Biomedical subjects

A S Hoffman

Publications and source records attributed to A S Hoffman.

At least 19 recordsLinked to original sources

PubMed1995

Control of protein-ligand recognition using a stimuli-responsive polymer.

Stimuli-responsive polymers exhibit reversible phase changes in response to changes in environmental factors such as pH or temperature. Conjugating such polymers to antibodies and proteins provides molecular systems for applications such as affinity separations, immunoassays and enzyme recovery and recycling. Here we show that conjugating a temperature-sensitive polymer to a genetically engineered site on a protein allows the protein's ligand binding affinity to be controlled. We synthesized a mutant of the protein streptavidin to enable site-specific conjugation of the responsive polymer near the protein's binding site. Normal binding of biotin to the modified protein occurs below 32 degrees C, whereas above this temperature the polymer collapses and blocks binding. The collapse of the polymer and thus the enabling and disabling of binding, is reversible. Such environmentally triggered control of binding may find many applications in biotechnology and biomedicine, such as the control of enzyme reaction rates and of biosensor activity, and the controlled release of drugs.

Acrylic Resins↗

PubMed1995

Graft copolymers that exhibit temperature-induced phase transitions over a wide range of pH.

There are many potential applications of 'intelligent' aqueous polymer systems in medicine, biotechnology, industry and in environmental problems. Many of these polymer systems undergo reversible phase transitions--for example, abrupt changes in volume--in response to external stimuli such as temperature, pH or the nature of the solvent. Most of the polymers studied previously are responsive to only one kind of stimulus. But for some applications, independent responsiveness to several factors, such as temperature and pH, may be required. Here we describe a polymer that undergoes marked solubility changes in water in response to temperature and/or pH changes. The polymer is prepared by grafting temperature-sensitive side chains onto a pH-sensitive backbone. We also find that block copolymers, in which the temperature- and pH-sensitive units alternate along the chain, show similar behaviour.

Acrylamides↗

PubMed1995

Platelet and monoclonal antibody binding to fibrinogen adsorbed on glow-discharge-deposited polymers.

The state of fibrinogen adsorbed on untreated and glow-discharge-treated surfaces was examined by measuring platelet adhesion, monoclonal antibody (mAb) binding, the amount of fibrinogen adsorbed, and the amount of adsorbed fibrinogen which could be eluted with sodium dodecyl sulfate (SDS). Tetrafluoroethylene (TFE) glow-discharge-treated polymers have a lower surface free energy (in air) and retain a larger fraction of adsorbed fibrinogen than untreated surfaces after SDS elution. Platelet adhesion was lowest on the TFE-treated surfaces which retain the highest amounts of fibrinogen after SDS elution. Fibrinogen may undergo unfolding or spreading on the TFE-treated surfaces to minimize interfacial free energy (in water) and maximize protein-surface interactions. When it is adsorbed on the TFE-treated surfaces, fibrinogen evidently assumes a state which somehow prevents its recognition and binding by platelet receptors. Monoclonal antibodies that bind to the three regions in fibrinogen thought to be involved in platelet adhesion were therefore used to detect changes in adsorbed fibrinogen. These regions and the antibodies which bind to them are: the COOH-terminal of the gamma-chain, mAb M1; the RGD peptide sequence at A alpha 95-98, mAb R1; the RGD sequence at A alpha 572-575, mAb R2. For fibrinogen adsorbed on the untreated or TFE-treated surfaces, M1 and R2 binding was relatively high compared to background, while R1 binding was low. However, the amount of binding of each mAb to fibrinogen adsorbed on the TFE-treated surfaces was equal to or greater than fibrinogen adsorbed to the untreated surfaces. Therefore, antibody-detectable changes in the platelet binding regions of adsorbed fibrinogen that might have been caused by conformational or orientational rearrangements were not observed for the TFE-treated surfaces. The data suggest that the tight binding of fibrinogen on a surface may directly affect the ability of the fibrinogen to interact with the platelet receptors--i.e., that fibrinogen must be loosely held to facilitate maximal interaction with platelet receptors.

Adhesiveness↗

PubMed1995

"Intelligent" polymers in medicine and biotechnology.

