Biomedical subjects

G M Whitesides

Publications and source records attributed to G M Whitesides.

At least 55 records · Page 3Linked to original sources

PubMed1998

Regulation of protrusion shape and adhesion to the substratum during chemotactic responses of mammalian carcinoma cells.

We report here the first direct observation of chemotaxis to EGF by rat mammary carcinoma cells. When exposed to a gradient of EGF diffusing from a micropipette, MTLn3 cells displayed typical ameboid chemotaxis, extending a lamellipod-like protrusion and moving toward the pipette. Using a homogeneous upshift in EGF to model stimulated lamellipod extension (J. E. Segall et al., 1996, Clin. Exp. Metastasis 14, 61-72), we analyzed the relationship between adhesion and chemoattractant-stimulated protrusion. Exposure to EGF led to a rapid remodeling of the adhesive contacts on adherent cells, in synchrony with extension of a flat lamellipod over the substratum. EGF-stimulated lamellipods still extended in the presence of adhesion-blocking peptides or over nonadhesive surfaces. They were, however, slightly shorter and retracted rapidly under those conditions. The major protrusive structure observed on well-spread, adherent cells, after EGF stimulation was a flat broad lamellipod, whether or not in contact with the substratum, while cells in suspension showed transient protrusive activity over the entire cell surface. We conclude that the initial adhesive status of the cell conditions the shape of the outcoming protrusion. Altogether our results suggest that, although adhesive contacts are not necessary for lamellipod extension, they play a role in stabilizing the protrusion as well as in the control of its final shape and amplitude.

Animals↗

PubMed1998

A trivalent system from vancomycin.D-ala-D-Ala with higher affinity than avidin.biotin.

Tris(vancomycin carboxamide) binds a trivalent ligand derived from D-Ala-D-Ala with very high affinity: dissociation constant (Kd) approximately 4 x 10(-17) +/- 1 x 10(-17) M. High-affinity trivalent binding and monovalent binding are fundamentally different. In trivalent (and more generally, polyvalent) binding, dissociation occurs in stages, and its rate can be accelerated by monovalent ligand at sufficiently high concentrations. In monovalent binding, dissociation is determined solely by the rate constant for dissociation and cannot be accelerated by added monomer. Calorimetric measurements for the trivalent system indicate an approximately additive gain in enthalpy relative to the corresponding monomers. This system is one of the most stable organic receptor-ligand pairs involving small molecules that is known. It illustrates the practicality of designing very high-affinity systems based on polyvalency.

Alanine↗

PubMed1998

Studying how different terminal groups change the motion of H2NSO2C6H4CONH(EG)3R when bound to the active site of human carbonic anhydrase II.

Molecular dynamics simulations have been used to explore the motions of series of ligands containing coupled benzenesulfonamide and oligoethylene glycol moieties (H2NSO2C6H4CONH(CH2-CH2OCH2CH2OCH2CH2)R+; R+ = NH3+, NHCOCH2NH3+, NHCOCH(CH2Ph)NH3+) bound at the active site of human carbonic anhydrase II (HCAII; E.C. 4.2.1.1). These complexes have been examined previously by X-ray crystallography; the locations of the terminal groups of these ligands were not defined in the crystal structures. These stimulations, carried out in the presence of water, provide dynamic information about the motion of the bound ligand that supplements the quasistatic information from crystallography. Our results suggested that the Gly and Phe groups of these ligands interacted weakly with the protein adjacent to the active site. Quantitative estimates of energies of binding did not correlate usefully with observed free energies of binding, but in the absence of information about entropies, it is not possible to tell if the lack of correlation between calculated energies and observed free energies represents inaccuracies in the energies, or a compensation between enthalpies and entropies. When the terminal Phe group was placed near a previously identified hydrophobic patch in the active site (Phe20 and Pro202) the average conformation of the ligand inferred from this simulation was inconsistent with that from the crystal structure; this result illustrates the problems of misleading local minima in these types of simulations.

