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PubMed2005

Effects of controlled fibronectin surface orientation on subsequent Staphylococcus epidermidis adhesion.

Several bacterial species, including Staphylococcus aureus and Staphylococcus epidermidis (SE) are known to express cell receptors that bind specifically to surface immobilized or extracellular matrix ligands, such as the protein fibronectin (FN). Yet, few existing studies have examined the effect of protein surface orientation on bacterial adhesion. We report here a substratum modification protocol that allows for the specific orientation of FN molecules on a surface at known levels of surface coverage. Monoclonal antibodies (Mabs), specific to either the COOH-terminus or NH3-terminus of FN, are conjugated to biotin, then immobilized to streptavidin-coated glass substrata. Specific orientation of the bound FN molecules is verified using the same Mabs in an ELISA. Bacterial adhesion of Staphylococcus epidermidis (SE) to FN bound by either its C-terminus or its NH3-terminus was quantified in batch static adhesion assays. Results indicate an increase in SE adhesion to FN-coated surfaces when the FN is bound by its C-terminus (NH3-terminus free), indicating SE receptor-specific adhesion to the FN NH3-terminus. These studies demonstrate that antifibronectin monoclonal antibodies can be used to specifically bind and orient fibronectin on a surface. In addition, adhesion of SE to these model substrata can be controlled by the orientation of the protein.

Antibodies, Monoclonal↗

PubMed2005

Adsorbed layers of oriented fibronectin: a strategy to control cell-surface interactions.

Fibronectin (Fn) is a matrix protein known to induce cell attachment and spreading through its cell binding site and related synergy sites. Fn-coated surfaces are therefore useful in tissue engineering and other cell contacting applications, but a problem with many immobilization strategies is a random distribution of molecular orientations. We sought to control Fn orientation, and thus enhance the availability of its cell binding site, by immobilizing Fn via a carboxymethyl dextran layer onto which are chemically attached monoclonal antibodies specific to a region near to Fn's C terminus (and thus away from the cell binding site). Using optical waveguide lightmode spectroscopy, we show the presence of chemically coupled antibodies to yield a considerably denser and thicker Fn layer, consistent with a more vertically aligned protein. Human umbilical vein endothelial cells spread significantly faster, and in a more spherically symmetric way, on an oriented Fn layer (i.e., in the presence of immobilized monoclonal antibodies) as compared with a control Fn layer (i.e., in the absence of bound antibodies). However, we observe human umbilical vein endothelial cell spreading on the oriented Fn layer to be similar to that on a Fn layer in the absence of a carboxymethyl dextran layer, suggesting that although orienting Fn is a promising strategy, coupling strategies using linkers other than dextran may be needed.

Adsorption↗

PubMed2007

Effects of collagen fiber orientation on the response of biologically derived soft tissue biomaterials to cyclic loading.

In the present study, the effects of initial collagen fiber orientation on the medium-term (up to 50 x 10(6) cycles) fatigue response of heart valve soft tissue biomaterials was investigated. Glutaraldehyde treated bovine pericardium (GLBP), preselected for uniform structure and collagen fiber orientation, was used as the representative heart valve biomaterial. Using specialized instrumentation, GLBP specimens were subjected to cyclic tensile loading to maximum stress levels of 500 +/- 50 kPa at a frequency of 22 Hz. Two sample groups were examined, one with the preferred collagen fiber direction parallel (PD) and perpendicular (XD) to the direction of applied strain. The primary findings indicated that GLBP fatigue response was highly sensitive to the direction of loading with respect to fiber orientation. Specifically, when loading perpendicular to the preferred collagen fiber orientation, fiber reorientation is the dominant mechanism. In contrast, when loaded parallel to the preferred fiber direction a reduction in both collagen fiber crimp and fiber reorientation occurred. Moreover, alterations in the degree and direction of mechanical anisotropy can be inducted by cyclic loading when specimens are loaded perpendicular to the preferred fiber direction. Fourier Transform Infrared Spectroscopy (FT-IR) results indicate that molecular-level damage to collagen occurs in both groups after only 20 x 10(6) cycles. Taken as a whole, the results of this study suggest that initial collagen orientation plays a critical role in bioprosthetic heart valve biomaterial fatigue response.

