Biomedical subjects

M T Hearn

Publications and source records attributed to M T Hearn.

At least 91 records · Page 5Linked to original sources

PubMed1995

Application of a sensitive HPLC-based fluorometric assay to determine the sialic acid content of human gonadotropin isoforms.

The human pituitary gonadotropins, follitropin (hFSH) and lutropin (hLH) are glycoproteins which are microheterogeneous in terms of their charge and molecular size, as well as their in vitro and in vivo bioactivities. The aim of this study was to determine the contribution of variations in sialic acid (N-acetyl neuraminic acid) content to the structural heterogeneity of these glycoproteins. Sialic acid (Neu5Ac) was released by partial acid hydrolysis (0.1 M TFA, 80 degrees C, 1 h) and derivatised with the fluorescent label DMB (1,2-diamino-4,5-methylenedioxybenzene) in conjunction with an internal standard (N-glycoyl-neuraminic acid). The derivatives were then separated by reversed-phase HPLC. This method allowed quantitation of the sialic acid content over a range of 5-100 pmol with between assay variation of < 6% for sialic acid released from approximately 100 ng (3 pmol) of hFSH or hLH. Comparison of the sialic acid contents of standard sialylated glycoproteins by either DMB-derivatisation or high-performance anion-exchange chromatography with pulsed amperometric detection yielded similar results, confirming the reliability of the fluorescence detection method. The sialic acid contents of 9 hFSH isoforms varied between 1.5-13.7 mol Neu5AC/mol FSH, whilst a range of 1.1-9.1 mol Neu5AC/mol LH was observed for 12 hLH isoforms. The sialic acid content of the hFSH isoforms was also observed to be related to the hormonal specific activity in a radioreceptor assay, confirming that alterations in the carbohydrate structure can influence the FSH-receptor interaction. In contrast, the sialic acid content of the hLH isoforms was found to be not related to specific activity at the receptor level.

Chromatography, High Pressure Liquid↗

PubMed1995

A simple and inexpensive sample-handling method for the semi-preparative RP-HPLC of polypeptides and non-polar peptide derivatives: pre-adsorption of samples.

A simple method is described for the application onto HPLC columns of very crude or alternatively poorly soluble polypeptide samples prior to their chromatographic purification. The procedure involves the batch pre-adsorption of the crude polypeptide mixture from a dilute solution onto an appropriate preparative-grade chromatographic adsorbent, removal of the solvent by rotary evaporation or lyophilisation and then dry-packing the pre-adsorbed chromatographic material into guard column cartridges of suitable dimensions. The polypeptide products can then be eluted either by isocratic or gradient elution methods through the cartridge coupled in tandem with prepacked semi-preparative HPLC columns. This method has been successfully utilised for the routine RP-HPLC purification of polar and hydrophobic polypeptides prepared by solid phase peptide synthesis (SPPS) methods as well as peptide derivatives and intermediates used as part of SPPS procedures.

Adsorption↗

PubMed1995

Investigations into the rheological characteristics of bovine amniotic fluid.

The rheological characteristics of bovine amniotic fluid have been studied at different shear rates. The viscosity of bovine amniotic fluid at 20 degrees C was found to increase with time at a constant low shear rate during the measurement. Additionally, the viscosity was observed to decrease with increasing shear rate, indicating that a shear thinning behaviour of the fluid was occurring. The log-log plot of shear stress versus shear rate yielded a straight line consistent with non-Newtonian behaviour of the fluid and characteristic of pseudoplastic liquids. The data of shear stress versus shear rate could be represented by a power law model. The treatment of amniotic fluid with cetylpyridinium chloride (CPC) resulted in the precipitation of a mixture of components, including complex sulphated polysaccharides and extracellular proteoglycans, with the viscosity of the resulting liquid similar to that of water at 20 degrees C. In addition, the viscosity of the CPC-pretreated fluid did not increase with time at constant shear rate and remained constant with the increase in shear rate. The apparent increase in viscosity with time and the shear thinning behaviour of the amniotic fluid can thus be attributed to pseudoplastic liquid behaviour associated with the presence of structurally complex polysaccharides and extracellular proteoglycans. The implications of this fluid viscosity behaviour are discussed in terms of their impact on the operation of packed or expanded (fluidized) chromatographic bed systems when amniotic fluid biofeedstocks are used as a source of commercially important proteins.

