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Biomedical subjects

S E Harding

Publications and source records attributed to S E Harding.

At least 91 records · Page 5Linked to original sources
PubMed1996

Cyclic AMP levels in ventricular myocytes from noradrenaline-treated guinea-pigs.

Chronic activation of the sympathetic nervous system in human heart failure is believed to cause cardiac beta-adrenoceptor desensitisation. We have investigated the relationship between beta-adrenoceptor desensitisation and cyclic AMP levels in cardiac myocytes isolated from the ventricle of guinea-pigs chronically infused with noradrenaline hydrochloride for 7 days. Functional beta-adrenoceptor desensitisation was confirmed by a significant decrease in the maximum isoprenaline-stimulated contraction amplitude and an increased EC50 for isoprenaline. In the absence of beta-adrenoceptor stimulation, basal cyclic AMP levels were significantly depressed in populations of myocytes from noradrenaline-treated animals compared to sham-operated controls, and this was not accounted for by myocyte hypertrophy or necrosis. Similarly, there was a significant decrease in cyclic AMP levels at maximally inotropic isoprenaline concentrations. Threshold and maximum inotropic concentrations of the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX), restored isoprenaline-stimulated cyclic AMP levels in noradrenaline-treated guinea-pig cardiac myocytes, although we have previously reported no increase in maximum inotropic effect of isoprenaline with these compounds.

Animals↗
PubMed1996

Characterization of pig colonic mucins.

Pig colonic mucins isolated from the adherent mucus gel in the presence of proteinase inhibitors were solubilized by homogenization and the component mucins fractionated by CsC1 density-gradient centrifugation. Polymeric and reduced pig colonic mucin were both largely excluded on Sepharose CL-2B, papain-digested colonic mucin was included. The M(r) values of polymeric, reduced and digested mucins were 5.5 x 10(6), 2.1 x 10(6) and 0.6 x 10(6) respectively. This suggests that pig colonic mucin is comprised of 2-3 subunits, each subunit containing 3-4 glycosylated regions. The intrinsic viscosities of polymeric, reduced and digested mucin were 240 ml.g-1, 100 ml.g-1 and 20 ml.g-1 respectively. Polymeric pig colonic mucin comprised 16% protein per mg of glycoprotein and was rich in serine, threonine and proline (43% of total amino acids). There were approx. 150 disulphide bridges and 53 free thiol groups per mucin polymer. A seventh of the protein content was lost on reduction. This protein was particularly rich in proline and the hydrophobic amino acids. Papain-digested pig colonic mucin contained 11% protein per mg of glycoprotein and was rich in serine, threonine, glutamate and aspartate. All types of amino acids with the exception of aspartate were lost on digestion. The amino acid analysis of the proteolytically digested regions of pig colonic mucin are markedly different to the tandem repeat regions of the human mucin genes shown to be expressed in the colon.

Amino Acids↗
PubMed1996

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure, backbone dynamics and protein association.

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155-270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of 1H-15N-correlated 2D HSQC, 15N-edited 3D TOCSY-HMQC, and 15N-edited 3D NOESY-HMQC spectra. By analysis of the alpha-proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain. To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.

Amino Acid Sequence↗
PubMed1996

A physico-chemical investigation of the self-association of the DNA binding domain of the yeast transcriptional activator GAL4.

It has previously been suggested that the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 exists in solution in dimeric form, with the region responsible for dimerisation somewhere between residues 74 and 147. In this study limited proteolysis and carboxy-terminal deletions of the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 followed by subsequent characterization by equilibrium sedimentation in the analytical ultracentrifuge have been used to define more precisely the regions required for DNA binding and protein self-association. Sedimentation equilibrium analyses confirmed that the 'hydrophobic region' of the protein (residues 54-97, which contains a larger proportion of alpha-helix), is essential for dimerisation, with an apparent dissociation constant K(D,app), of approximately 50 microM for the 1-94 residue peptide and approximately 20 microM for the 1-147 residue peptide. Our studies do not rule out the possible formation of small amounts of additional higher order complexes.

Amino Acid Sequence↗
PubMed1996

Acceleration of contraction by beta-adrenoceptor stimulation is greater in ventricular myocytes from failing than non-failing human hearts.

