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Biomedical subjects

S E Harding

Publications and source records attributed to S E Harding.

At least 109 records · Page 6Linked to original sources
PubMed1995

Abnormalities of the myocytes in ischaemic cardiomyopathy.

Acute myocardial ischemia and subsequent reperfusion result in biochemical and ionic changes in cardiac myocytes which cause contracture of the muscle and a reduced contractile force. Whether changes observed in single myocytes isolated from ischaemic ventricles are a direct consequence of the acute insult, or develop more slowly due to subsequent alterations in load and neurohumoural environment, is controversial. Myocytes from ischemic hearts have a similar contraction amplitude to those from non-failing hearts at physiological or maximally activating levels of ca2+. This could be partly due to the method of cell selection, or could represent the detection of a population of myocytes that have recovered from the original insult. However, there are significant decreases in the velocities of contraction and, particularly, relaxation in myocytes from the ischaemic heart. These resemble alterations caused by anoxia/reperfusion, but similar changes have also been observed in non-ischaemic causes of heart failure. Responses of beta-adrenoceptor stimulation are reduced in single cells from the failing heart, and a post-receptor defect has also been detected. Treatment with pertussis toxin, which reduces the activity of the inhibitory guanine-nucleotide binding protein (Gi) was able to restore beta-adrenoceptor responses to normal. The hypothesis that alterations in the beta-adrenoceptor/Gi/cAMP pathway represent the response of the myocyte to continued exposure to noradrenaline, because of the high sympathetic drive in these patients, is supported by the strong parallels observed with catecholamine-treated animals, and by the fact that non-ischemic aetiologies exhibit similar desensitization. It is concluded that the surviving myocytes in an ischaemic heart are damaged by the neurohumoral alterations that represent the body's attempt to restore cardiac output.

Cyclic AMP↗
PubMed1995

Cell geometry and contractile abnormalities of myocytes from failing human left ventricle.

OBJECTIVES: Systolic and diastolic dysfunction of the failing human heart may be due to changes in myocyte function, or to extracellular influences such as necrosis, fibrosis or repositioning of viable cells. In order to determine the contribution of cellular factors we have characterised the contraction amplitudes, and contraction and relaxation velocities of single myocytes isolated from failing human left ventricle. METHODS: Myocytes were enzymatically isolated from the left ventricles of 42 subjects, superfused at 32 degrees C and paced at 0.2 Hz. Using a video/edge tracking system we obtained contraction amplitude and contraction and relaxation velocities as well as times to peak contraction (TTP) and to 50% and 90% relaxation (R50 and R90). Concentration-response curves to Ca2+ were constructed for each cell. RESULTS: There was little difference in contraction amplitude at any Ca2+ concentration between cells from failing and non-failing hearts at this low frequency. At maximally activating Ca2+ concentrations (6-20 mM) there was a 30% slowing of relaxation velocity in myocytes from patients with both mild-moderate (P < 0.001) and severe (P < 0.001) congestive heart failure. Contraction and relaxation times were increased in myocytes from failing hearts [TTP: 0.46 +/- 0.02 s (n = 34 patients) vs. 0.35 +/- 0.02 s (n = 6), P < 0.01 and R50: 0.25 +/- 0.02 s (n = 34) vs. 0.16 +/- 0.02 s (n = 6), P < 0.001]. Impaired relaxation was seen with most etiologies, including ischemic and dilated cardiomyopathies and mitral valve disease. Myocytes from failing hypertrophied ventricles were more severely affected than those from failing non-hypertrophied hearts for both contraction and relaxation velocities. Cells from failing hypertrophied ventricles had a significantly larger area than from non-failing or failing non-hypertrophied ventricles, although cell length and sarcomere length were similar between groups. Larger myocytes did not show a more pronounced change in relaxation velocity than normally sized cells from the same hypertrophied ventricle. CONCLUSIONS: Significant impairment of relaxation can be observed in ventricular myocytes from failing human heart under conditions where contraction amplitude appears normal. The defect is not confined to one etiology of disease, but is exacerbated during hypertrophy. An increase in cell size, although observed in myocytes from hypertrophied ventricle, does not itself account for changes in relaxation. Cellular changes contribute to diastolic dysfunction in the failing human heart.