Binding Sites↗

PubMed1998

Affinity capillary electrophoresis: a physical-organic tool for studying interactions in biomolecular recognition.

Affinity capillary electrophoresis (ACE) is a technique that is used to measure the binding affinity of receptors to neutral and charged ligands. ACE experiments are based on differences in the values of electrophoretic mobility of free and bound receptor. Scatchard analysis of the fraction of bound receptor, at equilibrium, as a function of the concentration of free ligand yields the dissociation constant of the receptor-ligand complex. ACE experiments are most conveniently performed on fused silica capillaries using a negatively charged receptor (molecular mass < 50 kDa) and increasing concentrations of a low molecular weight, charged ligand in the running buffer. ACE experiments that involve high molecular weight receptors may require the use of running buffers containing zwitterionic additives to prevent the receptors from adsorbing appreciably to the wall of the capillary. This review emphasizes ACE experiments performed with two model systems: bovine carbonic anhydrase II (BCA II) with arylsulfonamide ligands and vancomycin (Van), a glycopeptide antibiotic, with D-Ala-D-Ala (DADA)-based peptidyl ligands. Dissociation constants determined from ACE experiments performed with charged receptors and ligands can often be rationalized using electrostatic arguments. The combination of differently charged derivatives of proteins - protein charge ladders - and ACE is a physical-organic tool that is used to investigate electrostatic effects. Variations of ACE experiments have been used to estimate the charge of Van and of proteins in solution, and to determine the effect of the association of Van to Ac2KDADA on the value of pKa of its N-terminal amino group.

Animals↗

PubMed1997

Using microcontact printing to pattern the attachment of mammalian cells to self-assembled monolayers of alkanethiolates on transparent films of gold and silver.

This paper describes a convenient methodology for patterning substrates for cell culture that allows the positions and dimensions of attached cells to be controlled. The method uses self-assembled monolayers (SAMs) of terminally substituted alkanethiolates (R(CH2)11-15S-) adsorbed on optically transparent films of gold or silver to control the properties of the substrates. SAMs terminated in methyl groups adsorb protein and SAMs terminated in oligo(ethylene glycol) groups resist entirely the adsorption of protein. This methodology uses microcontact printing (microCP)-an experimentally simple, nonphotolithographic process-to pattern the formation of SAMs at the micrometer scale; microCP uses an elastomeric stamp having at its surface a pattern in relief to transfer an alkanethiol to a surface of gold or silver in the same pattern. Patterned SAMs having hydrophobic, methyl-terminated lines 10, 30, 60, and 90 microm in width and separated by protein-resistant regions 120 microm in width were prepared and coated with fibronectin; the protein adsorbed only to the methyl-terminated regions. Bovine capillary endothelial cells attached only to the fibronectin-coated, methyl-terminated regions of the patterned SAMs. The cells remained attached to the SAMs and confined to the pattern of underlying SAMs for at least 5-7 days. Because the substrates are optically transparent, cells could be visualized by inverted microscopy and by fluorescence microscopy after fixing and staining with fluorescein-labeled phalloidin.

Actin Cytoskeleton↗

PubMed1997

On-line detection of nonspecific protein adsorption at artificial surfaces.

A detailed understanding of the interaction of proteins with artificial surfaces is essential for many applications in medicine and biochemistry. The affinity of surfaces toward proteins may, for instance, remove pharmacological proteins from media or control the adherence of pathogenic bacteria to protheses. Only a few analytical techniques now exist that can be used to study the binding process in real time, using unlabeled proteins. By investigating the adsorption kinetics of fibrinogen at differently terminated self-assembled monolayers (SAMs) of alkanethiols on thin gold films, it is demonstrated that acoustic plate-mode sensors are a promising analytical tool for studying the adsorption of proteins. In agreement with previous studies for fibrinogen, it is shown in situ that hexa(ethylene glycol)-terminated SAMs (HS(CH2)11 (OCH2CH2)6OH) exhibit very low protein adsorption and that methyl-terminated SAMs (HS(CH2)11CH3) tend to absorb large amounts of protein nonspecifically. The observed adsorption kinetics deviate from classical Langmuir behavior; these kinetics are compatible with a mechanism that involves an unfolding of fibrinogen after adsorption. Film quality is controlled by IR, XPS, and contact angle measurements.