Myocytes from failing human ventricle contract and relax more slowly than those from non-failing. This has been suggested to result from the lowering of basal cyclic AMP level in failing myocardium, and the consequent withdrawal of a tonic lusitropic effect. We present data to support this hypothesis by demonstrating that the acceleration of contraction and relaxation by beta-adrenoceptor stimulation is greater in myocytes from failing than non-failing heart. This is despite the desensitisation of the inotropic effect of isoprenaline in the same failing cells. Following beta-adrenoceptor stimulation, speeds of contraction and relaxation are normalised in myocytes from failing heart, with final values not significantly different from non-failing.

Adrenergic beta-Agonists↗
PubMed1996

Rapid size distribution and purity analysis of gastric mucus glycoproteins by size exclusion chromatography/multi angle laser light scattering.

The weight average molar masses and molar mass distributions of two commercial pig gastric mucins and two fresh mucin preparations were determined by the technique of size exclusion chromatography coupled on-line to multi angle laser light scattering (SEC/MALLS). Both commercial samples exhibited much lower molar mass averages than the freshly prepared material and contained more impurities. On the other hand, a fresh mucin preparation examined after being stored frozen for 18 months revealed a slight increase in molar mass.

Animals↗
PubMed1996

Maillard induced complexes of bovine serum albumin--a dilute solution study.

Association of bovine serum albumin (BSA) on heating in the presence and absence of 2% xylose has been studied using dynamic light scattering and sedimentation velocity. When 3% solutions of the protein alone are heated at 95 degrees C association products are formed with molar masses of approximately 2 x 10(6) g/mol, a value which is independent of the time of heating. These aggregates can be dissociated in solvents that disrupt non-covalent bonds. When the reducing sugar xylose is present there is a continuous change in the hydrodynamic properties with time. After 80 min a molar mass in excess of 7 x 10(6) g/mol is obtained. This increase in molar mass is attributed to additional non-disulphide linkages resulting from the Maillard reaction. Information about the gross conformation of the Maillard induced association products has been obtained from MHKS (Mark-Houwink-Kuhn-Sakarada) double logarithmic plots of D20,w and S20,w against molar mass. The values of the MHKS coefficients obtained are most consistent with a linear rod: i.e. the association is of an end-to-end type.

Animals↗
PubMed1996

Hydrodynamic examination of the dimeric cytoplasmic domain of the human erythrocyte anion transporter, band 3.

Solution studies of the cytoplasmic domain (molecular mass approximately 40kDa) of band 3, the anion exchanger from human erythrocyte membranes, previously suggested a dimeric molecule on the basis of the relative techniques of calibrated gel filtration and calibrated preparative ultracentrifugation. This dimeric behavior is firmly established on an absolute basis by a combination of calibrated gel chromatography and absolute ultracentrifugation techniques. Sedimentation velocity in the analytical ultracentrifuge combined with calibrated gel chromatography give a molecular mass M of (77 +/- 4) kDa, a value confirmed by low-speed sedimentation equilibrium. Velocity sedimentation in the analytical ultracentrifuge gave a single sedimenting species with an s o 20,w of (3.74 +/- 0.07)S. Sedimentation equilibrium analysis was also used to establish the strength of the binding via the dissociation constant Kd, with a value from direct fitting of the concentration distribution curves of (2.8 +/- 0.5) microM, confirmed by a value of approximately 3 microM obtained from fitting a plot of molecular weight Mw,app versus cell loading concentration. Hydrodynamic calculations based on the classical translational frictional ratio showed that the protein was highly asymmetric, with an axial ratio of approximately 10:1, consistent with observations from electron microscopy.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1996

Comparison of Quasielastic Light Scattering and Laser Diffractometry as Nondestructive Probes into the Structure of Bacillus sphaericus Spores Produced at Different Temperatures.

Quasielastic light scattering (QLS) and laser diffractometry (LD) are relatively novel nondestructive procedures for estimating the sizes of bacterial spores in suspension. This study for the first time directly compared the two with a destructive procedure, namely, scanning electron microscopy (SEM), for quasispherical spores of Bacillus sphaericus. Because of the different physical aspect measured, the sizes derived by QLS and LD are, as could be expected for spores with an exosporium, significantly different. The larger estimates obtained by QLS (1.70, 1.58, and 1.14 (mu)m for spores produced at 15(deg)C [BS15], 20(deg)C [BS20], and 30(deg)C [BS30], respectively) than by LD (0.56 [BS15], 0.58 [BS20], and 0.52 [BS30] (mu)m) and SEM (0.64 [BS15], 0.58 [BS20], and 0.70 [BS30] (mu)m) are explained in terms of the detection by QLS, LD, and SEM of different spore layers and the degree of nonsphericity of the latter.