Amniotic Fluid↗

PubMed1995

Requirement for heparan sulphate proteoglycans to mediate basic fibroblast growth factor (FGF-2)-induced stimulation of Leydig cell steroidogenesis.

This study reports that, in contrast to previous findings, basic fibroblast growth factor (FGF-2) stimulates immature Leydig cell steroidogenesis in the absence of luteinizing hormone (LH). Heparan sulphate proteoglycans (HSPGs) are essential for this action of FGF-2 and the data suggest that HSPG/FGF-2 interactions have a significant role in the maintenance of immature Leydig cell steroidogenesis. Culture conditions were established for the maintenance of immature rat Leydig cells steroidogenesis in vitro for at least 2 days. Under these conditions the effect of exposure to FGF-2 at doses ranging from 0.1-10 ng/ml was shown to cause a significant stimulation of basal, but not LH-stimulated, 5 alpha-androstane 3 alpha,17 beta-diol production over 24h in culture. This stimulatory action on basal steroidogenesis is mediated through HSPG, as it was blocked by the addition of heparin (100 micrograms/ml), sodium chlorate (25mM) and protamine sulphate (5 micrograms/ml). These data demonstrate the involvement of HSPG in regulating FGF-2 action on Leydig cells and a potential role for Leydig cell HSPG in mediating paracrine regulatory actions of other heparin binding growth factors.

Animals↗

PubMed1995

Synthesis of peptide amides using Fmoc-based solid-phase procedures on 4-methylbenzhydrylamine resins.

A two-step low-high protocol for the efficient synthesis of peptide amides is described. The protocol exploits the efficiency of Reagent K for side-chain deprotection with the capability of the hard acid trifluoromethane-sulfonic acid (TFSMA) for cleavage of the peptide from the benzhydrylamine resin. This procedure has proven to be an effective method for the synthesis of peptide amides. The formation of alpha-aminosuccinimide (Asu) derivatives were observed with aspartyl-containing peptides as a minor side reaction product of this procedure, but this Asp-->Asu rearrangement could be successfully suppressed by employing low temperature conditions. The N- to O-acyl rearrangement of threonine and/or serine residues also only occurred to a minor extent under these synthetic conditions.

Amides↗

PubMed1995

Structure and in vivo activity of hypoglycaemic analogues of human growth hormone (6-13).

A range of peptide analogues related to Asu11-human growth hormone (6-13) have been synthesized and tested for hypoglycaemic activity using in vivo insulin tolerance tests. Utilising an alanine scan procedure and a selective amino acid residue approach these structure-activity studies suggest that residues Phe10, Arg8 and the C-terminal beta-turn structure are important for the expression of biological activity.

Animals↗

PubMed1994

High-performance liquid chromatography of amino acids, peptides and proteins. CXXXIII. Peak tracking of peptides in reversed-phase high-performance liquid chromatography.

A peak tracking algorithm for peptide analysis has been developed based on a normalised spectral overlay method which directly compares the UV spectra of any two chromatographic peaks. Additionally, the algorithm compares the spectrum of each peak in the first chromatogram with the spectra of every peak in the second chromatogram to determine the best cross-match. The sensitivity of the technique was further enhanced by incorporation of the primary and secondary derivative spectra for cross-match normalisation. The utility of the software was demonstrated by its application to the analysis of tryptic digests of porcine growth hormone. Peptide solutes could be identified and tracked in chromatograms generated with various column types, gradient times, mobile phase types and temperatures. These results therefore constitute the initial stages of development of a more robust approach to the optimisation of the resolution, detection and characterisation of peptides and proteins separated by HPLC techniques.