Animals↗

PubMed2001

Object-oriented approach to drug design enabled by NMR SOLVE: first real-time structural tool for characterizing protein-ligand interactions.

As a result of genomics efforts, the number of protein drug targets is expected to increase by an order of magnitude. Functional genomics efforts are identifying these targets, while structural genomics efforts are determining structures for many of them. However, there is a significant gap in going from structural information for a protein target to a high affinity (K(d) < 100 nM) inhibitor, and the problem is multiplied by the sheer number of new targets now available. nature frequently designs proteins in classes that are related by the reuse, through gene duplication events, of cofactor binding domains. This reuse of functional domains is an efficient way to build related proteins in that it is object-oriented. There is a growing realization that the most efficient drug design strategies for attacking the mass of targets coming from genomics efforts will be systems-based approaches that attack groups of related proteins in parallel. We propose that the most effective drug design strategy will be one that parallels the object-oriented manner by which nature designed the gene families themselves. IOPE (Integrated Object-Oriented PharmacoEngineering) is such an approach. It is a three-step technology to build focused combinatorial libraries of potential inhibitors for major families and sub-families of enzymes, using cogent NMR data derived from representatives of these protein families. The NMR SOLVE (Structurally Oriented Library Valency Engineering) data used to design these libraries are gathered in days, and data can be obtained for large proteins (> 170 kDa). Furthermore, the process is fully object-oriented in that once a given bi-ligand is identified for a target, potency is retained if different cofactor mimics are swapped. This gives the drug design process maximum flexibility, allowing for the more facile transition from in vitro potency to in vivo efficacy.

Binding Sites↗

PubMed2005

Angular orientation of trabecular bone in the femoral head and its relationship to hip joint loads in leaping primates.

The elastic properties and mechanical behavior of trabecular bone are largely determined by its three-dimensional (3D) fabric structure. Recent work demonstrating a correlation between the primary mechanical and material axes in trabecular bone specimens suggests that fabric orientation may be used to infer directional components of the material strength and, by extension, the hypothetical loading regime. Here we quantify the principal orientation of trabecular bone in the femoral head and relate these principal fabric directions to loading patterns during various locomotor behaviors. The proximal femora of a diverse sample of prosimians were scanned using a high-resolution X-ray computed tomography scanner with resolution of better than 50 mum. Spherical volumes of interest were defined within the femoral heads and the 3D fabric anisotropy was calculated using the mean intercept length and star volume distribution methods. In addition to differences in bone volume and anisotropy, significant differences were found in the spatial orientation of the principal trabecular axes depending on locomotor behavior. The principal orientations for leapers (Galago, Tarsius, Avahi) are relatively tightly clustered (alpha(95) confidence limit: 8.2; angular variance s: 18.2 degrees ) and oriented in a superoanterior direction, while those of nonleapers are more variable across a range of directions (alpha(95): 16.8; s: 42.0 degrees ). The mean principal directions are significantly different for leaping vs. nonleaping taxa. These results further suggest a relationship between bone microstructure in the hip joint and locomotor behavior and indicate a similarity of loading across leapers despite differences in kinematics and phylogeny.

Animals↗

PubMed1988

Time-varying magnetic fields: effects of orientation on chondrocyte proliferation.

The purpose of this study was to determine the effect of orientation of pulsed electromagnetic fields (PEMFs) on cellular proliferation and extracellular matrix synthesis. Bovine articular chondrocytes were cultured in PEMFs (repetitive pulse at 72 Hz) generated using Helmholtz coils oriented either parallel (horizontal) or perpendicular (vertical) to the plane of cell adhesion. Dissipation of signal energy in the form of heat increased the temperature of the PEMF coils by 2 degrees C and the tissue culture medium by 1 degree C. Therefore, control coils, which emitted no PEMFs, were heated to the temperature of PEMF coils by circulating water. Chondrocytes were cultured in 16-mm-well culture plates, and the data for individual wells were pooled as triplicates. Although not observed by microscopic examination of individual wells, positionally dependent electric field effects may be minimized by this approach. PEMFs generated by coils oriented vertically significantly decreased chondrocyte proliferation. The effect was dependent on the concentration of serum in the culture media. At 3% serum concentration, the total cell number attained after 10 days of culture was reduced by 50% in stimulated cultures when compared with controls. At 5% serum concentration, there was no effect. PEMFs applied by coils oriented horizontally did not alter proliferation of articular chondrocytes. PEMFs had no effect on synthesis of extracellular matrix by chondrocytes plated at high density, irrespective of orientation.

