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Biomedical subjects

P D Allen

Publications and source records attributed to P D Allen.

At least 109 records · Page 6Linked to original sources
PubMed1991

Vascular smooth muscle expresses a truncated Na+, K(+)-ATPase alpha-1 subunit isoform.

By regulating transmembrane Na+ and K+ concentrations and membrane potential, the Na+,K(+)-ATPase plays an important role in regulating cardiac, skeletal, and smooth muscle function. A high degree of amino acid sequence and structural identity characterizes the three Mr 100,000 Na+,K(+)-ATPase alpha subunit isoforms expressed in cardiac and skeletal muscle. Strikingly, vascular smooth muscle utilizes alternative RNA processing of the alpha-1 gene to express a structurally distinct Mr approximately 65,000 isoform, alpha 1-T (truncated). Analysis of both its mRNA and protein structure reveals that alpha-1-T represents a major, evolutionarily conserved, truncated Na+,K(+)-ATPase isoform expressed in vascular smooth muscle. This demonstrates an unexpected complexity in the regulation of vascular smooth muscle Na+,K(+)-ATPase gene expression and suggests that a structurally novel, truncated alpha subunit may play a role in vascular smooth muscle active ion transport.

Amino Acid Sequence↗
PubMed1991

Troponin T isoform expression in humans. A comparison among normal and failing adult heart, fetal heart, and adult and fetal skeletal muscle.

The expression of troponin (Tn) T, a thin-filament regulatory protein, was examined in left ventricular myocardium from normal and from failing adult human hearts. The differences in isoform expression between normal and failing myocardium led us to examine the ontogenic expression of TnT in human striated muscle. Left ventricular samples were obtained from patients with severe heart failure undergoing cardiac transplantation and normal adult organ donors. Fetal muscle was obtained from aborted fetuses after 14-15 weeks of gestation, and adult skeletal muscle was obtained from surgical biopsies. Western blots of normal and failing adult heart proteins demonstrated that two isoforms, TnT1 and TnT2, are expressed in different amounts, with TnT2 being significantly greater in failing hearts (p less than 0.004). Western blots of two-dimensional gels of these proteins resolved two predominant spots of both TnT1 and TnT2 and several minor TnT species. Alkaline phosphatase treatment converted the two major spots of each isoform into the single more basic spots. A comparison of the ATPase activities and the TnT2 percentage of total TnT in individual failing and normal adult hearts demonstrated an inverse and negative relation (r = 0.7, p less than 0.02). In the fetal heart, four TnT isoforms were found, two of which had the same electrophoretic mobilities as the adult cardiac isoforms TnT1 and TnT2. Fetal skeletal muscle expressed two of the four fetal cardiac TnT isoforms, one of which comigrated with adult cardiac TnT1. These cardiac isoforms were expressed in low abundance in fetal skeletal muscle relative to seven fast skeletal muscle TnT isoforms. No cardiac isoforms were present in adult skeletal muscle. Because many etiologies caused heart failure in the transplant patients, we propose that the disease-associated increased expression of the TnT isoform TnT2 is an adaptation to the heart failure state and a partial recapitulation of the fetal expression of cardiac TnT isoforms.

Adult↗
PubMed1991

Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts.

Expression of the two sarcomeric actins, alpha-skeletal and alpha-cardiac, is regulated in the rodent heart in response to developmental, hormonal, and hemodynamic stimuli. Little is known in man, except that both isogenes were found to be coexpressed in three adult ventricles. In this report, we investigated the isoactin mRNA composition in ventricles from 21 control patients (4 fetal, 5 juvenile, 12 adult) and from 15 patients undergoing cardiac transplantation (5 idiopathic dilated cardiomyopathies, 5 ischemic myopathies with myocardial infarcts, 5 diverse etiologies) by two different and complementary techniques: RNA dot blot analysis with specific cDNA probes, and primer extensions with an oligonucleotide common to alpha-cardiac and alpha-skeletal actins. In the case of dot blot analysis, quantification of each isoform was performed by using as standards RNA transcripts obtained from cloned human alpha-actin sequences, and the total amount of sarcomeric actin mRNA was evaluated as a function of total poly(A+)RNA. We found that both isogenes are always coexpressed, and that the isoactin pattern changes during development. In utero and in neonatal hearts, alpha-skeletal actin mRNA represents less than or equal to 20% of sarcomeric actins, it increases to 48 +/- 6% during the first decade after birth and becomes the predominant isoform of adult hearts (60.4 +/- 8.5%). The 15 adult failing hearts exhibited the same isoactin pattern as the control ones (62.84 +/- 11.06%), and there was no difference in expression between patients with dilated cardiomyopathy or ischemic heart disease. These observations demonstrate that cardiac development in man, in contrast to rodent heart, is characterized by an up-regulation of the skeletal actin gene, the expression of which does not change in hypertrophied and failing hearts, and suggest that the actin and myosin heavy chain families are independently regulated in human heart.

