenous retrovirus was also observed. When specific sequences involved in these recombination events were eliminated, deletion variants still arose with the same kinetics upon virus passage and by apparently similar mechanisms, although at different locations in the vectors. Our results suggest that while lengthened, insert-containing genomes can be maintained over multiple replication cycles, preferential deletions resulting in loss of the inserted sequences confer a strong selective advantage.

3' Untranslated Regions↗
PubMed1991

Reduced Polyomavirus DNA replication as a consequence of a late-region deletion that results in early mRNA instability.

A Polyomavirus mutant with a 504 base pair deletion downstream of the polyadenylation signal for the early genes was constructed in vitro. This mutant showed a reduced synthesis of viral DNA. In cotransfection experiments, this defect was complemented by the presence of a wild-type genome. To define the sequences involved in the determination of this phenotype, a set of viral mutants was constructed. The properties of these mutants suggested that the deletion of a short DNA segment located 35 base pairs downstream of the early polyadenylation site affected the stability of early mRNA. The boundaries of the deletion were within the late coding sequences. However, the truncated form of the major capsid protein VP1 expressed by the mutant, did not influence the formation of early mRNA and the synthesis of viral DNA.

Base Sequence↗
PubMed2005

Lysolipid incorporation in dipalmitoylphosphatidylcholine bilayer membranes enhances the ion permeability and drug release rates at the membrane phase transition.

The enhanced permeability of lipid bilayer membranes at their gel-to-liquid phase transition has been explained using a "bilayer lipid heterogeneity" model, postulating leaky interfacial regions between still solid and melting liquid phases. The addition of lysolipid to dipalmitoylphosphatidylcholine bilayers dramatically enhances the amount of, and speed at which, encapsulated markers or drugs are released at this, already leaky, phase transition through these interfacial regions. To characterize and attempt to determine the mechanism behind lysolipid-generated permeability enhancement, dithionite permeability and doxorubicin release were measured for lysolipid and non-lysolipid, containing membranes. Rapid release of contents from lysolipid-containing membranes appears to occur through lysolipid-stabilized pores rather than a simple enhancement due to increased drug solubility in the bilayer. A dramatic enhancement in the permeability rate constant begins about two degrees below the calorimetric peak of the thermal transition, and extends several degrees past it. The maximum permeability rate constant coincides exactly with this calorimetric peak. Although some lysolipid desorption from liquid state membranes cannot be dismissed, dialyzation above T(m) and mass spectrometry analysis indicate lysolipid must, and can, remain in the membrane for the permeability enhancement, presumably as lysolipid stabilized pores in the grain boundary regions of the partially melted solid phase.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2026

Neocentromeres fail to maintain DNA methylation boundaries, driving CENP-A drift, instability, and chromosome missegregation.

Centromere identity is specified by CENP-A, a histone H3 variant that epigenetically defines centromere position. How CENP-A is maintained at one location in rapidly evolving centromeric DNA is unknown. Using single-cell-derived clones of human cell lines, we demonstrate heterogeneity in CENP-A position within cell populations at neocentromeres and a native centromere. CENP-A heterogeneity is accompanied by heterogeneous DNA methylation patterns, with DNA methylation shifting according to CENP-A position. We demonstrate centromere epigenetic plasticity over extended proliferation, with native centromeres maintaining stable DNA methylation boundaries, but neocentromeres exhibiting DNA methylation instability, boundary loss, and increased missegregation. Finally, we show that neocentromeres are more sensitive to DNA methylation inhibition than native centromeres, and that this inhibition is accompanied by expanded CENP-A-enriched domains and increased missegregation. This study supports a role for DNA methylation boundaries in maintaining centromere position, stability, and function and highlights the intrinsic instability of DNA methylation at neocentromeres.

CENP-A↗
PubMed1988

Characteristic features of thermal stability map of DNA in Escherichia coli and eukaryotic genes.