One can define "intelligent" polymers as those polymers that respond with large property changes to small physical or chemical stimuli. These polymers may be in various forms, such as in solution, on surfaces, or as solids. One may also combine intelligent aqueous polymer systems with biomolecules to yield a large family of polymers that respond intelligently to physical, chemical, or biological stimuli. This article overviews such interesting and versatile polymer systems.

Biochemical Phenomena↗

PubMed1994

Correlation between corneal epithelial cell outgrowth and monoclonal antibody binding to the cell binding domain of adsorbed fibronectin.

The ability of corneal epithelial cells to attach, spread, and migrate on synthetic surfaces is largely determined by the characteristics of the adsorbed protein layer. In previous studies we have described an in vitro model for quantitating epithelial cell outgrowth from explanted corneal buttons onto synthetic materials (Pettit et al., Invest. Ophthalmol. Vis. Sci., 31, 2269 [1990]). We have also described the role of fibronectin (fn) adsorption and binding strength on epithelial cell outgrowth (Pettit et al., J. Biomed. Mater. Res., 26, 1259 [1992]). In the current study we have used a monoclonal antibody against the RGD cell binding domain of fn (mAb 3E3) to further characterize the role of adsorbed fn in promoting epithelial cell outgrowth. Ten materials of diverse chemical and physical properties were adsorbed with fn (0.1 mg/ml) or mixtures of fn and albumin (concentrations totaling 0.1 mg/ml) and tested for antibody recognition of the cell binding domain. The surface density of bound anti-cell binding domain antibody varied from a low of 0.66 +/- 0.11 for fluorinated ethylene propylene copolymer (FEP) to a high of 1.90 +/- 0.26 for tissue culture polystyrene dish substrates (units are OD at 450 nm measured in the ELISA technique normalized to polyethylene). A general increase in cell outgrowth areas was noted, with increases in recognizable cell binding domain. However, several exceptions to this trend were noted as well (e.g., low cell outgrowth but high antibody recognizability for glass). These results suggest that, although the number of cell binding domains exposed on adsorbed fn molecules may influence cell outgrowth, other characteristics of the adsorbed protein, such as the binding strength to the underlying substrate, may be equally important in characterizing epithelial cell-substrate interactions.

Adsorption↗

PubMed1994

Novel delivery system for inducing quiescence in intestinal stem cells in rats by transforming growth factor beta 1.

BACKGROUND/AIMS: Intestinal mucosa, a tissue in a dynamic state of rapid cellular proliferation, is often adversely affected by cytotoxic drugs. The purpose of this study was to develop an oral delivery system targeting transforming growth factor (TGF) beta 1 locally and analyze its effects on the epithelial stem cells of gastrointestinal mucosa. METHODS: Rats were treated with recombinant TGF-beta 1 in alginate beads perorally or with recombinant TGF-beta 1 in phosphate-buffered saline perorally or intraperitoneally. Control animals received phosphate-buffered saline only. The size of the villi was measured. Proliferating and mitotic indices were determined by quantifying immunohistochemical staining for proliferating cell nuclear antigen. RESULTS: Alginate beads released no TGF-beta 1 in acid. However, in pH 7.4, TGF-beta 1 was released in an active form. Histomorphometrical analysis showed a marked reduction in villus height (50%-70%) in the intestinal mucosa of animals treated perorally with recombinant TGF-beta 1 in alginate beads. Also, the proliferating and mitotic indices were significantly reduced (P < 0.01) in these animals as compared with controls and other routes of administration. CONCLUSIONS: This study shows that recombinant TGF-beta 1 administered using a novel oral delivery system induces stem cell quiescence in the intestinal mucosa of the rat.

Administration, Oral↗

PubMed1994

The stabilization of a human IgM monoclonal antibody with poly(vinylpyrrolidone).