Actins↗
PubMed1991

Production of neutrons from water, polyethylene, tissue equivalent material and CR-39 irradiated with 2.5-30 MeV photons.

Photoneutron yields from water, polyethylene, tissue substitute and CR-39 have been calculated for the photon energy range of 2 to 30 MeV, using a previously established method and photoneutron production data on hydrogen, carbon, nitrogen and oxygen. The rarer isotopes of the constituent elements of these compounds, namely 2H, 13C, 15N, 17O and 18O, have been taken into account and neutrons are shown to be produced for photon energies above 2.2 MeV, the (gamma, n) threshold for 2H. The data are useful for estimating neutron production in materials located in the vicinity of a megavoltage radiotherapy beam. Substances such as those considered here are often used as filtration, phantom or scattering material and as components of neutron dosimetry detectors. Photoneutrons produced in such materials may need to be taken into consideration when carrying out neutron dosimetry in the presence of photons in this energy range, especially when the neutron flux is several orders of magnitude less than that of the photons.

Models, Structural↗
PubMed1990

The effects of extracellular magnesium on myoplasmic [Ca2+] in malignant hyperthermia susceptible swine.

It is now well established that the pathophysiology of the malignant hyperthermia (MH) syndrome is related to a malfunction of intracellular calcium homeostasis. Magnesium plays important roles in the basic contractile properties of muscle, and many of its actions are antagonistic to those of calcium. The aim of this study was to determine the effectiveness of magnesium sulphate to prevent the MH episode in susceptible animals and correlate this with its effects on the intracellular free calcium [( Ca2+]i). The experiments were carried out using six control (Yorkshire) and ten MH-susceptible crossbred swine (Poland China X Pietrain). After determination of resting concentrations of [Ca2+]i and [Mg2+]i, each animal was given either two iv bolus doses of 50 mg/kg or one iv bolus of 100 mg/kg of MgSO4. The resting [Ca2+]i and [Mg2+]i were determined by means of ion-selective microelectrodes. The resting [Ca2+]i in normal muscle fibers was 0.11 +/- 0.01 microM (mean +/- SEM), whereas in the MH muscles the resting [Ca2+]i was 0.36 +/- 0.01 microM. In neither group was the resting [Ca2+]i modified by MgSO4. This cumulative dose of MgSO4 (100 mg/kg) was not able to prevent the induction of an MH episode by 2% halothane. Although MgSO4 did not directly decrease [Ca2+]i, it did attenuate the increase in [Ca2+]i associated with the syndrome from 7.29 +/- 0.43 microM in untreated animals to 0.84 +/- 0.03 microM in MgSO4 pretreated swine. In addition, the limb rigidity that accompanies this increase in calcium was prevented by MgSO4 pretreatment. Baseline measurements of [Mg2+]i were not different in control and MH-susceptible muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Altered sarcoplasmic reticulum Ca2(+)-ATPase gene expression in the human ventricle during end-stage heart failure.