Algorithms↗

PubMed1994

In vitro inhibition of the actions of basic FGF by a novel 16 amino acid peptide.

A composite procedure involving molecular modelling and a property-pattern algorithm, the Resonant Recognition Model (RRM), has been applied to structure-function studies with basic fibroblast growth factor (bFGF). Property-pattern characteristics for biological activity and receptor recognition for a group of FGF-related proteins were defined and then used to aid the design of a set of peptides which can act as bFGF antagonists. Molecular modelling techniques were then employed to identify the peptide within this set with the greatest conformational similarity to the putative receptor domain of bFGF. This 16 amino acid residue peptide (16mer), which exhibits no sequence homology to bFGF, antagonised the stimulatory effect of bFGF on fibroblast [3H]thymidine incorporation and cell proliferation, but exerted no effect itself in these in vitro bioassays.

Algorithms↗

PubMed1994

Monoclonal anti-H1 histone autoantibodies from unimmunized Balb/c mice. Specificity and VH and VL domain sequences.

Examination of the nuclear reactivities of monoclonal IgM kappa autoantibodies, secreted by GFM-5 1B12 and NU-6 1F12 hybridomas derived from germ-free and nude mice, respectively, demonstrated homogeneous nuclear immunofluorescence staining patterns consistent with the recognition of histones. Under these conditions, GFM-5 1B12 and NU-6 1F12 mAbs produced species non-specific binding to components within the nuclei of mouse, human and Drosophila melanogaster cells. Immunoblotting confirmed the binding of these two autoantibodies to autologous H1 histones as well as bovine and insect H1 histones. Identification of the epitopes bound by GFM-5 1B12 and NU-6 1F12 mAbs within the D. melanogaster H1 histones was undertaken using 248 overlapping octapeptides encompassing the entire sequence of D. melanogaster H1 histones. GFM-5 1B12 mAbs bound several octapeptides derived from the amino- and carboxyl-terminal regions of D. melanogaster H1 histones with accessible KT, AT or VT amino acids. NU-6 1F12 mAbs, which stained nuclei within sections of D. melanogaster lavae, failed to bind to any of the 248 linear octapeptides, implying recognition of a conformational H1 histone epitope by this autoantibody. ELISA analysis of the polyspecific binding properties of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that both antibodies exhibited unique polyspecificity profiles. GFM-5 1B12 mAbs recognized bovine carbonic anhydrase and mouse IgG1, while NU-6 1F12 bound bovine cardiolipin, rat cytochrome c, Escherichia coli beta-galactosidase, toxoid from Clostridium tetani, mouse IgG1 and the haptens, DNP and FITC from the 24 antigen test panel. Comparison of the VH and VL domain sequences of GFM-5 1B12 and NU-6 1F12 mAbs demonstrated that the variations in autoreactivity and polyspecificity profiles resulted from amino acid variations in the CDRs of the VH and VL domains of these autoantibodies. Significantly, major differences in the VH domain sequences of the NU-5 1F12 and GFM-5 1B12 mAbs suggest that the VH domains may preferentially contribute to the unique specificities of the two anti-H1 histone autoantibodies.

Amino Acid Sequence↗

PubMed1994

Synthesis and conformation of constrained peptides with hypoglycaemic activity derived from human growth hormone.