Journal Article↗
PubMed1996

Beta-adrenoceptor function changes with age of subject in myocytes from non-failing human ventricle.

The contractile response to beta-adrenoceptor stimulation has been found to be reduced in single myocytes from failing human ventricle. A portion of that reduction was statistically related to the age of the patient. We have developed a method for obtaining viable contracting myocytes from small biopsies of human ventricle, enabling experiments to be performed on non-failing left ventricle from patients undergoing coronary artery surgery. These subjects are generally in an older age range than those we have previously obtained from donor hearts. The present study compares responses to isoproterenol and high Ca2+ between myocytes from older (> 50 years, n = 8) and younger (< 40 years, n = 5) subjects. Myocytes from older patients did not differ in their contraction amplitude (10.7 +/- 1.0 cf. 10.8 +/- 1.1% cell shortening), or contraction and relaxation velocities in high Ca2+ at a driving frequency of 0.2 Hz (32 degrees C). The sensitivity to isoproterenol, as determined from the EC50 value (concentration for half-maximal effect), was also similar between age groups (1.16 +/- 0.64 nM young, cf. 2.76 +/- 2.0 nM old), although higher than in myocytes from failing hearts (22.3 +/- 7.5 nM, n = 11). However, for the older group there was a significant depression in maximum contraction amplitude with isoproterenol (8.5 +/- 0.6 cf. 11.5 +/- 1.0% cell shortening, P < 0.05) and in the ratio between this and the maximum Ca2+ response (isoproterenol/Ca2+ ratio, 0.79 +/- 0.05 cf. 1.16 +/- 0.12, P < 0.05). Concomitantly, the maximum contraction and relaxation velocities achieved in the presence of isoproterenol were also depressed in older subjects (P < 0.02 for both). We conclude that age and/or coronary disease with unimpaired left ventricular function selectively reduces the maximum effect of isoproterenol but not the concentration at which this occurs.

Adrenergic beta-Agonists↗
PubMed1996

Tachycardia-induced failure alters contractile properties of canine ventricular myocytes.

OBJECTIVE: Rapid cardiac pacing has been used as a model for experimentally-induced cardiomyopathy. However, its relevance to human heart failure is not clear at present because little is known about changes in size and function of ventricular myocytes. We have therefore studied the responses to graded increases in frequency and calcium in canine ventricular myocytes from failing hearts. The aim of our study was to evaluate the resemblance between canine pacing-induced and human end-stage heart failure. METHODS: Myocytes were isolated from the left ventricular wall of dogs that were in heart failure after 6 weeks of pacing at 250 beats/min. Cell shortening was measured by edge detection. RESULTS: Clinical signs of failure included dyspnea, ascites, and heart dilatation; the hemodynamic parameters were: LVdP/dtmax 1613 +/- 149 vs. 4713 +/- 304 mmHg/s in 6 control dogs; LVEDP 17.2 +/- 4.4 vs 5.6 +/- 1.1 mmHg; LV volume 60.5 +/- 6.2 vs. 30-35 ml. Myocytes from failing hearts were longer and thinner than those from controls (from factor: 0.40 +/- 0.01 vs. 0.47 +/- 0.01, P < 0.001, > 30 cells/heart). With 6 mM Ca2+ and at 0.5 Hz, contraction amplitude was significantly attenuated in myocytes from failing hearts: 6.6 +/- 0.9% cell shortening vs. 10.0 +/- 0.8% in controls (P < 0.05). This deficit was exacerbated at higher stimulation rates. Time-to-peak contraction and time-to-50% relaxation were not altered. There was no difference in sensitivity to thapsigargin. CONCLUSION: As with cells from human failing hearts, contraction amplitude showed rate-dependent depression in this animal model, whereas features like slowing of contraction and relaxation and reduced sensitivity to thapsigargin, were not reproduced.

Animals↗
PubMed1995

Sensitization of human atrial 5-HT4 receptors by chronic beta-blocker treatment.