Animals↗

PubMed1991

Improvement of flow cytometry analysis and sorting of bull spermatozoa by optical monitoring of cell orientation as evaluated by DNA specific probing.

Flow cytometry is a potential method for the separation of X and Y bearing spermatozoa, on the basis of their relative DNA content evaluated by the fluorescence emission intensity due to specific fluorochrome DNA staining. However, spermatozoa DNA is highly condensed and nuclei exhibit flat non spherical shape, which can produce artefacts impeding accurate analysis. In order to avoid these limitations, decondensation of DNA performed by enzymatic treatment and a modification of the flow cytometer that orients the spermatozoa relative to the laser beam are generally used. In this work, we describe alternative methods and materials for selection of 1) decondensed and thus dead spermatozoa without orientation, sorted on the basis of only the 10% spermatozoa containing the least DNA (expected Y) and the 10% spermatozoa containing the more DNA (expected X), or 2) native spermatozoa homogeneously oriented using a simultaneous measurement of Axial light loss (extinction) and Forward angle light scatter. For testing enrichment of each selected fraction we have worked out a molecular hybridization procedure using X and Y specific DNA probes. We analyse and sort bull spermatozoa on these basis: the purity obtained for these fractions is 80% without orientation after enzymatic treatment, and 70% on live spermatozoa "optically" oriented.

Animals↗

PubMed2002

Orientational diffusion reflects fiber structure within a voxel.

Several new MR techniques have been introduced to infer direction through diffusion in multiple nerve fiber bundles within a voxel. To date, however, there has been no physical model reported to evaluate these methodologies and their ability to determine fiber orientation. In this article a model of diffusion analogous to nerve fibers is presented. Diffusion measurements at multiple closely spaced angles of 15 degrees in samples with different fiber orientations are compared with theoretical calculations for restricted diffusion in cylindrical geometry. Orientational diffusion measurements are shown to reflect fiber geometry and theoretical predictions to within 10%. Simulations of fiber crossings within a voxel suggest fiber orientation does not correspond to the direction of the largest measured diffusion coefficient, but theoretical knowledge of signal decay curves can predict the shape of these diffusion coefficient contours for given fiber orientation probabilities.

Anisotropy↗

PubMed2003

Mapping fiber orientation in human muscle by proton MR spectroscopic imaging.

Proton magnetic resonance spectroscopic imaging ((1)H-MRSI) was used to determine muscle fiber orientations in human calf muscles. The method is based on the fact that some resonances show orientation-dependent dipolar splitting, caused by incomplete motional averaging. This leads to proton spectra that depend strongly on the angle between muscle fibers and the magnetic field B(0). The orientation-dependent dipolar splittings were mapped using a fit with a basis set of predefined coupling patterns reflecting the fiber orientation. The fitted coupling patterns were displayed as images and assigned to different muscles based on segmented MR images. They showed gross differences in fiber orientation between some muscles, including m. soleus and m. tibialis anterior, for all subjects. In addition, smaller but significant differences between subjects were detected, which could be due to localization differences or real interindividual differences. Since dipolar splitting affects metabolite intensities, it is important to take this effect into account when calculating metabolite concentrations from MR spectra in muscle tissue. Spatial maps of the MR signals of trimethyl-ammonium groups and creatine/ phosphocreatine revealed significant differences in intensity between muscles.

Creatine↗

PubMed2005

Water in tendon: orientational analysis of the free induction decay.

