PubMed Health⌕ Search

Biomedical subjects

V Hlady

Publications and source records attributed to V Hlady.

15 recordsLinked to original sources
PubMed1998

Relative importance of surface wettability and charged functional groups on NIH 3T3 fibroblast attachment, spreading, and cytoskeletal organization.

Understanding the relationships between material surface properties, adsorbed proteins, and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In this study, we have prepared model surfaces with different functional groups to provide a range of surface wettability and charge. The cellular responses of attachment, spreading, and cytoskeletal organization have been studied following preadsorption of these surfaces with dilute serum, specific serum proteins, and individual components of the extracellular matrix. When preadsorbed with dilute serum, cell attachment, spreading, and cytoskeletal organization were significantly greater on hydrophilic surfaces relative to hydrophobic surfaces. Among the hydrophilic surfaces, differences in charge and wettability influenced cell attachment but not cell area, shape, or cytoskeletal organization. Moderately hydrophilic surfaces (20-40 degree water contact angle) promoted the highest levels of cell attachment. Preadsorption of the model surfaces with bovine serum albumin (BSA) resulted in a pattern of cell attachment very similar to that observed following preadsorption with dilute serum, suggesting an important role for BSA in regulating cell attachment to biomaterials exposed to complex biological media.

3T3 Cells↗
PubMed1997

Adsorption Kinetics, Conformation, and Mobility of the Growth Hormone and Lysozyme on Solid Surfaces, Studied with TIRF

Interactions of recombinant human growth hormone and lysozyme with solid surfaces are studied using total internal reflection fluorescence (TIRF) and monitoring the protein's intrinsic tryptophan fluorescence. The intensity, spectra, quenching, and polarization of the fluorescence emitted by the adsorbed proteins are monitored and related to adsorption kinetics, protein conformation, and fluorophore rotational mobility. To study the influence of electrostatic and hydrophobic interactions on the adsorption process, three sorbent surfaces are used which differ in charge and hydrophobicity. The chemical surface groups are silanol, methyl, and quaternary amine. Results indicate that adsorption of hGH is dominated by hydrophobic interactions. Lysozyme adsoption is strongly affected by the ionic strength. This effect is probably caused by an ionic strength dependent conformational state in solution which, in turn, influences the affinity for adsorption. Both proteins are more strongly bound to hydrophobic surfaces and this strong interaction is accompanied by a less compact conformation. Furthermore, it was seen that regardless of the characteristics of the sorbent surface, the rotational mobility of both proteins' tryptophans is largely reduced upon adsorption.

Journal Article↗
PubMed1996

Protein adsorption on solid surfaces.

The research field of protein adsorption on surfaces appears to be as popular as ever. In the past year, several hundred published papers tackled problems ranging from fundamental aspects of protein surface interactions to applied problems of surface blood compatibility and protein surface immobilization. Although some parts of the protein adsorption process, such as kinetics and equilibrium interactions, can be accurately predicted, other aspects, such as the extent and the rate of protein conformational change, are still somewhat uncertain. The whole field is ripe for a comprehensive theory on protein adsorption.

Journal Article↗
PubMed1996

A constant compliance force modulation technique for scanning force microscopy (SFM) imaging of polymer surface elasticity.

A new method of force modulation scanning force microscopy (SFM) imaging based on a constant compliance feedback loop is presented. The feedback adjusts the loading force applied by the SFM tip to the surface in order to maintain a constant compliance beneath the tip. The new method, constant compliance force modulation (CCFM), has the advantage of being able to quantify the loading force exerted by the tip onto the sample surface and thus to estimate the elastic modulus of the material probed by the SFM tip. Once the elastic modulus of one region is known, the elastic moduli of other surface regions can be estimated from the spatial map of loading forces using the Hertz model of deformation. Force vs. displacement measurements made on one surface locality could also be used to estimate the local modulus. Several model surfaces, including a rubber-toughened epoxy polymer blend which showed clearly resolved compliant rubber phases within the harder epoxy matrix, were analyzed with the CCFM technique to illustrate the method's application.

Compliance↗
PubMed1995

Fluorescence assay for measuring lipid deposits on contact lens surfaces.

A simple fluorescence assay for estimating the propensity of the contact lens surface towards the uptake of lipids and formation of lipid deposits has been developed. The assay is based on the incubation of the contact lenses with an artificial tear-lipid mixture, staining of the lenses with a fluorescent lipid probe, Nile Red, and subsequent macroscopic imaging of the fluorescent lipid deposits on the contact lens surface with a thermoelectrically cooled charge-coupled device (CCD) camera. The results of the assay show that statistically significant differences in the formation of lipid deposits exist between the surfaces of different contact lens materials.

Computer Simulation↗
PubMed1995

Effect of Tamm-Horsfall protein on calcium oxalate precipitation.