The alpha-aminosuccinimide (Asu11) octapeptide analogue of human growth hormone hGH[6-13] (Leu6-Ser-Arg-Leu-Phe-Asu-Asn-Ala13) has been reported [Robson et al. (1990) Biol. Chem. Hoppe Seyler 371, 423-431] to have hypoglycaemic activity whilst the corresponding peptide with Asp at position 11 is inactive. In order to determine whether this change in activity is caused by conformational and/or stereo-electronic effects, the incorporation of two different isomeric gamma-lactam structures at position 11 has been investigated. One lactam structure (i) is of the type developed by Freidinger and coworkers [Freidinger et al. (1982) J. Org. Chem. 47, 102-107], whilst the isomeric gamma-lactam structure (ii) represents a new type of constrained synthon for use in peptide synthesis. The chiral type-ii gamma-lactam was synthesized via a suitably protected desoxodipeptide prepared in several ways from L-aspartic acid. The solution conformations of the [Asu11]- and the [gamma-lactam11]-containing hGH[6-13] peptide analogues were investigated with the aid of two-dimensional NMR (COSY and NOESY) spectroscopy. Conformational similarities were found for these hGH[6-13] peptide analogues. For example, for all peptide analogues studied, weak NOEs were evident between the Phe10 ring protons and protons of the amino acid residues at the C-terminus. Overall, however, the NOESY NMR spectra of the [Asu11]- and the [gamma-lactam11]-containing peptides related to hGH[6-13] suggest the presence of an extended structure in solution with a possible weak type II' beta-turn at position 11. The extent of conformational constraint introduced into these hGH[6-13] peptide analogues by substitution of the Asu11 residue with either isomeric gamma-lactam structure was reflected as differences in their hypoglycaemic activity. In particular, the hGH[6-13] peptide analogue derived from the new chiral type-ii gamma-lactam exhibits both lower activity in intravenous insulin tolerance tests in vivo and weaker NOEs than the isomeric hGH[6-13] peptide analogue derived from the type (i) gamma-lactam structure. The relative change in blood glucose levels from 20 to 90 min for the racemic (R,S)-form of the type-ii gamma-lactam compared to the control values was approximately half that of the (S)-stereoisomer.

Amino Acid Sequence↗

PubMed1994

Monoclonal antibody to a Thy-1 related/associated molecule identifies a subpopulation of immature mouse thymocytes.

In this study, NMT-1 mAbs have been shown to identify a thymocyte cell surface molecule (Immature Thymocyte Marker-1, ITM-1) expressed on 86.2 to 90.8% of CD4+CD8+CD3-/lo/int cells within the thymus of Balb/c, CBA, C57B1/6, 129 Rej, A/J and AKR mice. Similarly, immunoprecipitation analysis of cell membrane extracts from 125Iodine-labelled thymocytes, using NMT-1mAbs, identified 14.5 and 18.3 kDa molecules from all strains of mice examined. Comparison of these immunoprecipitation profiles with those produced by rat IgG2b anti-Thy-1 antibodies indicated that NMT-1 mAbs recognized a subspecies of the Thy-1 molecule. Unlike the Thy-1 molecule, the expression of ITM-1 molecules in Balb/c, CBA, C57B1/6, 129Rej, A/J and AKR strains of mice was restricted to the thymus. The ITM-1 molecule was not expressed on lymphoid cells within the spleen, lymph node, bone marrow, peritoneal cavity and peripheral blood in these strains. Phenotypic characterization associated the expression of ITM-1 molecule with the CD4+CD8+CD3-/lo/int thymocyte subpopulation indicating that NMT-1 mAbs recognize an epitope on a distinct developmentally-related Thy-1 glycoform. The expression of the ITM-1 molecule on differentiating thymocytes may prove useful in further subdividing immature thymic subpopulations, particularly those implicated in positive and negative selection.

Animals↗

PubMed1993

High-performance liquid chromatography of amino acids, peptides, and proteins. 123. Dynamics of peptides in reversed-phase high-performance liquid chromatography.

The dynamics of several peptides in reversed-phase high-performance liquid chromatography (RP-HPLC) have been investigated on both n-octadecyl (C18) silica and n-butyl (C4) silica sorbents. In particular, the conformational interconversions and the relative rates of chromatographic relaxation of bombesin, glucagon, and beta-endorphin on both C18 and C4 n-alkylsilicas were monitored by examining changes in the experimental bandwidths of these peptides as a function of temperature and column residence time under linear gradient elution RP-HPLC conditions. The observed band-broadening trends were correlated with previously derived retention parameters and thermodynamic descriptors of the association process determined for bombesin, beta-endorphin, glucagon, and a control peptide, penta-L-phenylalanine. This study confirms that bandwidth measurements can be used as an integral experimental component to study the effect of the secondary structure of peptidic solutes on their RP-HPLC retention behavior. Further, the data demonstrate the utility of RP-HPLC as a tool to examine peptide conformational dynamics at hydrophobic surfaces. The relevance of these results to the general phenomenon of peptide-lipid interactions is discussed in terms of the associated evidence for lipid-induced changes in the conformation of these three bioactive peptides.

