Statistical method for surface pattern-matching between dissimilar molecules: electrostatic potentials and accessible surfaces

This paper outlines a statistical method for patternmatching between surfaces and is applicable to structural and energetic patterns found on molecular surfaces. Correlation coefficients generated for the pattern match are scale invariant. Regression analysis applied to the patterns reveals the scaling and displacement relationships. The method for measuring the similarities between molecular surfaces of two dissimilar molecules held infixed orientations is given explicitly. Implicit in this procedure is a method for studying the inverse phenomenon, namely complementarity between surface parameters at a binding site and its ligand. The method has been used to assess surface differences in structural similarities generated by computer fitting and by visual comparison. Various pitfalls likely to be encountered in evaluating molecular structural similarities are noted.     

Neurotrimin expression during cerebellar development suggests roles in axon fasciculation and synaptogenesis

We investigated the temporal expression of the neural cell adhesion molecule, neurotrimin, in the rat cerebellum and the brainstem from birth to adulthood using immunoreactive labeling. A wave of expression accompanied the development of projection pathways extending from brainstem nuclei (pons/inferior olive) through the cerebellar peduncles into the arbor vitae and disappeared with myelination by P14. Immuno-EM revealed expression of neurotrimin on the surface of unmyelinated axons but not on astrocytes or oligodendroglia. With the development of the molecular and internal granular layers, intense labeling occurred on the surface of parallel fiber bundles, granule cells and mossy fibers. With synaptogenesis, each excitatory junction was labeled by the immunoreaction. By P21, neurotrimin reactivity decreased on the surfaces of neuronal somata, dendrites and axons but remained at excitatory synaptic contact sites in both the molecular and granular layers. The spatial-temporal expression pattern of neurotrimin suggests that this adhesion molecule plays a role in axonal fasciculation of specific cerebellar systems and may also be involved in the formation of excitatory synapses and their stabilization into adulthood.     

Laser microfabricated model surfaces for controlled cell growth

The relatively recent applications of microelectronics technology into the biological sciences arena has drastically revolutionized the field. New foreseeable applications include miniaturized, multiparametric biosensors for high performance multianalyte assays or DNA sequencing, biocomputers, and substrates for controlled cell growth (i.e. tissue engineering). The objectives of this work were to investigate a new method combining microphotolithographical techniques with laser excimer beam technology to create surfaces with well defined 3-D microdomains in order to delineate critical microscopic surface features governing material-cell interaction. Another obvious application of this study pertains to the fabrication of cell-based biosensors. Microfabricated surfaces were obtained with micron resolution, by "microsculpturing" polymer model surfaces using a laser excimer KrF beam coupled with a microlithographic projection technique. The laser beam after exiting a mask was focused onto the polymer target surface via an optical setup allowing for a 10-fold reduction of the mask pattern. Various 3-D micropatterned features were obtained at the micron level. Reproducible submicron features could also be obtained using this method. Subsequently, model osteoblast-like cells were plated onto the laser microfabricated surfaces in order to study the effects of particular surface microtopography on preferential cell deposition and orientation. Preferential cell deposition was observed on surfaces presenting "smooth" microtopographical transitions. This system may provide an interesting model for further insights into correlations between 3-D surface microtopography and cell response with new applications in the field biosensor, biomaterial and pharmaceutical engineering sciences (e.g. new cell based biosensors, controlled synthesis of immobilized cell derived active ingredients).     

Analytically defined surfaces to analyze molecular interaction properties

Molecular surfaces are widely used for characterizing molecules and displaying and quantifying their interaction properties. Here we consider molecular surfaces defined as isocontours of a function (a sum of exponential functions centered on each atom) that approximately represents electron density. The smoothness is advantageous for surface mapping of molecular properties (e.g., electrostatic potential). By varying parameters, these surfaces can be constructed to represent the van der Waals or solvent-accessible surface of a molecular with any accuracy. We describe numerical algorithms to operate on the analytically defined surfaces. Two applications are considered: (1) We define and locate extremal points of molecular properties on the surfaces. The extremal points provide a compact representation of a property on a surface, obviating the necessity to compute values of the property on an array of surface points as is usually done; (2) a molecular surface patch or interface is projected onto a flat surface (by introducing curvilinear coordinates) with approximate conservation of area for analysis purposes. Applications to studies of protein-protein interactions are described.     

Approximation and characterization of molecular surfaces

The representation and characterization of molecular surfaces are important in many areas of molecular modeling. Parametric representations of protein molecular surfaces are a compact way to describe a surface, and are useful for the evaluation of surface properties such as the normal vector, principal curvatures, and principal curvature directions. Simplified representations of molecular surfaces are useful for efficient rendering and for the display of large-scale surface features. Several techniques for representing surfaces by expansions of spherical harmonic functions have been reported, but these techniques require that the radius function is single valued, that is, each ray from an origin inside the surface intersects the surface at one and only one point. A new technique is described that removes this limitation and can be used to compute surface shape properties. © 1993 John Wiley & Sons, Inc.     