The orientation dependence of the free induction decay (FID) of 1H NMR water signal in ex vivo bovine digital flexor tendon at the native level of hydration is reported. Residual dipolar coupling due to the overall tissue anisotropy produces a 6:1 change in the signal intensity as an angle between the long axis of a specimen and the external magnetic field is changed from the "magic angle" of 54.7 degrees to 0 degrees. The strength of residual dipolar interactions between water protons was estimated by orientational analysis of the signal intensity to be equal to 780 Hz. Apparent signal maxima are observed at orientations 8-13 degrees away from 54.7 degrees due to an inhomogeneous contribution to the decay. A small fraction of total water in tendon is detectable at all orientations and exhibits a shift in the precession frequency. It is hypothesized that this water fraction resides in the interconnecting gaps at the ends of collagen molecules. The gaps have a disordered environment that allows for a zero time average of dipolar interactions. Measured frequency and phase shifts are interpreted as signatures of the bulk magnetic susceptibility effect due to geometry of the cavity formed by adjacent gaps at the ends of the collagen molecules. The multiexponentiality of the FID decay is hypothesized to be due to the exchange between orientationally restricted water structured along the length of the collagen molecule and disordered water in the cavity.

Animals↗

PubMed1993

Orientation and rotational dynamics of spin-labeled phalloidin bound to actin in muscle fibers.

We have used electron paramagnetic resonance spectroscopy (EPR) to investigate the orientational distribution of actin in thin filaments of glycerinated muscle fibers in rigor, relaxation, and contraction. A spin-labeled derivative of a mushroom toxin, phalloidin (PHSL), was bound to actin in the muscle fibers (PHSL-fibers). The EPR spectrum of unoriented PHSL-labeled myofibrils consisted of three sharp lines with a splitting between the outer extrema (2T parallel') of 42.8 +/- 0.1 G, indicating that the spin labels undergo restricted nanosecond rotational motion within an estimated half-cone angle of 76 degrees. When the PHSL-fiber bundle was oriented parallel to the magnetic field, the splitting between the zero-crossing points (2T') was 42.7 +/- 0.1 G. When the fiber bundle was perpendicular to the magnetic field, 2T' decreased to 34.5 +/- 0.2 G. This anisotropy shows that the motion of the probe is restricted in orientation by its binding site on actin, so that the EPR spectrum of PHSL-fiber bundles would be sensitive to small changes in the mean axial orientation of the PHSL-actin interface. No differences in the EPR spectra were observed in fibers during rigor, relaxation, or contraction, indicating that the mean axial orientation of the PHSL binding site changes by less than 5 degrees, and that the amplitude of nanosecond probe rotational motion, which should be quite sensitive to the local environment of the phalloidin, changes by no more than 1 degree.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗

PubMed2001

In situ deprotection: a method for covalent immobilization of peptides with well-defined orientation for use in solid phase immunoassays such as enzyme-linked immunosorbent assay.

The orientation of an immobilized antigen is important for recognition by, e.g., an antibody. When noncovalent passive adsorption is used for immobilization, the number of ways that the antigen can attach to the surface is numerous and control of how the antigen orientates on the surface is limited. Covalent immobilization restricts the number of the ways that the antigen can be immobilized to the number of reactive groups on the antigen and, hence, the orientation of the immobilized antigen is more predictable. Peptide antigens were synthesized and purified with protection groups on the lysine and cysteine side chains. These peptides, which have only one good nucleophilic group (the N-terminal alpha-amino group), were immobilized covalently in microtiter plates supplied with tresyl groups on the surface and the protection groups were cleaved off in situ after immobilization. The controlled orientation of these peptides resulted in enhanced recognition by antibodies in general. An enzyme-linked immunosorbent assay for detection of antibodies against a peptide derived from outer surface protein C from Borrelia burgdorferi, found in Lyme borreliosis patients, was established using this strategy. Lyme borreliosis suspect patient sera showed up to a 10-fold increase in the signal when the orientation of the peptide antigen was controlled by the in situ deprotection strategy.

Antibodies↗

PubMed1998

A mathematical model for fibroblast and collagen orientation.