BACKGROUND: Chronic treatment of patients with beta-blockers induces beta 2-adrenergic receptor hyperresponsiveness in atrium and sinoatrial node. To investigate whether other atrial Gs protein-coupled receptors also become hyperresponsive after chronic treatment with beta-blockers, we investigated 5-HT4 receptors in tissues and myocytes, which mediate serotonin-evoked increases of both contractile force and cAMP levels. METHODS AND RESULTS: Isolated right atrial strips from patients who had been chronically treated or not treated with a beta-blocker were set up to contract. In tissues from beta-blocker-treated patients (n = 27), the maximum inotropic response to serotonin was 56 +/- 3% (mean +/- SEM) of the effect elicited by (-)-isoproterenol (200 mumol/L) compared with a response of 19 +/- 6% in tissues from non-beta-blocker-treated patients (n = 13) (P < .001). The responsiveness of the tissues to Ca2+ was unchanged by chronic beta-blocker treatment. Serotonin (1 and 10 mumol/L) increased tissue cAMP levels, the increase with 10 mumol/L being significantly greater (P < .05) in tissues from beta-blocker-treated (n = 9) than in non-beta-blocker-treated (n = 7) patients. In paced atrial myocytes, serotonin caused concentration-dependent increases in contraction. Myocytes obtained from atria of beta-blocker-treated patients were more sensitive (P < .01) to the effects of serotonin (-log EC50, 7.9 +/- 0.2 mol/L; n = 12) than myocytes obtained from non-beta-blocker-treated patients (-log EC50, 7.3 +/- 0.2 mol/L, n = 12). Chronic beta-blocker treatment had no effect on forskolin-evoked myocyte responses. Carbachol (1 mumol/L) suppressed the effects of both serotonin (n = 6) and (-)-isoproterenol (n = 6) in the same atrial myocyte. CONCLUSIONS: Chronic treatment of patients with beta-blockers causes atrial 5-HT4 receptor inotropic hyperresponsiveness and enhanced serotonin-evoked increases in cAMP levels. This may be due to modified cross talk between 5-HT4 receptors, beta-adrenergic receptors, and muscarinic receptors.

Adrenergic beta-Antagonists↗
PubMed1995

Reduced contraction and altered frequency response of isolated ventricular myocytes from patients with heart failure.

BACKGROUND: Previous work has failed to demonstrate reduced maximal contraction of isolated ventricular myocytes from failing human hearts compared with nonfailing control hearts. The effect of alterations in stimulation frequency and temperature on the contraction of isolated ventricular myocytes has been investigated. Left ventricular myocytes were isolated from the hearts of patients with severe heart failure undergoing heart transplantation and compared with myocytes isolated from myocardial biopsies from patients with coronary disease but preserved left ventricular systolic function or from myocytes from rejected donor hearts. METHODS AND RESULTS: Myocytes were exposed to either a maximally activating level of extracellular calcium at 37 degrees C or to 2 mmol/L calcium at 32 degrees C. There was no significant difference in the contraction amplitude between myocytes from failing and nonfailing hearts at 0.2 Hz. With increasing stimulation frequency, there was a reduction in contraction amplitude in cells from failing hearts relative to control hearts in both maximal calcium from 0.33 Hz (4.5% versus 6.6%) to 1.4 Hz (3.9% versus 8.8%) (ANCOVA, P < .001) and at 2 mmol/L calcium from 0.50 Hz (2.3% versus 3.5%) to 1.4 Hz (1.8% versus 3.9%) (ANCOVA, P < .001). The time to peak contraction and the times to 50% and 90% relaxation were prolonged in myocytes from failing hearts at stimulation rate of 0.2 Hz (P < .01), but only the time to 50% relaxation was prolonged at 1.0 Hz (P < .05). CONCLUSIONS: Reduced contraction, slowed relaxation, and impaired frequency response occurring at the level of the individual ventricular myocyte can be demonstrated in human heart failure. This demonstrates that disruption of myocyte function can contribute to both the systolic and the diastolic abnormalities that occur in the failing human heart.

Adult↗
PubMed1995

Identification of a flavin:NADH oxidoreductase involved in the biosynthesis of actinorhodin. Purification and characterization of the recombinant enzyme.