Adolescent↗
PubMed1994

Product-induced stabilization of tertiary and quaternary structure in Escherichia coli dehydroquinase.

The effects of irreversibly attaching product at the active site of type I dehydroquinase from Escherichia coli have been investigated at the level of the secondary, tertiary, and quaternary structure of the protein by spectroscopic and hydrodynamic techniques. The results agree with the previously identified stabilization of the enzyme but show for the first time that the protein dimer is also stabilized and suggest that the E. coli dehydroquinase subunit may be bilobal.

Circular Dichroism↗
PubMed1994

Effect of lemakalim on action potentials, intracellular calcium, and contraction in guinea pig and human cardiac myocytes.

OBJECTIVE: The aim was to investigate the effects of lemakalim on action potential duration, intracellular free calcium ([free Ca2+]i), and cell contraction in human and guinea pig cardiac myocytes. In addition, the possible modulation by pH of lemakalim induced activation of ATP sensitive potassium (KATP) channels was assessed. METHODS: Single ventricular myocytes were enzymatically dissociated from adult male guinea pigs (300-600 g). Single myocytes were isolated from human ventricular tissues. Cells were loaded with the acetoxymethyl ester form of fura-2 to monitor changes in [free Ca2+]i and subjected to conventional electrophysiological techniques. RESULTS: In guinea pig cells, lemakalim (3, 10, 30 microM) reduced action potential duration in a concentration dependent manner. This decrease was accompanied by hyperpolarisation of the resting membrane potential. Lemakalim (3, 10, 30 microM) reduced the systolic fura-2 fluorescence ratio without having a significant effect on diastolic fluorescence and also reduced the cell contraction in concentration dependent manner. Glibenclamide (1 microM), a specific inhibitor of KATP channels, did not affect action potential duration, fura-2 fluorescence ratio, or cell contraction in the absence of lemakalim. However, the same dose of glibenclamide markedly inhibited the lemakalim induced decrease in action potential duration, fura-2 fluorescence ratio, and cell contraction. Reducing extracellular pH enhanced the decrease in action potential duration induced by lemakalim. In human ventricular myocytes, lemakalim (3, 10 and 30 microM) caused a decrease in action potential duration and systolic fura-2 fluorescence ratio. The reduction in action potential duration and fura-2 fluorescence ratio was also reversed by glibenclamide (1 microM). CONCLUSIONS: These results suggest that lemakalim reduces systolic [free Ca2+]i by activating ATP sensitive potassium channels which results in a decrease of action potential duration in guinea pig and human ventricular myocytes. The reduction in [free Ca2+]i mediates the negative inotropic effect induced by lemakalim. In addition, pH may modulate the KATP channel activation by the channel opener.

Action Potentials↗
PubMed1994

Contractile function and response to agonists in myocytes from failing human heart.

Ventricular myocytes from failing human hearts have a similar maximum contraction amplitude in high Ca2+ to those from non-failing heart at low stimulation rates (0.2 Hz, 32 degrees C), but do not exhibit the same positive frequency-interval relationship. At higher stimulation rates (1 Hz) therefore, the amplitude is depressed in cells from failing hearts compared to controls. Slow relaxation is seen in myocytes from failing ventricle at all stimulation rates, and contraction velocity is also slightly reduced. beta-adrenoceptor desensitization is evident, and increases with severity of disease. There is also a post-receptor defect in myocytes from failing heart since responses to forskolin and cyclic AMP analogues are reduced, and this is accompanied by decreased cyclic AMP levels in myocardium from patients in end-stage disease. Pertussis toxin treatment, which inactivates Gi, reverses most of the alterations in the beta-adrenoceptor pathway. The role of the sympathetic system is indicated by the parallels between myocytes from failing human heart and those from the noradrenaline-treated guinea-pig, which show beta-adrenoceptor desensitization, a post-receptor defect and reduced basal cyclic AMP levels. However, relaxation velocities are not slowed in these guinea-pig myocytes, indicating that basal cyclic AMP does not have a tonic role in speeding relaxation.

Adrenergic beta-Agonists↗
PubMed1994

Characteristics of myocytes isolated from hearts of renovascular hypertensive guinea pigs.