A decrease in the myocardial level of the mRNA encoding the Ca2(+)-ATPase of the sarcoplasmic reticulum (SR) has been recently reported during experimental cardiac hypertrophy and failure. To determine if such a deficit occurs in human end-stage heart failure, we compared the SR Ca2(+)-ATPase mRNA levels in left (LV) and right ventricular (RV) specimens from 13 patients undergoing cardiac transplantation (6 idiopathic dilated cardiomyopathies; 4 coronary artery diseases with myocardial infarctions; 3 diverse etiologies) with control heart samples using a rat cardiac SR Ca2(+)-ATPase cDNA probe. We observed a marked decrease in the mRNA for the Ca2(+)-ATPase relative to both the 18S ribosomal RNA and the myosin heavy chain mRNA in LV specimens of patients with heart failure compared to controls (-48%, P less than 0.01 and -47%, P less than 0.05, respectively). The LV ratio of Ca2(+)-ATPase mRNA to 18S RNA positively correlated with cardiac index (P less than 0.02). The RV ratio correlated negatively with systolic, diastolic and mean pulmonary arterial pressures (P less than 0.02, P less than 0.02, and P less than 0.01, respectively). We suggest that a decrease of the SR Ca2(+)-ATPase mRNA in the myocardium plays an important role in alterations of Ca2+ movements and myocardial relaxation reported during human end-stage heart failure.

Adult↗
PubMed1990

Properties of adult rat ventricular cells in long-term culture.

Adult mammalian cardiac myocytes in long-term culture offer advantages over intact heart tissue and freshly isolated ventricular cell preparations for a variety of experimental studies. To characterize this preparation in detail, we have examined the physiological properties of isolated adult rat ventricular cells maintained in culture for 10-14 days. Adult rat myocytes in longterm culture contracted spontaneously, with electrical coupling of adjacent cells, at 1-3 Hz. Most myocytes showed myofibrils with well-developed mitochondria and transverse tubular systems. They showed predominantly the V1 type myosin heavy chain isoform. In standard physiological superfusion media (pH 7.35), the intracellular pH of cultured cells measured with 2',7'-bis-carboxyethyl-carboxyfluorescein (BCECF) was 7.26 +/- 0.03, and was regulated by an amiloride-sensitive Na-H exchanger. The time-averaged free intracellular Ca2+ level of cultured adult rat myocytes measured with fura-2 at an extracellular Ca2+ level of 1 mM was 99.0 +/- 16.8 nM. Ouabain, Bay k 8644 or isoproterenol caused a significant rise in time-averaged intracellular [Ca2+], while the dihydropyridine Ca channel blocker nifedipine induced a decrease in intracellular [Ca2+]. Measurements of contractile state with an optical-video system demonstrated that ouabain. Bay k 8644, isoproterenol, or elevated extracellular [Ca2+] increased the amplitude of cell motion and the rates of both shortening and relaxation, while nifedipine lowered them. Microelectrode impalements indicated a resting potential of -75 +/- 1 mV and an action potential amplitude of 100 +/- 2 mV. Exposure of cultured adult rat cardiocytes to the thyroid hormone triiodothyronine (10 nM) for 48 hours resulted in a 2-fold increase in NaK-ATPase alpha-1 catalytic subunit mRNA accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Abnormal intracellular calcium handling, a major cause of systolic and diastolic dysfunction in ventricular myocardium from patients with heart failure.

Intracellular Ca2+ release and reuptake are necessary for normal contraction and relaxation of the human heart. Intracellular Ca2+ transients were recorded with aequorin during isometric contraction of myocardium from patients with end-stage heart failure. In contrast to controls, contractions and Ca2+ transients of muscles from failing hearts were markedly prolonged, and the Ca2+ transients exhibited two distinct components. Muscles from the failing hearts showed a diminished capacity to restore a low resting Ca2+ level during diastole. These data obtained in actively contracting human myocardium suggest that intracellular Ca2+ handling is abnormal and might cause both systolic and diastolic dysfunction in heart failure. The inotropic effectiveness of drugs that act to increase intracellular levels of cyclic adenosine monophosphate (AMP), such as beta-adrenergic agonists and phosphodiesterase inhibitors, was markedly reduced in muscles from patients with heart failure. In contrast, the effectiveness of inotropic stimulation with drugs that act by cyclic AMP-independent mechanisms, such as the cardiotonic steroids and DPI 201-106, were preserved. Stimulation of intracellular cyclic AMP production by the adenylate cyclase activator forskolin restored the inotropic response to phosphodiesterase inhibitors. These studies indicate that an abnormality in cyclic AMP production may be a fundamental defect in patients with end-stage heart failure that may markedly diminish the effectiveness of agents that depend on generation of this nucleotide for a positive inotropic effect. Moreover, deficient production of cyclic AMP seems, at least in part, to account for the reversal of the force-frequency relation that characterizes failing myocardium. Of interest, direct measurement of total cellular cyclic AMP content and protein kinase activity did not reveal significant differences between the control and myopathic tissue, suggesting the presence in human ventricular muscle of physiologically distinct compartmentalized pools of cyclic AMP. Finally, changes in the sensitivity of the contractile apparatus to Ca2+ also seem to play an important role in the differential responsiveness to drugs of myopathic versus normal human myocardium.