The biosynthesis of the polyketide antibiotic actinorhodin by Streptomyces coelicolor involves the oxidative dimerization and hydroxylation of a precursor, most likely dihydrokalafungin, as the final steps in its formation. Mutations in the actVB gene block these last steps, and the mutants secrete kalafungin as a shunt product. To investigate the role of the actVB gene in these transformation, we have overexpressed the gene in Escherichia coli and purified and characterized the recombinant protein. ActVB was shown to catalyze the reduction of FMN by NADH to give NAD and FMNH2, which, unusually, is released into solution. The protein contains no chromogenic cofactors and exhibits no requirements for added metal ions. The reaction obeys simple kinetics and proceeds through the formation of a ternary complex; Km values for FMN and NADH are 1.5 and 7.3 microM, respectively, and kcat is about 5 s-1. FAD and riboflavin are also substrates for the enzyme, although they have much higher Km values. The subunit structure of the enzyme was investigated by analytical ultracentrifugation, which showed the protein to exist in rapid equilibrium between monomer and dimer forms. The possible role of this oxidoreductase in the oxidative chemistry of actinorhodin biosynthesis is discussed.

Amino Acid Sequence↗
PubMed1995

Inversion formulae for ellipsoid of revolution macromolecular shape functions.

Explicit relations linking ellipsoid of revolution shape parameters with the axial ration (a/b) have been available for a long time. It is not possible to directly invert these functions to give a/b as a function of the shape parameter, so in the past graphical or tabular interpolation methods have been necessary. By the use of simple unconstrained polynomial expansions this restriction is no longer necessary. For the two salient hydration-dependent shape functions (P from the frictional ratio and v from the intrinsic viscosity) and the four salient hydration-independent shape functions (beta and R, both from sedimentation and viscosity measurements, and Lambda and Pi from combining viscosity measurements with respectively fluorescence depolarization or thermodynamic second virial coefficient measurements) the polynomial expansion coefficients (to no more than seventh order) are given for the ranges 1.1 < a/b < 2.0, 2 < 2/b < 10, and 10 < a/b < 100. The polynomial coefficients give an excellent fit to the real data values, except for four uninteresting cases (where the shape function is too insensitive for practical use).

Computer Simulation↗
PubMed1995

Decreased contractile responses to isoproterenol in isolated cardiac myocytes from aging guinea-pigs.

We have characterized the age-related changes of contractility and beta-adrenoceptor function in isolated cardiac myocytes from guinea-pigs. We used either adult animals from 2 to 14 weeks of age, where body weight increases linearly with age, or senescent ones aged between 53-65 weeks. There was some indication of a decrease in contractility in maximum Ca2+ with age, with significant differences between a young (< or = 4 weeks, weight < 400 g) and aged (> or = 8 weeks, weight > 600 g) group in contraction amplitude expressed as percentage shortening (but not when expressed as micron change in length) or contraction and relaxation velocities. This decline was continued into senescence, and ANOVA showed a significant difference between the three groups for contraction amplitude (percentage shortening, 12.2 +/- 0.9%, young, n = 31; 9.5 +/- 0.6%, n = 28 aged; 6.7 +/- 0.8%, n = 6, senescent; P = 0.005), and contraction or relaxation velocities (P < 0.001). There was a more pronounced decline in maximum response to isoproterenol with age. ANOVA for the maximum isoproterenol response for the three divisions showed significant differences for percentage shortening (11.8 +/- 0.7%, n = 30, young; 7.9 +/- 0.5%, n-28, aged and 5.5 +/- 1.1%, n = 6, senescent; P < 0.001), velocities of contraction (P < 0.001) and relaxation (P < 0.001), and normalized velocities of contraction (P < 0.001) and relaxation (P < 0.01) at maximum isoproterenol, as well as in ISO EC50 (P < 0.001) and isoproterenol/Ca2+ ratio (P < 0.02). A general decrease in contractility of the myocyte occurs as the animal ages, with maximum contraction amplitude being reduced and velocity of contraction and relaxation slowed. The effect was more pronounced for beta-adrenoceptor stimulation than for high Ca2+, suggesting a specific lesion in the adenylate cyclase related pathway. Much of the change occurred between the young adult (< or = 4 weeks) and the aged adult (> or = 8 weeks), although the trend was continued in senescent animals (> 52 weeks).

Adrenergic beta-Agonists↗