A model of renovascular hypertension has been developed in the guinea pig using the Goldblatt (2-kidney, 1-clip) operation. Systolic and diastolic blood pressures were significantly increased 3 and 7 wk after the operation, but levels fell to control values at 11 wk. The two-dimensional areas of myocytes isolated from the hearts of Goldblatt-operated (GB) animals were larger than those in control cells at 3 wk (cf. 3,397 +/- 87 and 2,208 +/- 125 microns 2, P < 0.01), and the difference was maintained at 7 and 11 wk. No change in cell contraction or relaxation characteristics were seen at either 3 or 7 wk after clipping. Myocytes from the 11-wk GB group showed a significantly reduced contraction amplitude and velocity at 32 degrees C in maximally activating Ca2+ or isoproterenol concentrations (%cell shortening in Ca2+, cf. 6.8 +/- 0.4 and 10.0 +/- 0.9, P < 0.01). Concentrations eliciting 50% of maximal response for Ca2+ or isoproterenol were unchanged, as was the ratio of isoproterenol to Ca2+ effect in the same cell. Increases in time to peak contraction (TTP) and time to 50% relaxation (R50) were observed in 11-wk GB myocytes, but only at room temperature. There was no lengthening of TTP or R50 of the Ca2+ transient, nor was there any change in Ca2+ current density or inactivation kinetics in these myocytes.

Animals↗
PubMed1994

Long-term exposure to norepinephrine results in down-regulation and reduced mRNA expression of pulmonary beta-adrenergic receptors in guinea pigs.

We studied the down-regulation of beta 2-adrenoceptors in guinea pig lung after chronic exposure to a beta-agonist. Guinea pigs were exposed to either norepinephrine (NE) or vehicle for 7 days. The density and affinity of beta-adrenoceptors were determined by Scatchard analysis of [125I]cyanopindolol binding in a lung membrane preparation and beta 1- and beta 2-adrenoceptor subtypes were studied in the presence of 0.1 microM ICI 118,551, a selective beta 2-adrenoceptor antagonist, or 0.1 microM CGP 20712 A, a selective beta 1-adrenoceptor antagonist, respectively. beta 2-Adrenoceptor mRNA was examined by Northern blot analysis. The tissue distribution of beta 2-adrenoceptors and of beta 2-adrenoceptor mRNA in lung after NE infusion were determined using receptor autoradiography and in situ hybridization, respectively. The functional significance of the decrease in beta 2-adrenoceptor was tested by measuring the relaxation response to a beta 2-agonist in isolated parenchymal strips. NE treatment resulted in decreases of 75 +/- 9%, 84 +/- 4%, and 66 +/- 9% of total beta-, beta 1-, and beta 2-adrenoceptors in the lung, compared with control animals. The administration of NE had only minimal effects on the dissociation constant (Kd) of the receptor for the radioligand. beta 2-Adrenoceptors mRNA was decreased by 46 +/- 13% after NE treatment. There was a correspondence between the distribution of beta 2-adrenoceptors and beta 2-adrenoceptor mRNA localization in both control and NE-treated guinea pigs. NE treatment reduced alveolar beta 2-adrenoceptors by 70 +/- 3% and its mRNA expression by 78 +/- 5%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The multicatalytic proteinase complex (proteasome): structure and conformational changes associated with changes in proteolytic activity.

The multicatalytic proteinase complex or proteasome is a high-molecular-mass multisubunit proteinase which is found in the nucleus and cytoplasm of eukaryotic cells. Electron microscopy of negatively stained rat liver proteinase preparations suggests that the particle has a hollow cylindrical shape (approximate width 11 nm and height 17 nm using methylamine tungstate as the negative stain) with a pseudo-helical arrangement of subunits rather than the directly stacked arrangement suggested previously. The side-on view has a 2-fold rotational symmetry, while end-on there appears to be six or seven subunits around the ring. This model is very different from that proposed by others for the proteinase from rat liver but resembles the structure of the simpler archaebacterial proteasome. The possibility of conformational changes associated with the addition of effectors of proteolytic activity has been investigated by sedimentation velocity analysis and dynamic light-scattering measurements. The results provide the first direct evidence for conformational changes associated with the observed positive co-operativity in one component of the peptidylglutamylpeptide hydrolase activity as well as with the stimulation of peptidylglutamylpeptide hydrolase activities by MnCl2. In the latter case, there appears to be a correlation between changes in the shape of the molecule and the effect on activity. KCl and low concentrations of SDS may also act by inducing conformational changes within the complex. Sedimentation-velocity measurements also provide evidence for the formation of intermediates during dissociation of the complex by urea, guanidinium chloride or sodium thiocyanate. Dissociation of the complex either by these agents or by treatment at low pH leads to inactivation of its proteolytic components. The results suggest that activation and inhibition of the various proteolytic activities may be mediated by measurable changes in size and shape of the molecules.