An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50 degrees C. A variety of excipients was tested for their ability to inhibit antibody aggregation. The amount of 4B9 aggregation, which was determined by analysis on a size-exclusion HPLC, was significantly reduced in the presence of low concentrations [between 0.1 and 1.0% (w/v)] of poly(vinylpyrrolidone) (PVP) molecules ranging in molecular weight from 10 to 40 kDa. When the PVP concentration was greater than 1.0%, antibody aggregation was enhanced, and with the highest molecular weight PVP, antibody precipitation occurred. HPLC was used to show that more PVP was associated with the 4B9 at 50 degrees C than at 25 degrees C. Differential scanning calorimetry revealed that PVP concentrations greater than 2.0% decreased the antibody thermal transition temperature. Enzyme-linked immunosorbent assays were used to assess the effects of PVP on the antigen binding capacity of 4B9 and on 4B9 quantitation. At 4 degrees C, PVP solutions of up to 5.0% had no effect on either 4B9 quantitation or antigen binding. At 50 degrees C, however, less 4B9 was detected in the 5.0% PVP solution. The heat stabilization of the 4B9 antibody by low concentrations of PVP can be explained by a weak binding of PVP to the native protein. The PVP may sterically interfere with protein-protein interactions, thus reducing aggregation. Higher concentrations of PVP lead to protein aggregation and precipitation, probably by a volume-exclusion mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1994

Synthesis of carboxylated poly(NIPAAm) oligomers and their application to form thermo-reversible polymer-enzyme conjugates.

A thermo-reversible poly(N-isopropylacrylamide) poly(NIPAAm) oligomer with a carboxyl functional end group has been synthesized by radical polymerization using beta-mercaptopropionic acid as a chain transfer reagent. This polymer has been conjugated to an enzyme, beta-D-glucosidase, to form a thermo-reversible water soluble-insoluble polymer-enzyme conjugate. This conjugate can be used for separation, recovery and recycle of an enzyme simply by applying small temperature changes to the reaction medium. In contrast to the random polymer-enzyme conjugates reported in the literature, in this study the enzyme is coupled to each polymer chain by a single end attachment. These preliminary studies show that the conjugated enzyme exhibits very high retention of activity (> 90%) compared to the native enzyme and shows improved thermal stability.

Acrylic Resins↗

PubMed1994

Activated, N-substituted acrylamide polymers for antibody coupling: application to a novel membrane-based immunoassay.

A room-temperature-precipitable, activated terpolymer consisting of N-isopropylacrylamide (NIPAAm)/N-n-butylacrylamide(nBAAm)/N-acryloxysuccinimide (NASI) (LCST = 7-13 degrees C) at a monomer feed ratio of 60:40:2.5, respectively, was prepared and conjugated to an antibody. The conjugate was evaluated in a novel cellulose acetate (CA) membrane-based immunoassay which utilizes the especially strong physical attachment of the polymer to CA to bind and concentrate the polymer attached protein onto the membrane. When compared in the CA membrane immunoassay to the antibody-poly(NIPAAm) conjugate prepared via anhydrous copolymerization of NIPAAm and NASI at the monomer feed ratio of 40:1, respectively, the performance of the NIPAAm/nBAAm/NASI terpolymer was superior to that of the NIPAAm/NASI copolymer (LCST = 32 degrees C) when the studies were carried out at room temperature. However, the terpolymer and copolymer gave equivalent performance when the assay mixture was heated to 45 degrees C. These results indicate the importance of the LCST of the polymer component of the Ab-polymer conjugate to its adsorption and binding on the CA membrane.

Acrylamides↗

PubMed1994

Dextran permeation through poly(N-isopropylacrylamide) hydrogels.

The permeation of macromolecules such as fluoroescein-labeled dextran fractions through thermally reversible hydrogels has been investigated. A permeation model has been formulated, which takes into account hydrogel porosity and tortuosity as well as the combined effect of a geometric restraint for a relatively large solute molecule at a pore entrance and the friction between solute molecules moving through the pores and pore walls. Based on this model, we have estimated the tortuosity and average pore size of a swollen hydrogel, poly(N-isopropylacrylamide) [poly(NIPAAm)] and a swollen heterogel, poly(N-isopropylacrylamide-co-vinyl-terminated dimethylsiloxane) [poly(NIPAAm-co-VTPDMS)]. The permeation data for dextran molecules up to the size of 43.5 A in radius show good agreement with the values predicted from the model.

Acrylic Resins↗

PubMed1994

Temperature-dependent absorption/desorption behavior of lower critical solution temperature (LCST) polymers on various substrates.