The effect of Tamm-Horsfall protein isolated from urine of healthy subjects on calcium oxalate precipitation was studied in model systems of precipitation. The study was performed using following conditions: concentrations of calcium chloride 10 mmol/l, sodium chloride 150 mmol/l, oxalic acid 300 mumol/l; pH 6.0, and temperature 310 K. The concentration of Tamm-Horsfall protein varied between 1-10 mg/l. The kinetics of calcium oxalate precipitation was observed by measuring the number and volume of particles in the suspension, and the precipitate composition by an optic microscope. In all the studied systems, the precipitate morphology corresponded to pure calcium oxalate monohydrate. Tamm-Horsfall protein was found to inhibit the growth of calcium oxalate monohydrate crystals and stimulate their aggregation in the given experimental conditions. Both effects were enhanced by increase in the concentrations of Tamm-Horsfall protein and were most pronounced at the concentration of Tamm-Horsfall protein of 10 mg/l.

Adult↗
PubMed1992

Adsorption of complement proteins on surfaces with a hydrophobicity gradient.

Activation of the complement system is recognized as one of the major problems with respect to biocompatibility of biomaterials. The binding of C3 (central component of complement) and B (factor B, an activator of C3), and H (factor H, an inhibitor of C3 activation) plays a crucial role in the activation of the alternative pathway of complement on the surfaces of biomaterials during extracorporeal procedures. Here we report on the adsorption of C3, B or H on to the silica surface with a hydrophobicity gradient. The amount of native 125I-C3 bound to both hydrophilic and hydrophobic surfaces was very similar (0.8 and 0.9 micrograms/cm2; 4 x 10(-12) mol/cm2). Neither factor H nor factor B was able to displace already adsorbed 125I-C3 from either of the surfaces. The extent of binding of factors B and H to preadsorbed C3 was a function of the surface hydrophobicity: more 125I-B or 125I-H was bound to C3 adsorbed at the hydrophilic end than at the hydrophobic end of the gradient surface. The binding of 125I-B or 125I-H to preadsorbed C3 appeared to be influenced by the availability of their binding sites on adsorbed C3 molecules rather than by the amount of surface-bound C3. At the hydrophobic end of the gradient surface the molar binding ratio of B/C3 was considerably smaller than the molar binding ratio of H/C3. It can be speculated that the hydrophobicity of the surface determines orientation and/or conformation of adsorbed C3 molecule; when adsorbed at the hydrophobic end of the gradient, C3 molecule predominantly exposes the binding site to which only factor H can bind.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1991

Interaction of plasma proteins with heparinized gel particles studied by high-resolution two-dimensional gel electrophoresis.

In order to further the understanding of protein-surface interactions in the coagulation system, we have chosen to study plasma protein adsorption onto heparin-immobilized surfaces. Heparin-binding proteins are abundant in plasma: a search of amino acid sequences revealed that many plasma proteins have possible heparin binding sites. Plasma protein adsorption to the heparinized surfaces is monitored by a novel technique in which the solution depletion of proteins is analytically determined using quantitative two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). This method enables simultaneous, quantitative detection of the majority of plasma proteins before, during, and after their adsorption onto high surface area adsorbents. Using computerized densitometry of silver-stained 2-D PAGE gels, the amount of each protein can be determined from the integrated optical density of each protein "spot." Kinetics of adsorption and adsorption isotherms of four important heparin binding proteins, antithrombin III (ATIII), complement factor C3 (C3), apolipoprotein AI (Apo-AI) and apolipoprotein AIV (Apo-AIV) are reported in this paper. From the adsorption isotherms, the apparent binding constants of each protein-immobilized heparin complex, Ka, were calculated. The surface binding constants were of the same order of magnitude as the respective solution binding constants in the literature. The surface binding constants followed the same order as the respective solution binding constants: Ka (ATIII) greater than Ka (Apo-AIV) greater than Ka (C3) greater than Ka (Apo-AI), indicating that protein binding to the immobilized heparin used is not essentially different from solution binding.

Adsorption↗
PubMed1991

Vroman effects, techniques, and philosophies.

Leo Vroman's work on blood-materials interaction over the years has motivated and influenced much of our work in this field. Here we show how most of our studies on proteins at interfaces can be traced to Vroman's ideas presented in Blood over 25 years ago. Specifically, we briefly discuss simple proteins at simple interfaces, complex interfaces, complex proteins at interfaces, multi-parameter phenomena, and scientific communication and education.

Biocompatible Materials↗
PubMed1990

Spatially resolved detection of antibody-antigen reaction on solid/liquid interface using total internal reflection excited antigen fluorescence and charge-coupled device detection.

Spatially-resolved detection of antibody-antigen reactions at the solid/liquid interface was investigated by total internal reflection excited fluorescence from large area flat surfaces. Anti-HSA immunoglobulin G (IgG) antibody was immobilized at four spatially distinct spots. Binding of fluorescein-labeled human serum albumin (HSA) from the solution to immobilized antibody was detected by a cooled charge-coupled device (CCD) as a charge in the fluorescence intensity. A two-dimensional representation of the fluorescence was obtained during the binding reaction time of 25 mins. The contributions from bound and free antigen to the total signal were evaluated. The influence of the scattered excitation light and the normalization of fluorescence signal with respect to the two-dimensional incident light intensity distribution are discussed.

Antigen-Antibody Reactions↗
PubMed1987

Plasma protein adsorption: the big twelve.