Distribution of double-helix thermal stability of Escherichia coli and eukaryotic DNAs was analyzed. The results confirmed the previous propositions based on the study of the stability distribution in phage DNAs: (1) stability fluctuation appears near the boundaries of protein coding regions (PCRs) and non protein coding regions (NPCRs); (2) PCRs have less fluctuation than NPCRs. The present analysis also revealed that the local G + C content is lower in the beginning of PCRs of E. coli than the average G + C content of PCR and that deviations in the amino acid composition and the third letter usage PCRs are involved in the low G + C content; the biological meaning of this is discussed in relation to mRNA structure.

Base Composition↗
PubMed2003

Multireflection boundary conditions for lattice Boltzmann models.

We present a general framework for several previously introduced boundary conditions for lattice Boltzmann models, such as the bounce-back rule and the linear and quadratic interpolations. The objectives are twofold: first to give theoretical tools to study the existing link-type boundary conditions and their corresponding accuracy; second to design boundary conditions for general flows which are third-order kinetic accurate. Using these new boundary conditions, Couette and Poiseuille flows are exact solutions of the lattice Boltzmann models for a Reynolds number Re=0 (Stokes limit) for arbitrary inclination with the lattice directions. Numerical comparisons are given for Stokes flows in periodic arrays of spheres and cylinders, linear periodic array of cylinders between moving plates, and for Navier-Stokes flows in periodic arrays of cylinders for Re<200. These results show a significant improvement of the overall accuracy when using the linear interpolations instead of the bounce-back reflection (up to an order of magnitude on the hydrodynamics fields). Further improvement is achieved with the new multireflection boundary conditions, reaching a level of accuracy close to the quasianalytical reference solutions, even for rather modest grid resolutions and few points in the narrowest channels. More important, the pressure and velocity fields in the vicinity of the obstacles are much smoother with multireflection than with the other boundary conditions. Finally the good stability of these schemes is highlighted by some simulations of moving obstacles: a cylinder between flat walls and a sphere in a cylinder.

Journal Article↗
PubMed2005

Nematic-nematic phase separation in binary mixtures of thick and thin hard rods: results from Onsager-like theories.

The fundamental nature of the nematic-nematic phase separation in binary mixtures of rigid hard rods is analyzed within the Onsager second-virial theory and the extension of Parsons and Lee which includes a treatment of the higher-body contributions. The particles of each component are modeled as hard spherocylinders of different diameter , but equal length . In the case of a system which is restricted to be fully aligned (parallel rods), we provide an analytical solution for the spinodal boundary for the limit of stability of demixing; only a single region of coexistence bounded at lower pressures (densities) by a critical point is possible for such a system. The full numerical solution with the Parsons-Lee extension also indicates that, depending on the length of the particles, there is a range of values of the diameter ratio where the phase coexistence is closed off by a critical point at lower pressure. A second region of coexistence can be found at even lower pressures for certain values of the parameters; this region is bounded by an "upper" critical point. The two coexistence regions can also merge to give a single region of coexistence extending to very high pressure without a critical point. By including the higher-order contributions to the excluded volume (end effects) in the Onsager theory, we prove analytically that the existence of the lower critical point is a direct consequence of the finite size of the particles. A new analytical equation of state is derived for the nematic phase using the Gaussian approximation. In the case of Onsager limit (infinite aspect ratio), we show that the phase behavior obtained using the Parsons-Lee approach substantially deviates from that with the Onsager theory for the transition due to the nonvanishing third and higher order virial coefficients. We also provide a detailed discussion of the phase behavior of recent experimental results for mixtures of thin and thick rods of the same length, for which the Onsager and Parsons-Lee theories can provide a qualitative description.

Journal Article↗
PubMed2000

Surface geometric analysis of anatomic structures using biquintic finite element interpolation.