Animals↗
PubMed1993

Expression, purification and characterization of B72.3 Fv fragments.

The Fv fragment of the antibody B72.3 has been produced by expression in both a mammalian and microbial system, namely Chinese hamster ovary (CHO) cells and Escherichia coli. In both cases secretion of the Fv into the culture medium was achieved, with equivalent amounts of Vh and Vl produced. The yield of Fv from CHO cells was 4 mg/l in roller-bottle culture. E. coli proved to be a more productive system with yields of 40 mg/l in shake flasks rising to 450 mg/l in fermentations. B72.3 Fv from both sources was capable of binding to antigen with similar binding ability to the Fab' fragment. A detailed sedimentation analysis, both by velocity and equilibrium techniques, revealed that the two domains of Fv are associated at high concentrations at pH values close to neutral, but dissociate at concentrations lower than approx. 0.5 mg/ml. Individual Vh or Vl polypeptides are not able to bind to the antigen and thus these results suggest that the antigen promotes assembly of Fv at the low concentrations used in the antigen-binding assays. At a pH value of 1.9, Vh and Vl are completely dissociated even at very high concentrations and are apparently unfolded at low solute concentrations. Small-angle X-ray scattering was used to measure a radius of gyration of 1.75 +/- 0.2 nm (17.5 +/- 2 A) for Fv.

Animals↗
PubMed1993

Methylmalonyl-CoA mutase from Propionibacterium shermanii: characterization of the cobalamin-inhibited form and subunit-cofactor interactions studied by analytical ultracentrifugation.

A large proportion of adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermannii is isolated in an inactive form which contains a tightly bound cobalamin. Even when the enzyme was denatured in 5.0 M guanidine hydrochloride the cobalamin remained associated with the protein. However, when dithiothreitol was added to the denatured protein, the pink inhibitor was rapidly converted into a yellow-brown compound which could be removed by dialysis. Enzyme activity could be recovered after removal of the denaturant, although surprisingly this did not depend on prior treatment with dithiothreitol. The interaction between the protein and inhibitor was investigated by using analytical ultracentrifugation under denaturing conditions. The sedimentation coefficient s20,w was measured in various concentrations of guanidine hydrochloride. A complicated picture emerged in which at low denaturant concentrations subunit dissociation, partial unfolding and aggregation occur, whereas at high concentration the protein behaves as a monodisperse species. No major differences in sedimentation were observed between the enzyme-cobalamin complex and the cobalamin-free enzyme, suggesting that the inhibitor does not significantly stabilize higher-order structure within the protein.

Dithiothreitol↗
PubMed1993

Hydrodynamic characterization of chitosans varying in degree of acetylation.

The molecular weights and gross aqueous solution conformation of two chitosans of different degrees of acetylation, 'Sea Cure +210' (11% acetylated) and KN50 (58% acetylated) were characterized by viscometry, analytical ultracentrifugation and dynamic light scattering. The hydrodynamic parameters obtained were used to determine both the molecular weights and the gross solution conformation of the two chitosans. Using the Wales-Van Holde ratio of sedimentation coefficient concentration regression coefficient (ks) to intrinsic viscosity [eta], the Sea Cure +210 chitosan, which is much less acetylated than the KN50, is highly asymmetric in conformation. This, in conjunction with the charge on the molecule, would suggest a rod-like conformation in solution. The two largest KN50 chitosans have widely differing values for the Wales-Van Holde ratio, suggesting different solution conformation. However, when the series is examined as a whole using the Mark-Houwink-Kuhn-Sakurada relationships relating molecular weight to both intrinsic viscosity and translational diffusion coefficient, then a more spheroidal structure, approximating to a random coil, is predicted.

Acetylation↗