Due to the increasing importance of the extracellular matrix in many biological problems, in this paper we develop a model for fibroblast and collagen orientation with the ultimate objective of understanding how fibroblasts form and remodel the extracellular matrix, in particular its collagen component. The model uses integrodifferential equations to describe the interaction between the cells and fibers at a point in space with various orientations. The equations are studied both analytically and numerically to discover different types of solutions and their behavior. In particular we examine solutions where all the fibroblasts and collagen have discrete orientations, a localized continuum of orientations and a continuous distribution of orientations with several maxima. The effect of altering the parameters in the system is explored, including the angular diffusion coefficient for the fibroblasts, as well as the strength and range of the interaction between fibroblasts and collagen. We find the initial conditions and the range of influence between the collagen and the fibroblasts are the two factors which determine the behavior of the solutions. The implications of this for wound healing and cancer are discussed including the conclusion that the major factor in determining the degree of scarring is the initial deposition of collagen.

Animals↗

PubMed1998

The Development and Validation of Scales Assessing Students' Achievement Goal Orientations

Achievement goal theory has emerged as a major new direction in motivational research. A distinction is made among conceptually different achievement goal orientations including the goal to develop ability (task goal orientation), the goal to demonstrate ability (ability-approach goal orientation), and the goal to avoid the demonstration of lack of ability (ability-avoid goal orientation). Scales assessing each of these goal orientations were developed over an eight year period by a group of researchers at the University of Michigan. The results of studies conducted with seven different samples of elementary and middle school students are used to describe the internal consistency, stability, and construct validity of the scales. Comparisons of these scales with those developed by Nicholls and his colleagues provide evidence of convergent validity. Confirmatory factor analysis attests to the discriminant validity of the scales. Copyright 1998 Academic Press.

Journal Article↗

PubMed2002

Occupational dreams, choices and aspirations: adolescents' entrepreneurial prospects and orientations.

The present study examined possible early antecedents of entrepreneurship of 14-17-year-old 10th grade students (n=320). We hypothesized that Entrepreneurial Orientation (interest and self-efficacy), together with Willingness to Expend Effort, would be an important predictor of an adolescent's Entrepreneurial Prospects, i.e. prospects of becoming self-employed in the future. Furthermore, personality and the model of self-employed family were expected to predict the level of Entrepreneurial Orientation. The same relationships were investigated separately for students who were more or less willing to expend effort. Among students more willing to expend effort, levels of Entrepreneurial Orientation were higher for those who were conscientious, self-efficient, open to new experiences, and low in agreeableness. Among students less willing to expend effort, a high need for social recognition predicted higher levels of Entrepreneurial Orientation. In addition, parents' model for them was connected with lower levels of Entrepreneurial Orientation. A moderating effect of Willingness to Expend Effort was supported by the results for parents' model and need for social recognition.

Achievement↗

PubMed2002

Rheological properties and orientational distributions of dilute ferromagnetic spherocylinder particle dispersions. Part II. Analysis for the two typical magnetic field directions.

We have investigated the orientational distributions and rheological properties of dilute colloidal dispersions, which consist of ferromagnetic spherocylinder particles. First, the governing equation of the orientational distribution function has been derived for the typical two cases of magnetic field directions: the direction parallel to the shear flow and the direction parallel to the angular velocity vector of the shear flow. The equation has been solved approximately by Galerkin's method. With these numerical solutions we have obtained the results of the orientational distribution and viscosity. The results obtained for the magnetic field in the shear flow direction are summarized as follows. In the case of a weak magnetic field, the particle tends to orient nearly toward the shear flow direction and its opposite direction. As the magnetic field increases, the orientation of the particle is restricted and the viscosity increases significantly. As the influence of the magnetic field becomes dominant, an overshoot in the viscosity curve appears. This is due to the fact that there is a maximum deviation of the averaged particle direction from the magnetic field direction. When the strength of the magnetic field increases significantly, the particle inclines close to the magnetic field direction and the viscosity converges to a constant value. Particles with a larger aspect ratio give rise to a larger increment in the viscosity since such elongated particles induce larger resistance in a flow field. We also have obtained results for the case of the magnetic field in the direction parallel to the angular velocity vector of the shear flow. When the flow field is dominant over both the rotational Brownian motion and the magnetic interaction, the particle rotates in the plane nearly perpendicular to the magnetic field direction. As the magnetic field increases, the particle inclines toward the magnetic direction. For this direction of field, the viscosity is independent of the magnetic field and is always zero.