Adsorption↗

PubMed1997

Studies on alpha-sialylation using sialyl donors with an auxiliary 3-thiophenyl group.

Reaction of the methyl ester of 2-chloro-3-S-phenyl-3-thiosialic acid (4) with sodium thiomethoxide in acetonitrile at 0 degrees C affords the methyl ester of 2-S-methyl-3-S-phenyl-2,3-di-thio-alpha-sialic acid (6a) in quantitative yield. Sialylation of tetrahydropyran-2-methanol (7) and 2-(trimethylsilyl)ethyl 2,2'3,6,6'-penta-O-benzyl-alpha-lactoside (8) with 6a in the presence of phenylsulfenyl triflate (PST) as promotor in CH3 CN at -40 degrees C gives alpha-sialosides 9 and 10 in good yield and excellent stereoselectivity. No beta-sialosides are formed in either case. Acetylation of product 10, and the subsequent reductive removal of the 3-thiophenyl group using Ph3SnH, affords 12--protected GM3 trisaccharide--in 82% yield after two steps. Sialylation of acceptor 8 with chloride 4 using silver triflate as promotor afforded 10 in 48% yield after two days at -15 degrees C in THF. A possible mechanism of sialylation with 6a that involves intermediate alpha- and beta-nitrilium ions is discussed.

Carbohydrate Sequence↗

PubMed1997

Representing primary electrophoretic data in the 1/time domain: comparison to representations in the time domain.

A plot of absorbance vs 1/time (the "1/time domain") is a more useful representation of the primary data in capillary electrophoresis than traditional plots of absorbance vs time (the "time domain") in a wide set of circumstances, especially when comparing electropherograms in which the rate of electroosmotic flow is not precisely the same. The quantity that is of fundamental interest in capillary electrophoresis (CE) is the electrophoretic mobility of an analyte. The electrophoretic mobility of a species is nonlinearly proportional to time and, therefore, not linearly represented in the time domain: that is, the distance between two peaks along the time axis is not linearly related to the difference in their electrophoretic mobilities. In contrast, the electrophoretic mobility is linearly proportional to 1/time, and the distance between two peaks along the 1/time axis is linearly related to the difference in electrophoretic mobilities. Plots in the 1/time domain are similar to the familiar plots in the time domain (each analyte is represented by a peak, and the order of peaks corresponds to the order in which these analytes reach the detector), but the spacing between the peaks corresponds linearly to differences in mobility. This article derives this useful, visually appealing, and broadly applicable plotting strategy and illustrates common situations in which these plots are more useful than plots in the time domain.

Absorption↗

PubMed1997

Geometric control of cell life and death.

Human and bovine capillary endothelial cells were switched from growth to apoptosis by using micropatterned substrates that contained extracellular matrix-coated adhesive islands of decreasing size to progressively restrict cell extension. Cell spreading also was varied while maintaining the total cell-matrix contact area constant by changing the spacing between multiple focal adhesion-sized islands. Cell shape was found to govern whether individual cells grow or die, regardless of the type of matrix protein or antibody to integrin used to mediate adhesion. Local geometric control of cell growth and viability may therefore represent a fundamental mechanism for developmental regulation within the tissue microenvironment.

Animals↗

PubMed1997

Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins.