We have been studying adsorption and retention (resistance to desorption) behavior of temperature sensitive LCST polymers on different substrates as a function of temperature. According to our studies with Poly 64 (a copolymer of 60% (mol) NIPAAm and 40% (mol) NnBAAm, LCST = 8.5 degrees C in water), the copolymer retention depends on the rinse temperature. When the rinse temperature is above the LCST, the polymer adheres well to most surfaces. On the contrary, at rinse temperatures below the LCST, most of the adsorbed polymer is easily rinsed off. These studies are relevant to our work on the thermally reversible adsorption of LCST polymers conjugated to peptides and proteins, such as affinity ligands, for uses in immunoassays and affinity separations. The interaction between the LCST polymer and most hydrophobic polymer surfaces is mainly due to hydrophobic interactions, and the critical surface tension (gamma c) and the solubility parameter (delta) of the solid polymer substrate are the most important factors which influence the LCST polymer adsorption and retention. The critical surface tension appears to correlate best with the LCST polymer adsorption levels on different substrates, while the solubility parameter correlates best with the retention of the adsorbed polymer. According to our preliminary study, n-butyl groups probably interact more strongly with the substrates than isopropyl groups because of the greater hydrophobic surface area of the former groups.

Acrylamides↗

PubMed1994

Effects of branching and molecular weight of surface-bound poly(ethylene oxide) on protein rejection.

To understand better the origin of protein rejection observed with surface-bound poly(ethylene oxide) (or PEO), we have measured fibrinogen adsorption for a series of linear and branched, low-molecular-weight PEOs bound to solid polystyrene surfaces. The results show that a dependence on molecular weight is found below 1500 g mol-1 for linear PEO. Branched PEOs are less effective at protein rejection than linear PEOs. The branched PEOs have smaller exclusion volumes (from GPC) than the corresponding linear PEOs, consistent with restriction in conformational freedom for the branched compounds. The protein rejection results are interpreted in terms of entropy changes that result upon protein adsorption. In addition, some practical problems in preparation of PEO glycidyl ethers have been clarified, thus making these PEO derivatives more useful for surface modification.

Adsorption↗

PubMed1994

Attempts to stabilize a monoclonal antibody with water soluble synthetic polymers of varying hydrophobicity.

Proteins are subject to a variety of physical and chemical reactions that lead to a loss of activity. These reactions are a particular problem in controlled-release devices, where temperatures and protein concentrations are high. Current approaches to increasing protein stability include the addition of saccharides, amino acids, or polymers. New synthetic polymers may be promising protein stabilizers because properties such as molecular weight and side-chain composition can be controlled. In this study, the stability of a murine monoclonal antibody, BR96, was evaluated in solution at 37 degrees C. The antibody was incubated in the presence of a series of synthetic polymers that included poly(glucosylethyl methacrylate) (GEMA) and copolymers of N-vinylpyrrolidone (NVP) and methyl methacrylate (MMA). Samples were taken periodically up to 30 days. The formation of precipitated antibody in particulate aggregates was measured with a Coulter counter, and the molecular-weight distribution of soluble antibody was measured by size-exclusion chromatography. Two trends were evident. First, with poly(GEMA) and copolymers of NVP and MMA, protein aggregation increased at higher polymer concentrations. Second, higher molecular weights of the poly(NVP) homopolymer also led to increases in protein aggregation. Effects of polymer hydrophobicity were more complex. A copolymer containing 9 mol% MMA caused immediate protein precipitation, while a copolymer containing 21 mol% MMA did not. The effects of the copolymer containing 21% MMA were strongly concentration dependent. At 1 wt%, the polymer reduced aggregation, but aggregation increased strongly between concentrations of 2 and 3 wt%.

Animals↗

PubMed1994

Formation of poly(glucosyloxyethyl methacrylate)-concanavalin A complex and its glucose-sensitivity.