Characterisation of lithographically patterned organosilane monolayers by preferential adsorption of dye molecules

A method for the visualisation of micropatterned spots of molecular organosilane monolayers by adsorption of dyes is presented. Therefore, microspot arrays consisting of different types of organosilane monolayers are exposed to dye solutions. After this immersion, the chips are rinsed in a solvent, resulting in a locally differentiated fluorescence intensity. The contrast between silanized and non-silanized surface areas depends on the applied dyes and their solution concentrations. Charge and polarity seems to play an important role in the adsorption. In result, the pattern of molecular organosilane monolayers can easily be identified by intensity differences of fluorescence radiation from the different regions of chip surfaces.     

Bacterial adhesion at synthetic surfaces.

A systematic investigation into the effect of surface chemistry on bacterial adhesion was carried out. In particular, a number of physicochemical factors important in defining the surface at the molecular level were assessed for their effect on the adhesion of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli. The primary experiments involved the grafting of groups varying in hydrophilicity, hydrophobicity, chain length, and chemical functionality onto glass substrates such that the surfaces were homogeneous and densely packed with functional groups. All of the surfaces were found to be chemically well defined, and their measured surface energies varied from 15 to 41 mJ. m(-2). Protein adsorption experiments were performed with (3)H-labelled bovine serum albumin and cytochrome c prior to bacterial attachment studies. Hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption; however, our studies also showed that the effectiveness of poly(ethyleneoxide) (PEO) polymers was not simply a result of its hydrophilicity and molecular weight alone. The adsorption of the two proteins approximately correlated with short-term cell adhesion, and bacterial attachment for L. monocytogenes and E. coli also correlated with the chemistry of the underlying substrate. However, for S. aureus and S. typhimurium a different pattern of attachment occurred, suggesting a dissimilar mechanism of cell attachment, although high-molecular-weight PEO was still the least-cell-adsorbing surface. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups may be the best method for preventing cell adsorption to synthetic substrates provided they can be grafted uniformly and in sufficient density at the surface.     

The beauty of molecular surfaces as revealed by self-organizing neural networks

The Kohonen neural network is a self-organizing network that can be used for the projection of the surface properties of molecules. This allows one to view properties on a molecular surface, like the electrostatic potential in a single picture. These maps are useful for the comparison of molecules and provide a new definition of molecular similarity.     

Pattern recognition based on color-coded quantum mechanical surfaces for molecular alignment

A pattern recognition algorithm for the alignment of drug-like molecules has been implemented. The method is based on the calculation of quantum mechanical derived local properties defined on a molecular surface. This approach has been shown to be very useful in attempting to derive generalized, non-atom based representations of molecular structure. The visualization of these surfaces is described together with details of the methodology developed for their use in molecular overlay and similarity calculations. In addition, this paper also introduces an additional local property, the local curvature (C _L), which can be used together with the quantum mechanical properties to describe the local shape. The method is exemplified using some problems representing common tasks encountered in molecular similarity. Figure Molecular surfaces for Lorazepam (left) and Diazepam (right)     

Macromolecular solvation energies derived from small molecule crystal morphology.

The morphology of small molecule crystals provides a model for evaluating surface solvation energies in a system with similar packing density to that observed for amino acid residues in proteins. The solvation energies associated with the transfer of methylene and carboxyl groups between vacuum and aqueous phases are estimated to be approx. $40 and -260 cal/A2, respectively, from an analysis of the morphology of succinic acid crystals. These solvation energies predict values for contact angles in reasonable agreement with measurements determined from macroscopic monolayer surfaces. Transfer free energies between vapor and water phases for a series of carboxylic acids are also predicted reasonably well by these solvation energies, provided the surface exposure of different groups is quantitated with the molecular surface area rather than the more traditional accessible surface area. In general, molecular surfaces and molecular surface areas are seen to have important advantages for characterizing the structure and energetics of macromolecular surfaces. Crystal faces of succinic acid with the lowest surface energies in aqueous solution are characteristically smooth. Increasing surface roughness and apolarity are associated with higher surface energies, which suggests an approach for modifying the surface properties of proteins and other macromolecules.     

Electrochemically controllable conjugation of proteins on surfaces

The rational design of surfaces for immobilization of proteins is essential to a variety of biological and medical applications ranging from molecular diagnostics to advanced platforms for fundamental studies of molecular and cell biology. We have developed an advanced electrochemically based approach for site-selective and reaction-controlled immobilization of proteins on surfaces. When a molecular monolayer of 4-nitrothiophenol on gold electrode surfaces is reduced electrochemically in a selective fashion at its nitro groups, to afford amino groups by potentiometric scans, the amine can be employed to orchestrate the immobilization of proteins to the surface. This protein immobilization strategy could allow one to fabricate intricate protein structures on surfaces for addressing fundamental and applied problems in biology and medicine.     