Amino Acid Sequence↗

PubMed1993

High-performance liquid chromatography of amino acids, peptides and proteins. CXXVIII. Effect of D-amino acid substitutions on the reversed-phase high-performance liquid chromatography retention behaviour of neuropeptide Y[18-36] analogues.

The reversed-phase high-performance liquid chromatographic (RP-HPLC) gradient elution behaviour of a series of peptides related to Neuropeptide Y (NPY) has been investigated. The peptides studied included NPY, NPY[13-36], NPY[18-36] and a series of 16 analogues of NPY[18-36], each with a single D-amino acid substitution. Chromatographic parameters which relate to the interactive contact area and the binding affinity have been evaluated with two different stationary phase ligands and two organic modifiers. The results demonstrate that D-amino acid substitutions in the sequence region encompassing amino acid residues NPY[27-31] of these NPY[18-36] peptides significantly influence the interactive behaviour of these peptides relative to the unsubstituted NPY[18-36] molecule, while substitutions in the N- and C-terminal regions had little effect. Further, these results indicate that, in hydrophobic environments, NPY[18-36] adopts a significant degree of secondary structure which is severely disrupted by the presence of the D-amino acids in the central portion of the molecule.

Acetonitriles↗

PubMed1993

High-performance liquid chromatography of amino acids, peptides and proteins. CXXVI. Modelling of protein adsorption with non-porous and porous particles in a finite bath.

Analytical solutions for a mathematical model describing dynamic adsorption processes of proteins onto non-porous adsorbent particles in a finite bath are presented. The model, based on the Langmuir adsorption isotherm, has been applied to experimental data obtained with affinity and ion-exchange adsorbents. The external film mass transfer resistance, as well as the rate of surface interaction between proteins and adsorbents, have been taken into account. The model has been extended to the case of adsorption onto porous particles by employing a linear driving force approximation for describing mass transfer in the pore fluid. This approach enables the derivation of an effective overall liquid phase mass transfer coefficient, permitting subsequent adaptation of the analytical solutions developed for non-porous particles. The evaluation of the effective liquid phase mass transfer coefficients is also described. Examples of a comparison between predicted and experimental dynamic adsorption curves for both dye-affinity and ion-exchange systems are presented. The application of the model for predicting the optimum operating conditions is discussed.

Adsorption↗

PubMed1993

High-performance liquid chromatography of amino acids, peptides and proteins. CXXXII. Optimisation of operating parameters for protein purification with chromatographic columns.

In large-scale chromatography, process optimisation is one of the key elements for success. This paper presents a method for determining the optimum operating parameters for affinity and ion-exchange chromatographic columns when used for protein purification. Based on a mathematical model developed as part of our association investigations, computer programs have been developed to describe the dynamic relationships acting within the chromatographic system. Two basic operating parameters, the flow-rate and the effluent concentration at which the adsorption stage is terminated, can be optimised to give a maximum production rate. The sample loading volume and the processing time then can be determined. The effect of washing conditions on the production rate and the yield is also discussed. Examples are given for a specific system where the optimisation is based on the yield and the percentage utilisation of the column capacity. Contour plots are generated to aid the determination of the range of controlling parameters, and to guide further system design.

Chromatography, Affinity↗

PubMed1993

High-performance liquid chromatography of amino acids, peptides and proteins. CXXXI. O-phosphoserine as a new chelating ligand for use with hard Lewis metal ions in the immobilized-metal affinity chromatography of proteins.