The adsorption of proteins with net positive charges (pI > pH) on the walls of fused-silica capillaries is a common problem in the analysis of proteins by capillary electrophoresis. This paper explores the use of polycationic polymers as noncovalent coatings to limit this problem. The behavior of three sets of proteins was compared using uncoated and coated capillaries: (i) a protein charge ladder obtained by acetylation of lysozyme (EC 3.2.1.17); (ii) a protein charge ladder obtained by acetylation of carbonic anhydrase II (EC 4.2.1.1); (iii) a test panel of proteins with a range of values of molecular weight and pI. Four polycationic polymers were examined: polyethylenimine (PEI; MWav = 15000), Polybrene (MWav = 25000), poly(methoxyethoxyethyl)ethylenimine (MWav = 64000), and poly(diallyldimethylammonium chloride) (MWav = 10000). Detection of proteins with high pI was readily achieved using the first three of these polycationic polymer coatings but not with the poly(diallyldimethyl-ammonium chloride). Examination of the stability of these coatings indicates that they are robust: the change in electroosmotic flow was less than 10% for 25 replications of the same separations, using capillaries coated with PEI or Polybrene. This study demonstrates that the charge ladder obtained by acetylation of lysozyme is a good model with which to test the efficiency of polycationic coatings. A study of the electrophoretic mobilities of the members of this charge ladder at pH 8.3 determined the effective charge of lysozyme (Zp(0) = +7.6 +/- 0.1) and established the acidity of the alpha-ammonium group of lysozyme (pKa = 7.8 +/- 0.1). Results from the test panel of proteins suggest that protein adsorption is mainly driven by electrostatic interactions.

Acetylation↗

PubMed1997

Using protein charge ladders to estimate the effective charges and molecular weights of proteins in solution.

This paper describes the use of capillary electrophoresis (CE) and protein charge ladders to estimate values of effective charge (Z) and molecular weight of proteins under nondenaturing conditions. A panel of 14 proteins with a range of charges and shapes was modified by acetylation with acetic anhydride to yield protein charge ladders. A protein charge ladder is a family of derivatives of a protein that differ in integral units of charge, but minimally in hydrodynamic drag; this mixture of proteins appears in electrophoresis as a set of peaks with regular spacings. Analysis of the electrophoretic mobilities of the members of these charge ladders yields values of Z and electrophoretic coefficients: for a description of mobility based on the equation mu = CPZ(MW)-alpha, CP = 6.3 cm2 min-1 kV-1 charge-1 kD0.48, alpha = 0.48; for mu = C(r)Z[r(1 + kappa r)]-1, C(r) = 55 cm2 min-1 kV-1 charge-1 A (r is the spherical radius of the protein and kappa is a function of ionic strength). The primary usefulness of charge ladders is in measuring the effective charge, Z, of proteins in solution; this information is difficult to obtain by any other procedure. A secondary value of the method is to estimate values of molecular weight. Although less general and convenient than SDS-PAGE, this method allows estimates of molecular weight of nondenatured proteins and is thus applicable to oligomers, noncovalent aggregates, proteins with multiple, non-cross-linked chains, and other systems to which SDS-PAGE is not applicable. The values of molecular weight calculated using the electrophoretic mobilities of proteins in solution and the above constants agreed with literature values to within 20% (with an ambiguous result for ovalbumin). A combination of this technique and SDS-PAGE will be useful in estimating the number of subunits or stage of aggregation of proteins in solution.

Acetic Anhydrides↗

PubMed1997

A miniaturized arrayed assay format for detecting small molecule-protein interactions in cells.