The surface geometry of anatomic structures can have a direct impact upon their mechanical behavior in health and disease. Thus, mechanical analysis requires the accurate quantification of three-dimensional in vivo surface geometry. We present a fully generalized surface fitting method for surface geometric analysis that uses finite element based hermite biquintic polynomial interpolation functions. The method generates a contiguous surface of C2 continuity, allowing computation of the finite strain and curvature tensors over the entire surface with respect to a single in-surface coordinate system. The Sobolev norm, which restricts element length and curvature, was utilized to stabilize the interpolating polynomial at boundaries and in regions of sparse data. A major advantage of the current method is its ability to fully quantify surface deformation from an unstructured grid of data points using a single interpolation scheme. The method was validated by computing both the principal curvature distributions for phantoms of known curvatures and the principal stretch and principal change of curvature distributions for a synthetic spherical patch warping into an ellipsoidal shape. To demonstrate the applicability to biomedical problems, the method was applied to quantify surface curvatures of an abdominal aortic aneurysm and the principal strains and change of curvatures of a deforming bioprosthetic heart valve leaflet. The method proved accurate for the computation of surface curvatures, as well as for strains and curvature change for a surface undergoing large deformations.

Animals↗
PubMed2026

Dynamic Centromeres Under Epigenetic Constraint.

Centromeres are essential chromosomal loci specified epigenetically by CENP-A chromatin, yet they undergo rapid sequence turnover, structural remodeling, and occasional repositioning. In this review, we integrate recent advances enabled by long-read genome assemblies and high-resolution chromatin mapping to synthesize current understanding of centromere organization across taxa. We examine how satellite repeats, transposable elements, molecular drive, and meiotic conflict generate extreme centromere diversity. We further explore how DNA methylation and H3K9me3 heterochromatin constrain CENP-A positioning, stabilize centromeric domains, and shape boundary dynamics during centromere drift, duplication, and de novo formation. Together, these perspectives show how centromeres accommodate evolutionary change while preserving the stringent requirements of faithful chromosome segregation.

Journal Article↗
PubMed2003

Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix-helix interactions.

Calmodulin is an EF-hand calcium-binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N- and C-terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N-domain fragment of Paramecium CaM (PCaM1-75), the addition of linker residues 76 to 80 (MKEQD) raised the Tm by 9 degrees C and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., [Biochemistry 2002;41:15-20]), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1-74 to PCaM1-80). For the first 4, PCaM1-74 through PCaM1-77, single amino acid additions at the C-terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5 degrees C in Tm and 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1-77 through PCaM1-80 were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N-domain fragments. Comparisons of NMR (HSQC) spectra of 15N-PCaM1-74 and 15N-PCaM1-80 and analysis of high-resolution structural models suggest these residues are latched to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains.

Amino Acid Sequence↗
PubMed2006

Resonance frequency analysis of implants in the guinea pig model: influence of boundary conditions and orientation of the transducer.

The goal of this study was to identify the parameters that must be controlled during in vivo resonance frequency measurements with a custom Osstell transducer for custom implants in the guinea pig animal model. A numerical study and in vitro measurements were performed to determine the influence of the boundary conditions as well as the transducer orientation on the resonance frequency measured by the custom Osstell transducer. In the reported guinea pig model, the type of boundary condition, the orientation of the transducer (parallel or perpendicular to the long axis of the bone) and the length of the modelled bone have a large influence on the resonance frequency values. This implies that a follow-up in time of the stability of an implant requires the boundary conditions applied to the bone in which the implant is installed as well as the orientation of the transducer to be highly repeatable. Applying controlled boundary conditions during in vivo measurements had a highly positive influence on the repeatability of the Osstell measurements. This improves the possibility of the technique to measure changes in the implant-bone interface during healing of the implant.

Animals↗
PubMed2004

Nonlinear stability of oscillatory wave fronts in chains of coupled oscillators.

We present a stability theory for kink propagation in chains of coupled oscillators and a different algorithm for the numerical study of kink dynamics. The numerical solutions are computed using an equivalent integral equation instead of a system of differential equations. This avoids uncertainty about the impact of artificial boundary conditions and discretization in time. Stability results also follow from the integral version. Stable kinks have a monotone leading edge and move with a velocity larger than a critical value which depends on the damping strength.

Journal Article↗
PubMed2002

Matrix methods for the calculation of stability diagrams in quadrupole mass spectrometry.