Calcium↗
PubMed1989

High-performance ion-exchange chromatography of myosin using a DEAE-5PW column.

High-performance ion-exchange chromatography of myosin using a DEAE-5PW packing was used to purify myosin from skeletal, cardiac and smooth muscle. This method produces high-speed resolution (30-min analysis) of myosin from contaminating myofibrillar proteins. The column has a high capacity for binding myosin (up to 1 g) and can be used for small-scale preparation of highly purified myosin. Gel analysis in the presence of sodium dodecyl sulfate showed recovery of myosin with very little contamination of other myofibrillar proteins. Myosin was also recovered from small biopsy samples (0.1 g) by a direct extraction technique with recovery of biological ATPase activity.

Animals↗
PubMed1989

Distribution and function of human ventricular beta adrenergic receptors in congestive heart failure.

This study examined the characteristics and distribution of sarcolemmal and light vesicular beta-adrenergic receptors (BAR) in left ventricular myocardium from 15 adults (aged 17 to 58 years) without left ventricular dysfunction or coronary artery disease and 29 patients (aged 14 to 53 years) with end-stage congestive heart failure (CHF). Sarcolemmal and intracellular fractions were prepared by 40,000 x g and 108,000 x g centrifugation, respectively. Agonist and antagonist binding properties were assessed by nonlinear computer modelling of isoproterenol-125I-pindolol (IPIN) displacement curves. Adenylate cyclase activity was also examined. Distribution of intracellular and sarcolemmal BAR was similar in normal and failing left ventricular myocardium, with intracellular BAR comprising 4.5 +/- 2.2% of total BAR in normal human heart and 5.7 +/- 5.1% of total BAR in CHF patients. For sarcolemmal BAR, antagonist affinity was similar for normal and CHF patients (KD IPIN in normals, 21.7 +/- 2.6 pM; KD IPIN in CHF, 20 +/- 2.3 pM). Agonist affinity was somewhat higher in CHF patients (KD isoproterenol in normals, 33 +/- 4.9 nM; KD isoproterenol in CHF, 6.2 +/- 1.5 nM). Sarcolemmal BAR number was reduced in CHF from 21.4 +/- 2.9 to 16.4 +/- 1.3 fmol/mg protein (P less than 0.04). Cyclic AMP production (pmol/mg protein/min above basal) was less in CHF after Gpp(NH)p stimulation (normals, 82 +/- 20; CHF, 27 +/- 9; P less than 0.01) and after stimulation with Gpp(NH)p + isoproterenol (normals, 129 +/- 25; CHF, 56 +/- 13; P less than 0.02). Stimulation with manganese + forskolin resulted in similar levels of cyclic AMP production in normals and in CHF patients. We conclude that: (a) sarcolemmal BAR number is reduced in CHF, but BAR are not redistributed intracellularly and (b) beta-adrenergic transmembrane signalling in CHF is also impaired at the level of the guanine nucleotide regulatory proteins.

Adenylyl Cyclases↗
PubMed1989

Developmental regulation of cardiac calcium channels and contractile sensitivity to [Ca]o.