BACKGROUND: Two complementary approaches to studying the cellular function of proteins involve alteration of function either by mutating protein-encoding genes or by binding a small molecule to the protein. A mutagen can generate millions of genetic mutations; correspondingly, split-pool synthesis can generate millions of unique ligands attached to individual beads. Genetic screening of mutations is relatively straightforward but, in contrast, split-pool synthesis presents a challenge to current methods of screening for compounds that alter protein function. The methods used to screen natural products are not feasible for large libraries composed of covalently immobilized compounds on synthesis beads. The sheer number of compounds synthesized by split-pool synthesis, and the small quantity of individual compound attached to each bead require assay miniaturization for efficient screening. RESULTS: We present a miniaturized cell-based technique for the screening of ligands prepared by split-pool synthesis. Spatially defined droplets with uniform volumes of approximately 50-150 nanoliters (depending on well dimensions) are arrayed on plastic devices prepared using a combination of photolithography and polymer molding. Using this microtechnology, approximately 6,500 assays using either yeast cells or mammalian tissue culture can be performed within the dimensions of a standard 10 cm petri dish. We demonstrate that the biological effect of a small molecule prepared by split-pool synthesis can be detected in this format following its photorelease from a bead. CONCLUSIONS: The miniaturized format described here allows uniformly sized nanodroplets to be arrayed on plastic devices. The design is amenable to a large number of biological assays and the spatially arrayed format ensures uniform and controlled ligand concentrations and should facilitate automation of assays. The screening method presented here provides an efficient means of rapidly screening large numbers of ligands made by split-pool synthesis in both yeast and mammalian cells.

Animals↗

PubMed1997

Stimulation of human cytotoxic T cells with HIV-1-derived peptides presented by recombinant HLA-A2 peptide complexes.

HLA-A2 heavy chain and beta 2-microglobulin were expressed in Escherichia coli, and refolded in the presence of peptides derived from HIV-1 RT and gag proteins. When recombinant HLA-A2 molecules were attached to cells lacking HLA-A2, the cells became susceptible to lysis by HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones specific for peptides derived from RT and gag proteins. Limiting dilution analyses of peripheral blood mononuclear cells from HIV-1-infected individuals showed that the recombinant HLA-A2 peptide complexes covalently immobilized on microspheres stimulated the development of HLA-A2 peptide-specific CTL. Preformed HLA-peptide complexes may provide an alternative to immunization procedures that depend upon intracellular processing of antigen to elicit T cell responses.

Antigen Presentation↗

PubMed1996

Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold.

This paper describes a method based on experimentally simple techniques--microcontact printing and micromolding in capillaries--to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. The technique uses replica molding in poly(dimethylsiloxane) molds having micrometer-scale relief patterns on their surfaces to form a contoured film of polyurethane supported on a glass slide. Evaporation of a thin (< 12 nm) film of gold on this surface-contoured polyurethane provides an optically transparent substrate, on which SAMs of terminally functionalized alkanethiolates can be formed. In one procedure, a flat poly(dimethylsiloxane) stamp was used to form a SAM of hexadecanethiolate on the raised plateaus of the contoured surface by contact printing hexadecanethiol [HS(CH2)15CH3]; a SAM terminated in tri(ethylene glycol) groups was subsequently formed on the bare gold remaining in the grooves by immersing the substrate in a solution of a second alkanethiol [HS(CH2)11(OCH2CH2)3OH]. Then this patterned substrate was immersed in a solution of fibronectin, the protein adsorbed only on the methyl-terminated plateau regions of the substrate [the tri(ethylene glycol)-terminated regions resisted the adsorption of protein]; bovine capillary endothelial cells attached only on the regions that adsorbed fibronectin. A complementary procedure confined protein adsorption and cell attachment to the grooves in this substrate.

Animals↗

PubMed1996

Screening derivatized peptide libraries for tight binding inhibitors to carbonic anhydrase II by electrospray ionization-mass spectrometry.

This paper describes the use of electrospray ionization-mass spectrometry (ESI-MS) to screen two libraries of soluble compounds to search for tight binding inhibitors for carbonic anhydrase II (EC 4.2.1.1). The two libraries, H2NO2SC6H4C(O)NH-AA1-AA2-C(O)NHCH2CH2CO2H where AA1 and AA2 are L-amino acids (library size: 289 compounds) or D-amino acids (256 compounds), were constructed by attaching tripeptides to the carboxyl group of 4-carboxybenzenesulfonamide. Screening of both libraries yielded, as the tightest binding inhibitor, compound 1 (AA1 = AA2 = L-Leu; binding constant Kb = 1.4 x 10(8) M-1). The ability of ESI-MS to estimate simultaneously the relative binding affinities of a protein to soluble ligands in a library, if general, should be useful in drug development.