Journal Article↗

PubMed1998

Orientation, positional, additivity, and oligomerization-state effects of interhelical ion pairs in alpha-helical coiled-coils.

The role of interhelical g-e' ion pairs in the dimerization specificity and stability of alpha-helical coiled-coils is highly controversial. Synthetic 35-residue coiled-coils based on the heptad repeat QgVaGbAcLdQeK f were used to investigate the effect of orientation of interhelical ion pairs between lysine and glutamic acid residues on coiled-coil stability. Stability was estimated from urea denaturation at 20 degreesC, monitoring unfolding with circular-dichroism spectroscopy. Double mutant cycles were employed to estimate the net interaction energy, Delta DeltaGuint, for the two orientations of the ion pair; Ee-Kg and Ke-Eg. Delta DeltaGuint was found to be about 1.4-fold higher for the Ee-Kg orientation in a coiled-coil containing an N-terminal disulfide bridge. The Delta DeltaGuint value was similar whether obtained from the middle heptad or averaged over all five heptads of the coiled-coil, suggesting that ion pairs contribute additively to coiled-coil stability. The effect of uncompensated charges was also illustrated by single substitutions of Gln with either Lys or Glu, resulting in Lys-Gln or Glu-Gln g-e' pairs. These substitutions were found to be twice as destabilizing at position g as at position e, and Lys was about twice as destabilizing as Glu at both positions e and g. In the absence of an interhelical disulfide bridge, Glu and Lys substitutions in the middle heptad were equally destabilizing at positions e and g (Lys continued to be more destabilizing than Glu) and the Delta DeltaGuint value for Lys-Glu ion pairs was not orientation dependent. These and previous results suggest the non-covalently-linked synthetic coiled-coils behave as molten globules, whereas a disulfide-bridge may "lock in" the structural differences between positions of the heptad repeat. Interhelical Lys-Glu ion pairs in either orientation promoted the formation of trimeric coiled-coils (in the absence of a disulfide bridge) while Gln-Gln g-e' interactions led to dimer formation. The results support a role for g-e' ionic attractions in controlling coiled-coil specificity, stability and oligomerization state, possibly through effects on the side-chain packing at the subunit interface.

Amino Acid Sequence↗

PubMed1998

Magic angle-oriented sample spinning (MAOSS): A new approach toward biomembrane studies.

The application of magic angle sample spinning (MAS) NMR to uniformly aligned biomembrane samples is demonstrated as a new general approach toward structural studies of membrane proteins, peptides, and lipids. The spectral linewidth from a multilamellar lipid dispersion is dominated, in the case of protons, by the dipolar coupling. For low-gamma or dilute spins, however, the chemical shift anisotropy dominates the spectral linewidth, which is reduced by the two-dimensional order in a uniformly aligned lipid membrane. The remaining line broadening, which is due to orientational defects ("mosaic spread") can be easily removed at low spinning speeds. This orientational order in the sample also allows the anisotropic intermolecular motions of membrane components (such as rotational diffusion, tauc = 10(-10) s) for averaging dipolar interactions to be utilized, e.g., by placing the membrane normal parallel to the rotor axis. The dramatic resolution improvement for protons which are achieved in a lipid sample at only 220 Hz spinning speed in a 9.4 T field is slightly better than any data published to date using ultra-high fields (up to 17.6 T) and high-speed spinning (14 kHz). Additionally, the analysis of spinning sidebands provides valuable orientational information. We present the first 1H, 31P, and 13C MAS spectra of uniformly aligned dimyristoylphosphatidylcholine (DMPC) bilayers. Also, 1H resolution enhancement for the aromatic region of the M13 coat protein reconstituted into DMPC bilayers is presented. This new method combines the high resolution usually achieved by MAS with the advantages of orientational constraints obtained by working with macroscopically oriented samples. We describe the general potential and possible perspectives of this technique.

Anisotropy↗