Conditions for the immobilization of O-phosphoserine (OPS) to epoxy-activated Sepharose CL-4B are described. The binding behaviour of OPS and iminodiacetic acid (IDA) immobilized onto Sepharose CL-4B, toward the hard Lewis metal ions Al3+, Fe3+, Ca2+ and Yb3+, and Cu2+ ion as a borderline metal ion control, over the pH range pH 4.0 to pH 8.0, was examined. Immobilized OPS shows a stronger affinity for Fe3+ and Al3+ ions but a lower affinity for Cu2+ and Yb3+ ions, compared to immobilized iminodiacetic acid (IDA), over the equilibrating range examined. Immobilized OPS-Mn+ was screened for protein binding using as model proteins tuna heart cytochrome c (THCC), horse myoglobin (HMYO) and hen egg while lysozyme (HEWL) over the pH range 5.5 to 8.0. Immobilized OPS-Fe3+ bound THCC under all the examined equilibrating conditions, bound HMYO between pH 5.5 and pH 7.0 and did not bind HEWL under any condition examined. Immobilized OPS thus presents an additional mode of metal ion and protein selectivity in immobilized-metal affinity chromatography.

Amino Acids↗

PubMed1993

High-performance liquid chromatography of amino acids, peptides and proteins. CXXIX. Ceramic-based particles as chemically stable chromatographic supports.

Porous zirconia based particles have been modified using different derivatisation procedures. The modified particles were characterised in terms of their accessible surface areas and degree of surface coverage of the bounded or physicoated phases utilising the strong and specific adsorption of phosphate ions to the zirconia surface. The hydroxyl group density was determined by a 1H NMR technique. The particles were modified by immobilising different silanes to introduce either hydrophobic ligands or reactive groups onto the zirconia surface. In the latter case, various ligands were then covalently attached to the activated supports. Using this type of modification, n-octadecyl- (C18), carbohydrate- and Cibacron Blue F3GA-modified zirconia particles were produced. Furthermore, polymeric coated particles were prepared either by using polybutadiene or by cross-linking the carbohydrate modified sorbents. The pH stability of the different sorbents were determined in batch experiments and under chromatographic conditions. The leakage of ligands was monitored by UV absorption and by employing radioactively labelled ligands. The performance of the C18 reversed-phase modified zirconia in packed columns was also used as an indicator of changes in the surface chemistry following pH stability tests. The experimental results indicate that the Cibacron Blue F3GA dye-modified sorbent was stable up to pH 10.5, the C18 reversed-phase packing up to pH 13 and the carbohydrate-bonded phase up to pH 12. These investigations substantiate the favourable chemical and physical characteristics anticipated for surface modified zirconias for potential use as chromatographic adsorbents.

Carbohydrates↗

PubMed1993

High-performance liquid chromatography of amino acids, peptides and proteins. CXXX. Modified porous zirconia as sorbents in affinity chromatography.

The utilisation of organosilanes to introduce active chemical groups onto zirconia surfaces, suitable for the subsequent immobilisation of proteins or other biomimetic ligands, is described. Two different types of porous zirconia-based particles with nominal pore diameters of 160 and 1000 A pore size were modified with two different affinity ligands. In the first case, methods to immobilise iminodiacetic acid-Cu(II) and its application in Cu(II) immobilised metal ion affinity chromatography (IMAC) were established. In the second series of experiments, concanavalin A was immobilised and the interaction of this lectin with the enzyme horseradish peroxidase examined. For both systems, adsorption isotherms were recorded as batch experiments. In each case, the experimental results could be fitted to langmuirean type adsorption isotherms, indicating that under the chosen conditions only one type of interaction is present, with nonspecific interactions with the support surface playing an insignificant role. These studies document the potential of surface modified zirconia particles for the immobilisation of chemical ligands or proteins for use in biospecific affinity chromatography and immobilised enzyme bioreactors.

Adsorption↗