To test the hypothesis that alterations in Ca-channel properties are a major factor in developmental changes in Ca sensitivity of the heart, we quantitated Ca-channel pharmacological properties during development of the chick heart and related pharmacological properties of Ca channels to the ability of channels to modulate contractility. Force-pCa curves were determined for chick hearts during development. From 3 to 5 days in ovo to 3-4 days posthatched, there was more than 1 log decrease in sensitivity to extracellular calcium concentration [( Ca]o) PN200-100 ligand-binding studies in cardiac membranes revealed no change in dissociation constant (KD; 660-1,040 pM; P = NS) during development, but between 18 days in ovo and 3-4 days posthatched, there was an increase in the number of binding sites from 2,180 +/- 260 to 3,530 +/- 410 fmol/mg (P less than 0.01). This change could not be attributed to nonspecific change in membrane composition. For intact ventricular strips, there was a developmental decrease in contractile sensitivity to the Ca-channel agonist BAY K 8644 despite the increase in Ca channels as identified by ligand binding. Thus abundance of dihydropyridine binding sites increases during chick ventricular development, but this is dissociated from the physiological response to a Ca-channel agonist.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Standards and calibration facilities of the Australian Radiation Laboratory for ionizing radiation beams.

The Australian Radiation Laboratory (ARL) maintains national standards of exposure and working standards of absorbed dose. Some of the exposure standards maintained by ARL have been compared recently with those of the Bureau International des Poids et Mesures (BIPM) for 100 to 250 kV x-rays and 60Co gamma rays. The absorbed dose standard for 60Co gamma rays is compared regularly with other standards through the International Atomic Energy Agency (IAEA) and World Health Organization (WHO). All results show good agreement. A range of calibration qualities is offered at ARL, extending from 10 kV x-rays to 60Co gamma rays. A system of regional facilities has been developed for radiation protection calibrations; each facility is supplied with a set of 137Cs sources and an ionization chamber and electrometer for use with x-rays. The system is now almost complete throughout Australia.

Australia↗
PubMed1988

Myoplasmic free [Ca2+] during a malignant hyperthermia episode in swine.

Malignant hyperthermia (MH) is a genetic syndrome usually initiated by exposure to volatile anesthetic agents or depolarizing neuromuscular blocking agents. We have used Ca2+-selective microelectrodes to measure in vivo the intracellular ionized calcium ([Ca2+]i) in skeletal muscle fibers of MH-susceptible swines before and during hyperthermic episodes and also after dantrolene administration. The animals were anesthetized with thiopental and fentanyl and maintained with a mixture of nitrous oxide (66%) and oxygen (34%). The malignant hyperthermic episode was triggered by exposure to halothane. Determinations of [Ca2+]i during the episode show an increase from 0.44 +/- 0.01 microM +/- SEM, n = 20) to 8.44 +/- 0.68 microM (mean +/- SEM, n = 10). Administration of dantrolene (2 mg/kg) during the hyperthermic episode reduces [Ca2+]i to 0.17 + 0.01 microM (mean +/- SEM, n = 10) and reverses the clinical symptoms. These results show that the MH episode is associated with an increase in the myoplasmic free Ca2+ concentration and that the therapeutic effect of dantrolene is related to a decrease in [Ca2+]i.

Animals↗
PubMed1988

Photoneutron production in tissue during high energy bremsstrahlung radiotherapy.

The total yield of photoneutrons from soft-tissue-equivalent material irradiated with bremsstrahlung of end-point energy from 6 to 28 MeV has been measured using the 35 MeV University of Melbourne Betatron and a 4 pi Halpern-type neutron detector. The results are compared with calculations based on the cross sections for neutron production of the constituent elements of tissue (H, C, N and O). It is shown that the less common isotopes of the constituent elements (2H, 13C, 15N, 17O, 18O) must be included to obtain agreement with the measured yields. The dose imparted to tissue by the photoneutrons is calculated for the energy range from 6 to 30 MeV and is compared with other available estimates. Further, the dose equivalent due to all photonuclear reactions in tissue is estimated for energies of 24 and 30 MeV.