Amino Acid Sequence↗

PubMed1996

Evaluating electrostatic contributions to binding with the use of protein charge ladders.

Electrostatic interactions between charges on ligands and charges on proteins that are remote from the binding interface can influence the free energy of binding (delta Gb). The binding affinities between charged ligands and the members of a charge ladder of bovine carbonic anhydrase (CAII) constructed by random acetylation of the amino groups on its surface were measured by affinity capillary electrophoresis (ACE). The values of delta Gb derived from this analysis correlated approximately linearly with the charge. Opposite charges on the ligand and the members of the charge ladder of CAII were stabilizing; like charges were destabilizing. The combination of ACE and protein charge ladders provides a tool for quantitatively examining the contributions of electrostatics to free energies of molecular recognition in biology.

Acetylation↗

PubMed1996

A self-assembled monolayer for the binding and study of histidine-tagged proteins by surface plasmon resonance.

This paper reports the generation of a self-assembled monolayer (SAM) that selectively binds proteins whose primary sequence terminates with a His-tag: a stretch of six histidines commonly incorporated in recombinant proteins to simplify purification. The SAM was prepared by the adsorption onto a gold surface of a mixture of two alkanethiols: one thiol that terminated with a nitrilotriacetic acid (NTA) group, a group that forms a tetravalent chelate with Ni(II), and a second thiol that terminated with a tri(ethylene glycol) group, a group that resists protein adsorption. His-tagged proteins bound to the SAM by interaction of the histidines with the two vacant sites on Ni(II) ions chelated to the surface NTA groups. Studies with model proteins showed the binding was specific for His-tagged proteins and required the presence of Ni(II) on the surface. Immobilized His-tagged proteins were kinetically stable in buffered saline at pH 7.2 but could be desorbed by treatment with 200 mM imidazole. Surface plasmon resonance studies for two model systems showed that His-tagged proteins adsorbed on the NTA-SAM retained a greater ability to participate in binding interactions with proteins in solution than protein immobilized in a thin dextran gel layer by covalent coupling.

Histidine↗

PubMed1996

Optically controlled collisions of biological objects to evaluate potent polyvalent inhibitors of virus-cell adhesion.

BACKGROUND: The biochemical and biomechanical determinants of adhesion between two biological objects following a collision are complex, and may vary from one system to another. We wished to develop an assay in which all the relevant factors, including the components of the solution, the relative orientation and the relative collision velocity, are under the user's control. RESULTS: A new assay is described in which two mesoscale particles are caused to collide using two independently controlled optical tweezers (optically controlled collision, OPTCOL). This assay enables precise examination of the probability of adhesion under biologically relevant conditions. The OPTCOL assay was used to evaluate the probability of adhesion of a single erythrocyte to a single virus-coated microsphere, in the absence and presence of a sialic acidbearing inhibitor. Inhibition constants for the most effective inhibitors could not be measured using other types of assays. The best inhibitor prevented attachment 50 % of the time at a sialic acid concentration of 35 pmol l-1; it is the most potent known inhibitor of attachment of influenza virus to erythrocytes. CONCLUSIONS: OPTCOL is a versatile new bioassay for studying dynamic interactions in biochemistry. It offers an approach to investigating interactions between moving biological objects that is both quantitative and interpretable. The simplicity of the OPTCOL technique suggests broad applicability to the study of adhesion of mesoscale (1-100 microm) objects in the areas of cell biology, microbiology, medicinal chemistry, and biophysics.

Cell Adhesion↗