The theory of the computer calculation of the stability of ion motion in periodic quadrupole fields is considered. A matrix approach for the numerical solution of the Hill equation and examples of calculations of stability diagrams are described. The advantage of this method is that it can be used for any periodic waveform. The stability diagrams with periodic rectangular waveform voltages are calculated with this approach. Calculations of the conventional stability diagram of the 3-D ion trap and the first six regions of stability of a mass filter with this method are presented. The stability of the ion motion for the case of a trapping voltage with two or more frequencies is also discussed. It is shown that quadrupole excitation with the rational angular frequency omega = Nomega/P (where N, P are integers and omega is the angular frequency of the trapping field) leads to splitting of the stability diagram along iso-beta lines. Each stable region of the unperturbed diagram splits into P stable bands. The widths of the unstable resonance lines depend on the amplitude of the auxiliary voltage and the frequency. With a low auxiliary frequency splitting of the stability diagram is greater near the boundaries of the unperturbed diagram. It is also shown that amplitude modulation of the trapping RF voltage by an auxiliary signal is equivalent to quadrupole excitation with three frequencies. The effect of modulation by a rational frequency is similar to the case of quadrupole excitation, although splitting of the stability diagram differs to some extent. The methods and results of these calculations will be useful for studies of higher stability regions, resonant excitation, and non-sinusoidal trapping voltages.

Journal Article↗
PubMed2005

Absolute instability of the von Kármán, Bödewadt and Ekman flows between a rotating disc and a stationary lid.

The stability of a family of boundary-layer flows, which includes the von Kármán, Bödewadt and Ekman flows for a rotating incompressible fluid between a rotating disc and a stationary lid, is investigated. Numerical computations with the use of a spectral method are carried out to analyse absolute and convective instability. It is shown that the stability of the system is enhanced with a decrease in distance between the disc and the lid.

Journal Article↗
PubMed1997

When a module is also a domain: the rôle of the N terminus in the stability and the dynamics of immunoglobulin domains from titin.

In the course of a structural study of titin, a giant modular protein from muscle, we have reported that N-terminal extension of immunoglobulin-like (Ig-like) domains from titin stabilizes this fold. In order to investigate the structural basis of such an effect, we have solved the structure of NEXTM5, which has six amino acids added to the sequence of M5, a domain for which full structure determination has been previously achieved. In the present work, the structures and the dynamics of M5 and NEXTM5 are compared in the light of data collected for these and other titin domains. In NEXTM5, three out of the six added residues are structured and pack against the nearby BC and FG loops. As a consequence, three new backbone hydrogen bonds are formed with the B strand, extending the A strand by two residues and decreasing the exposed surface area of the loops. Additional contacts which involve the side-chains give rise to a remarkable pH dependence of the stability. Interestingly, no correlation is observed on the NMR time-scale between the overall dynamics of the extended domain and its increased stability. The most noticeable differences between the two constructs are localised around the N terminus, which becomes more rigid upon extension. Since a similar pattern of contacts is observed for other domains of the immunoglobulin I-set, our results are of general relevance for this protein family. Our work might also inspire a more rational approach to the investigation of domain boundaries and their influence on module stability.

Algorithms↗
PubMed2006

Time-to-boundary measures of postural control during single leg quiet standing.

A novel approach to quantifying postural stability in single leg stance is assessment of time-to-boundary (TTB) of center of pressure (COP) excursions. TTB measures estimate the time required for the COP to reach the boundary of the base of support if it were to continue on its instantaneous trajectory and velocity, thus quantifying the spatiotemporal characteristics of postural control. Our purposes were to examine: (a) the intrasession reliability of TTB and traditional COP-based measures of postural control, and (b) the correlations between these measures. Twenty-four young women completed three 10-second trials of single-limb quiet standing on each limb. Traditional measures included mean velocity, standard deviation, and range of mediolateral (ML) and anterior-posterior (AP) COP excursions. TTB variables were the absolute minimum, mean of minimum samples, and standard deviation of minimum samples in the ML and AP directions. The intrasession reliability of TTB measures was comparable to traditional COP based measures. Correlations between TTB and traditional COP based measures were weaker than those within each category of measures, indicating that TTB measures capture different aspects of postural control than traditional measures. TTB measures provide a unique method of assessing spatiotemporal characteristics of postural control during single limb stance.

Adult↗