The complex formation between Concanavalin A (Con A) and a polymer having pendant glucose groups was studied in order to design a glucose-sensitive polymer. The polymer having pendant glucose (poly(glucosyloxyethyl methacrylate) or (poly(GEMA)) forms a complex with Con A in tris HCl buffer (pH = 7.5). The solution then becomes turbid due to the multiple associations between poly(GEMA) and Con A. When free glucose or mannose are added to the turbid solution, the solution becomes transparent again. However, the addition of galactose does not cause the solution to be transparent. This indicates that Con A prefers to form a complex with free glucose or mannose (but not galactose) rather than with the pendant glucose in poly(GEMA). Therefore, the complex between poly(GEMA) and Con A is expected to be glucose- and mannose-sensitive. The apparent dissociation constants of the complexes between saccharide (poly(GEMA), glucose, and mannose) and Con A were also determined by affinity electrophoresis.

Biosensing Techniques↗

PubMed1993

Thermal cycling effects on the bioreactor performances of immobilized beta-galactosidase in temperature-sensitive hydrogel beads.

The enzyme beta-galactosidase was immobilized in thermally reversible hydrogel beads that exhibit a reversible expansion and collapse of the gel volume in response to temperature. The kinetic performances of immobilized enzyme bead reactors were studied during thermal cycling operation. A periodic cycling of temperature for the packed-bed reactor induced a cyclic swelling and deswelling of the hydrogel beads. A temperature cycling operation around the phase transition temperature of the gel matrix enhanced the overall enzymatic conversion of the substrate, compared to its upper and lower isothermal operations. The effects on the overall conversion of various thermal cycling operational conditions, such as cycling range and heating and cooling rates, were investigated.

Biotechnology↗

PubMed1993

The feasibility of smoking bans on psychiatric units.

We conducted a prospective study of a smoking ban on a general inpatient psychiatry service in response to staff concerns about the feasibility of a proposed hospital-wide ban. Demographic information, smoking history, and DSM III-R diagnoses were obtained for consecutively admitted patients during two study conditions: smoking and nonsmoking. A log of p.r.n. medication, seclusion, restraint, elopement, incident reports, and smoking-related discharges was kept for each patient. Chi-square analysis of 232 patients for whom demographic, smoking, diagnostic, and log data were complete showed no significant differences between study conditions for demographic or diagnostic variables. Two-tailed t-test analysis of the log data for these 232 patients showed no significant difference in disruptive incidents during smoking and nonsmoking conditions (p = 0.183). Fifty staff members answered pre- and post-ban questionnaires. Paired t-test analysis demonstrated a significant change in staff attitude toward supporting the ban. These data indicate that smoking can be stopped on inpatient psychiatry units without increases in unit disruption or adverse effects on staff morale.

Adult↗

PubMed1993

Synthesis and characterization of a soluble, temperature-sensitive polymer-conjugated enzyme.

The enzyme, alkaline phosphatase, has been conjugated to a temperature-sensitive polymer which exhibits a lower critical solution temperature (LCST). A series of copolymers containing different molar ratios of N-isopropylacrylamide(NIPAAm) and N-acryloxysuccinimide(NAS) were synthesized and then conjugated to the enzyme. These polymer-enzyme conjugates precipitate and flocculate in aqueous solution above the LCST, and redissolve when cooled below that temperature. The kinetics of the conjugated enzymes have been characterized as a function of temperature and compared to free enzyme. The effect of the conjugation degree between polymer and enzyme on the activity of the conjugated enzymes was also investigated.

Acrylates↗

PubMed1993

Introduction of amine groups on poly(ethylene) by plasma immobilization of a preadsorbed layer of decylamine hydrochloride.

In order to introduce amine groups on poly(ethylene) (PE) surface, PE surfaces were preadsorbed with decylamine hydrochloride (DA.HCl) and subsequently treated with an argon plasma. It was shown by XPS (X-ray Photoelectron Spectroscopy), that approximately half of the preadsorbed (mono)layer was immobilized and that a substantial part (60-70%) of the incorporated nitrogen containing groups were amine groups. The availability of the surface amine groups for reactions was investigated by applying a gas phase reaction with 4-trifluoromethylbenzaldehyde and by a reductive methylation reaction in aqueous solution with 14C formaldehyde. A maximal number of reactive amine groups was found after a plasma treatment time of 2 s. The reductive methylation reaction was used to estimate the surface concentration of amine groups resulting in a typical surface concentration of 1 x 10(-6) mol/m2 after a plasma treatment time of 2 s.

Adsorption↗