Models, Structural↗
PubMed1988

Changes in myofibrillar content and Mg-ATPase activity in ventricular tissues from patients with heart failure caused by coronary artery disease, cardiomyopathy, or mitral valve insufficiency.

Force development and shortening by cardiac muscle occur as a result of the interaction between actin and myosin within the myofibrillar lattice. This interaction is dependent upon intracellular ionized calcium and is controlled by the troponin-tropomyosin regulatory proteins situated along the actin filament. In this study, we compared the myofibrillar content and myofibrillar Mg-ATPase activity of normal human ventricular muscle with that of ventricular muscle from patients in end-stage failure caused by coronary artery disease or cardiomyopathy and ventricular muscle from patients with heart failure due to mitral valve insufficiency. The results show that the amount of myofibrillar protein (mg/g wet wt ventricle) in hearts in end-stage failure (coronary artery disease and cardiomyopathy) is significantly lower compared with normal hearts and hearts in failure due to mitral valve insufficiency. However, the Mg-ATPase activity of myofibrils from hearts in both end-stage failure and failure due to mitral valve insufficiency is significantly lower compared with myofibrils from normal hearts. The data suggest that the reduction in the amount of myofibrillar protein in ventricular tissue is a pivotal event that may be responsible for the progression of heart disease to the point of end-stage failure.

Ca(2+) Mg(2+)-ATPase↗
PubMed1988

Modulation by the thyroid state of intracellular calcium and contractility in ferret ventricular muscle.

The purpose of this study was to determine whether the cardiac contractile abnormalities induced by hyperthyroidism or hypothyroidism are caused by changes in intracellular Ca2+ handling or by alterations in the distribution of isoenzymes of ventricular myosin. Right ventricular papillary muscles obtained from euthyroid ferrets and ferrets treated with L-thyroxine (hyperthyroid) or methimazole (hypothyroid) were loaded with the calcium indicator aequorin for recording intracellular Ca2+ levels during isometric contraction. In muscles from the hypothyroid ferrets, peak tension was reduced and the duration of contraction prolonged compared to the controls; these changes were associated with a Ca2+ transient of decreased amplitude and prolonged duration. Hyperthyroidism produced opposite changes in the time course of the Ca2+ transient and the associated isometric contraction. Native polyacrylamide gel electrophoresis was performed on myosin extracted from the right ventricular free wall of control and treated ferrets. The hyperthyroid state was associated with new myosin formation as indicated by the appearance of three myosin bands on the pyrophosphate gel. Gels of myosin from hypothyroid and euthyroid ferrets showed a single band that migrated with the slowest of the three bands from the hyperthyroid ferrets. These results suggest that changes in both Ca2+ handling and myosin isoenzymes may contribute to the contractile abnormalities observed in hyperthyroidism. Alterations in intracellular Ca2+ handling alone may account for the contractile changes induced by hypothyroidism.

Animals↗
PubMed1988

An alternate method for the determination of functional residual capacity in a plethysmograph.

The validity of measuring thoracic gas volume using a single inspiratory effort against an occlusion (Vtginsp) was determined in children and young adults (8 normal control subjects and 17 patients with cystic fibrosis [CF] or asthma). In addition, the validity of the measurement of Vtg at FRC in children at a low panting frequency (Vtgpant) was also determined. During both the panting (encouraged to be about 1 Hz) and inspiratory maneuvers, mouth pressure (Pm) and esophageal pressure (Pes) were measured simultaneously. Hence, Vtgpant and Vtginsp were determined using both delta Pm and delta Pes. Vtginsp using delta Pm was found to be similar to Vtginsp using delta Pes. Values for Vtgpant using either delta Pm or delta Pes were also found to be similar. The percent difference between Vtginsp (using delta Pm) and the average of Vtgpant and Vtginsp (using delta Pes for both) was calculated as an indication of the error of the inspiratory method. The error ranged from -13 to +13% and did not correlate with indices of air-flow limitation or hyperinflation. We have found that Vtginsp can be used to determine FRC in normal children and young adults as well as in those with CF or asthma. In addition, we have also validated the use of Vtgpant at a low panting frequency in these subjects.

Adolescent↗