Probing depth in monocular images

It is generally expected that depth (distance) is the internal representational primitive that corresponds to much of the perception of 3D. We tested this assumption in monocular surface stimuli that are devoid of distance information (due to orthographic projection and the chosen surface shape, with perspective projection used as a control) and yet are vividly three-dimensional. Slant judgments were found to be in close correspondence with the actual geometric slant of the stimuli; the spatial orientation of the surfaces was perceived accurately. The apparent depth in these stimuli was then tested by superimposing a stereo depth probe over the monocular surface. In both the perspective and orthographic projection the gradient of perceived depth, measured by matching the apparent depth of the stereo probe with that of the monocular surface at a series of locations, was substantial. The experiments demonstrate that in orthographic projection the visual system can compute from local surface orientation a depth quantity that is commensurate with the relative depth derived from stereo disparity. The depth data suggests that, at least in the near field, the zero value for relative depth lies at the same absolute depth as the stereo horopter (locus of zero stereo disparity). Relative to this zero value, the depth-from-slant computation seems to provide an estimate of distance information that is independent of the absolute distance to the surface.Supproted by Office of Naval Research Contract N00014-K-84-0533. We gratefully acknowledge the suggestions of Jacob Beck regarding the experimental design, and the assistance provided by Cathryn Stanford     

Microexudates from Cells Grown in Tissue Culture

Cellular substrata of known molecular structure and measurable dimensions can be constructed as transferred films from Langmuir troughs or as adsorbed films. In addition, large molecules in culture media form measurable adsorbates. With the techniques of ellipsometry and surface chemistry it is possible to characterize and measure (within ± 3A) as a function of several parameters a microexudate of molecular dimensions deposited when tissue cultured cells contact certain substrata. The selective attraction of substratum and cell for microexudate has been determined, and the time course of deposition in Eagle's medium is characterized by a rapid initial accretion of material. During this period, microexudate can diffuse several cell diameters and cannot be detected in the culture medium. In Eagle's medium the cells cannot be detached from glass surfaces by versene or trypsin unless the surface of cell or substratum is coated with certain molecules. Trypsin becomes adsorbed to cell surfaces, continues to be enzymatically active on the surface, and digests protein components of microexudate and substratum. Microexudate appears to be a complex mosaic of molecules (including protein) synthesized within or on the surfaces of cells and secreted by cells or transferred from their surfaces to specific substrata. It is proposed that this mosaic plays, on the molecular level, a significant role in cell-to-cell interactions, cell locomotion and adhesion, and the selective application and spreading of cells on various surfaces.     

Shape from texture

A central goal for visual perception is the recovery of the three-dimensional structure of the surfaces depicted in an image. Crucial information about three-dimensional structure is provided by the spatial distribution of surface markings, particularly for static monocular views: projection distorts texture geometry in a manner tha depends systematically on surface shape and orientation. To isolate and measure this projective distortion in an image is to recover the three dimensional structure of the textured surface. For natural textures, we show that the uniform density assumption (texels are uniformly distributed) is enough to recover the orientation of a single textured plane in view, under perspective projection. Furthermore, when the texels cannot be found, the edges of the image are enough to determine shape, under a more general assumption, that the sum of the lengths of the contours on the world plane is about the same everywhere. Finally, several experimental results for synthetic and natural images are presented.     

Projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the moth Manduca sexta

Octopod (Octo) is a mutation of the moth Manduca sexta, which transforms the first abdominal segment (A1) in the anterior direction. Mutant animals are characterized by the appearance of homeotic thoracic-like legs on A1. We exploited this mutation to determine what rules might be used in specifying the fates of sensory neurons located on the body surface of larval Manduca. Mechanical stimulation of homeotic leg sensilla did not cause reflexive movements of the homeotic legs, but elicited responses similar to those observed following stimulation of ventral A1 body wall hairs. Intracellular recordings demonstrated that several of the motoneurons in the A1 ganglion received inputs from the homeotic sensory hairs. The responses of these motoneurons to stimulation of homeotic sensilla resembled their responses to stimulation of ventral body wall sensilla. Cobalt fills revealed that the mutation transformed the segmental projection pattern of only the sensory neurons located on the ventral surface of A1, resulting in a greater number with intersegmental projection patterns typical of sensory neurons found on the thoracic body wall. Many of the sensory neurons on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons: an anteriorly directed projection terminating in the third thoracic ganglion (T3). Once this projection reached T3, however, it mimicked the projections of the thoracic leg sensory neurons. These results demonstrate that the same rules are not used in the establishment of the intersegmental and leg-specific projection patterns. Segmental identity influences the intersegmental projection pattern of the sensory neurons of Manduca, whereas the leg-specific projections are consistent with a role for positional information in determining their pattern. © 1995 John Wiley & Sons, Inc.     