We have discussed the general principles of protein adsorption at solid-liquid interfaces from single component and multicomponent solutions, based on qualitative kinetic models that include mass transport considerations, initial interaction energies, surface-dependent conformational changes, and possible desorption processes. We have surveyed plasma protein components greater than one milligram per milliliter in concentration, which we call "The Big Twelve." We considered their size, concentration, diffusion coefficient, structure and function, and methods of estimating their "surface denaturability" by using bulk solution measures of denaturation and conformational change. We have suggested that the role of the carbohydrate moieties in plasma proteins may have some bearing on their adsorption properties. We further suggest that lipoproteins, because of their lipid phase transition and conformational lability at body temperature, may tend to dominate the adsorption process, particularly on mobile elastomeric polymer surfaces. We suggest that detailed consideration of the structure and characteristics of each of the proteins involved is necessary in order to begin to understand plasma adsorption processes. Detailed characterization and understanding of the solid surface in the aqueous and protein environments are also required.

Adsorption↗
PubMed1987

Simultaneous measurement of calcium transients and motion in cultured heart cells.

The fluorescent Ca2+ probe indo-1 is a new intracellular Ca2+ concentration [( Ca2+]i) indicator that may be suitable for measurement of [Ca2+]i transients in intact heart cells. We exposed spontaneously contracting cultured chick embryo ventricular cells (37 degrees C) to the membrane-permeable indo-1-acetoxymethyl ester (indo-1 AM). Indo-1 loading was associated with a decrease in the amplitude of contraction measured with a video motion detector, but contractility returned to control levels during a subsequent 30-min wash. Analysis of emission spectra of dye obtained by digitonin permeabilization of cells loaded in indo-1 AM showed that the active intracellular dye was not pure indo-1 but probably includes partially deesterified molecules. With the use of an inverted X40 objective epifluorescence system, washed cells containing indo-1 were excited at 360 nm, and fluorescence intensity was measured at 410 nm (increases with increasing [Ca2+]) and 480 nm (decreases with increasing [Ca2+]). Calibration of the [Ca2+]i signals, reflected by the ratio of 410 to 480 nm fluorescence, was achieved by use of ethylen-glycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-Ca2+ buffered solutions containing the nonfluorescent Ca2+ ionophore Bromo-A23187. Average end-diastolic and peak-systolic [Ca2+]i were 328 +/- 32 and 813 +/- 72 nM (means +/- SE, n = 8). The onset of the [Ca2+]i transient preceded motion by 27 +/- 5 ms (means +/- SE, n = 4), but generally resembled the motion signals in contour. These findings indicate that indo-1 may be used to detect [Ca2+]i transients in isolated ventricular cells without causing significant alterations in mechanical performance.

Animals↗
PubMed1986

Human haptoglobin adsorption by a total internal reflection fluorescence method.

Haptoglobin (Hp) is one of the major protein constituents of plasma. Three different forms are found in the population. The 1-1 and 2-2 forms adsorb similarly onto hydrophobic silica [treated with dimethyl dichlorosilane (DDS)] and onto clean silica, although the affinities on the silica surface are lower at 60 minutes contact time. The two forms desorb differently from silica, but desorb similarly from DDS-silica. Adsorption is less reversible on the hydrophobic surface. Due to its adsorbtion tendencies and its high concentration in plasma, the adsorption of Hp may be important in blood interaction at solid-liquid interfaces.

Adsorption↗
PubMedDate not supplied

Human low density lipoprotein and human serum albumin adsorption onto model surfaces studied by total internal reflection fluorescence and scanning force microscopy.

The adsorption of low density lipoprotein (LDL) and human serum albumin (HSA) to model surfaces of different hydrophobicities has been studied using two, surface-sensitive, real-time, in situ techniques: total internal reflection fluorescence (TIRF) and scanning force microscopy (SFM). The model surfaces used were: (1) hydrophilic negatively charged silica (TIRF) and mica (SFM) surfaces, (2) hydrophobic octadecyldimethylsilyl-(ODS)-modified silica (TIRF) and ODS-modified oxidized silicon (SFM) surfaces and (3) amphiphilic ODS-silica gradient surfaces (TIRF). The kinetics of fluorescein isothiocyanate-LDL adsorption onto the ODS-silica gradient surface from FITC-LDL solution and from a solution mixture of LDL and HSA showed that a transport-limited process on the clean silica changed into an adsorption-limited process with increasing surface coverage of ODS chains. SFM analysis of the in situ adsorption of LDL on hydrophilic mica demonstrated a steady increase in surface coverage with time which was somewhat lower than determined by TIRF for FITC-LDL adsorption on silica. The adsorption behavior of a binary mixture of HSA and LDL suggested that lateral interactions between HSA and LDL affect the adsorption process. The diameter of LDL adsorbed on mica and ODS-modified silicon has been determined using SFM to be approximately 55 nm. Tetrameric LDL aggregates were observed on all of the surfaces in addition to some dimers and trimers. Imaging LDL and HSA adsorption on clean oxidized silicon surfaces using "contact mode' SFM techniques was hindered by probe manipulation of the proteins.

Adsorption↗