Behavioural and physical reactions of the Colorado potato beetle, Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae) walking on a slanted surface

 A natural reaction of the adult Colorado potato beetle [Leptinotarsa decemlineata (Say)] is to walk uphill on sloped surfaces. The geotaxis reaction of the beetle was observed on slopes of 20° or steeper. It was demonstrated that the uphill orientation behaviour was not a consequence of physical limitation for across-slope locomotion. The walking speed of insects deviating from the fall line did not change within the range of slope angles tested. The speed of adult beetles decreased with an increase in the slope of the substrate as a reaction to the increased gravitational force vector opposing uphill movement. The larger size of the hind legs might make uphill locomotion more efficient than traversing a sloped surface. As the angle of the slope increased, the gait changed from a 3/3 to a 5/1, as did the posterior and anterior extreme position of the legs. This behaviour might be triggered by the need to maintain balance on slanted surfaces as the vertical projection of the centre of mass on the substrate moved outside the support base pattern at the steeper angles. In one experiment beetles were made to pull a load when walking over a horizontal surface. The loads pulled were equivalent to the gravitational loads opposing forward motion when walking up a slope. No differences in forward speed or gait were observed at the lower-angle equivalent compared to beetles walking on slopes. Differences in speed were noted at slope angles higher than 40° indicating that adaptation of the walking strategy might be needed on steeply slanted surfaces. Received: 2 May 2000 / Accepted in revised form: 11 September 2000     

The molecular surface package

The Molecular Surface Package is a reimplementation, in C, of a set of earlier FORTRAN programs for computing analytical molecular surfaces, areas, volumes, polyhedral molecular surfaces, and surface curvatures. The software does not do interactive molecular graphics, but it will produce pixel maps of smooth molecular surfaces. The polyhedral molecular surfaces are suited to display on graphics systems with real-time rendering of polyhedra.     

Segmentation of protein surfaces using fuzzy logic

An algorithm has been developed that can be used to divide triangulated molecular surfaces into distinct domains on the basis of physical and topographical molecular properties. Domains are defined by a certain degree of homogeneity concerning one of these properties. The method is based on fuzzy logic strategies, thus taking into consideration the smooth changes of the properties considered along complex macromolecular surfaces. Scalar qualities assigned to every node point on a triangulated surface are translated into linguistic variables, which can then be processed using a special fuzzy dissimilarity operator.Possible applications are demonstrated using surface segmentation for properties like electrostatic potential, lipophilicity and shape for the analysis of serine proteinase substrate/inhibitor specificity.     

A photo-electroactive surface strategy for immobilizing ligands in patterns and gradients for studies of cell polarization

We report a combined photochemical and electrochemical method to pattern ligands and cells in complex geometries and gradients on inert surfaces. This work demonstrates: (1) the control of density of immobilized ligands within overlapping photopatterns, and (2) the attached cell culture patterned onto ligand defined gradients for studies of directional cell polarity. Our approach is based on the photochemical activation of benzoquinonealkanethiols. Immobilization of aminooxy terminated ligands in selected region of the quinone monolayer resulted in patterns on the surface. This approach is unique in that the extent of photochemical deprotection, as well as ligand immobilization can be monitored and quantified by cyclic voltammetry in situ. Furthermore, complex photochemical patterns of single or multiple ligands can be routinely generated using photolithographic masks. Finally, this methodology is completely compatible with attached cell culture and we show how the subtle interplay between cell-cell interactions and underlying peptide gradient influences cell polarization. The combined use of photochemistry, electrochemistry and well defined surface chemistry provides molecular level control of patterned ligands and gradients on surfaces.     

Hyaluronan conformations on surfaces: effect of surface charge and hydrophobicity

Extended, relaxed, condensed, and interacting forms of the polysaccharide hyaluronan have been observed by atomic force microscopy (AFM). The types of images obtained depend on the properties of the surfaces used. We have investigated several different surface conditions for HA imaging, including unmodified mica, mica chemically modified with two different kinds of amino-terminated silanes (3-aminopropyltriethoxysilane and N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride), and highly oriented pyrolytic graphite. We found the degree of HA molecular extension or condensation to be variable, and the number of bound chains per unit area was low, for all of the mica-based surfaces. HA was more easily imaged on graphite, a hydrophobic surface. Chains were frequently observed in high degrees of extension, maintained by favorable interaction with the surface after molecular combing. This observation suggests that the HA macromolecule interacts with graphite through hydrophobic patches along its surface. AFM studies of HA behavior on differing surfaces under well-controlled environmental conditions provides useful insight into the variety of conformations and interactions likely to be found under differing physiological conditions.