Bilateral traumatic posterior hip dislocation: CT findings.
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Accidents, Traffic↗
Biomedical subjects
P J O'Brien
Publications and source records attributed to P J O'Brien.
The remodelling of skeletal muscle for indefatigable hemodynamic work.
Skeletal muscle possesses inherent plasticity of gene expression. Low frequency pulse-train stimulation can remodel the biochemical machinery that confers physiological expression and fatigue resistance approaching that of the myocardium. This fatigue-resistant muscle can generate sufficient force to meet the power requirements for useful cardiac work. This ultimate goal is currently being pursued in models of cardiomyoplasty and muscle-powered cardiac assist devices. In this article, we review the three major subcellular systems subserving canine skeletal muscle transformation and compare them to those of cardiac muscle. The magnitude of the problem of clinical heart failure and the feasibility of fatigue-resistant skeletal muscle joining the therapeutic armamentarium are addressed. The adaptation and transformation of fast-twitch skeletal muscle in response to chronic electrical stimulation augers therapeutic potential as an endogenous, readily available power source for myocardial assistance. The basis mechanisms of skeletal muscle fatigue require elucidation to gain a complete and thorough understanding of how to manipulate this property to provide continuous hemodynamic work.
Animals↗
Cardiac and muscle fatigue due to relative functional overload induced by excessive stimulation, hypersensitive excitation-contraction coupling, or diminished performance capacity correlates with sarcoplasmic reticulum failure.
The development of muscle fatigue due to exhaustive exercise is associated with impaired sarcoplasmic reticulum (SR) Ca-transport activity. This study tested the hypothesis that SR failure is a consistent feature of cardiac and skeletal muscle fatigue owing to relative functional overload regardless of the method of induction: excessive stimulation, diminished performance capacity, or excessive excitation-contraction coupling. The Ca-transport activity was determined using three unique models of muscle fatigue: chronic and rapid ventricular pacing in dogs; metabolic inhibition caused by global cardiac ischemia in swine; and the hypermetabolic syndrome of porcine malignant hyperthermia (MH). Both pacing- and ischemia-induced fatigue resulted in reduction of SR Ca-transport ATPase activity: from 275 +/- 58 to 159 +/- 57 nmol.min-1.mg-1 (mU/mg) and from 577 +/- 82 to 177 +/- 133 mU/mg, respectively. Both pacing-induced fatigue and halothane-induced MH resulted in reduction of Ca-sequestration activity of muscle homogenates from 5.95 +/- 2.4 to 3.11 +/- 0.67 nM/s at 300 nM Ca and 38.7 +/- 10.5 to 16.3 +/- 8.0 nM/s at 1500 nM Ca, respectively (all p less than 0.01). The isolated SR Ca-ATPase activity correlated with Ca-sequestration activity of myocardial homogenates (r = 0.76; p less than 0.005). Different models were used to study the relationship of Ca-transport activity with relaxation function, degree of acidosis, and ionized Ca concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Myocardial calcium cycling defect in furazolidone cardiomyopathy.
We have previously demonstrated that in furazolidone-induced congestive heart failure in turkeys the specific Ca(2+)-ATPase activity of myocardial sarcoplasmic reticulum (SR) is 60% increased in compensation for a 50% depression in net Ca(2+)-sequestration activity. This study tested the hypothesis that SR Ca(2+)-uptake and Ca(2+)-ATPase activities were uncoupled in this cardiomyopathy because of increased Ca(2+)-release channel activity. A novel microassay was used to monitor Ca2+ transport by myocardial homogenates using the fluorescent Ca2+ dye indo 1 to indicate extravesicular ionized Ca2+. The method is applied to cyropreserved biopsy specimens of myocardium and requires only 50 mg tissue. Both SR Ca(2+)-pump and SR Ca(2+)-channel activity were estimated using the channel-inhibitor ruthenium red (RR) and the mitochondrial inhibitor sodium azide. The specificity of the RR inhibition was confirmed using ryanodine. Cardiomyopathy was induced in 2-week-old turkey poults by the addition of 0.07% furazolidone to their feed for 4 weeks. Compared with controls, myocardial maximal Ca(2+)-channel activity relative to maximal Ca(2+)-pump activity was 22% greater and duration of Ca(2+)-channel activity was 100% increased. However, the heart failure birds had 43 and 53% decreases in absolute maximal Ca(2+)-pumping and Ca(2+)-channel activities, respectively. The abnormal Ca(2+)-channel activity resulted in 200% greater time before initiation of net Ca2+ sequestration and 700% greater final myocardial Ca2+ concentrations. For all birds, the Ca(2+)-accumulating activity was highly correlated with Ca(2+)-release activity (all p less than 0.05). These data indicate that in this animal model of congestive heart failure there is defective SR Ca(2+)-channel function resulting in abnormal Ca2+ homeostasis.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Measurement of insulin-like growth factor-II in physiological fluids and tissues. I. An improved extraction procedure and radioimmunoassay for human and rat fluids.
The measurement of serum insulin-like growth factors (IGFs) in serum is complicated by the presence of high affinity IGF-binding proteins. The accurate measurement of IGFs by radioligand binding assays requires that the interference from binding proteins be eliminated. Acid-gel chromatography, the standard method for removing binding proteins, is laborious and time consuming. Alternative methods for extracting serum IGFs include the use of HCl-ethanol treatment and reverse phase minicolumns. However, these methods are unsuitable for use with serum for some species, such as rat and sheep, due to incomplete removal of binding proteins. We developed a fast protein liquid chromatography size-exclusion chromatographic method for characterizing the presence of IGF-binding proteins in physiological fluids and used this method to systematically investigate different combinations of acids and organic solvents as potential extraction methods for IGFs. We developed and validated an improved extraction procedure that uses formic acid, Tween-20, and acetone. The new extraction method was used in conjunction with purified biosynthetic human IGF-II and a commercially available anti-IGF-II monoclonal antibody in the development of an improved RIA for IGF-II. The new RIA is sensitive (5.0 pg/tube), specific (IGF-I cross-reactivity, less than 1%), and reproducible [interassay precision (coefficient of variation), less than 9.2%). We measured the serum concentrations of IGF-II in adults and found a significant difference between normal subjects and individuals with insulin-dependent diabetes mellitus.
Adult↗
Molecular mechanism for prevention of N-acetyl-p-benzoquinoneimine cytotoxicity by the permeable thiol drugs diethyldithiocarbamate and dithiothreitol.
The present study was carried out to elucidate the mechanism by which the permeable thiol drug diethyldithiocarbamate (DEDC) exhibited an antidotal effect against acetaminophen-induced hepatotoxicity in vivo. DEDC was found to act as an antidote against acetaminophen-induced cytotoxicity in hepatocytes isolated from a pyrazole-pretreated rat without affecting cytochrome P-450 levels. The mechanism of protection exhibited against reactive intermediate N-acetyl-p-benzoquinoneimine (NAPQI)-induced cytotoxicity by DEDC was then investigated and compared with that exhibited by the permeable thiol-reductant dithiothreitol (DTT). Cytotoxicity induced by the dimethylated analogue 2,6-dimethyl-N-acetyl-p-benzoquinoneimine (2,6-diMeNAPQI) was prevented if the hepatocytes were preincubated with DEDC for 5 min and removed before addition of 2,6-diMeNAPQI. Both DEDC and DTT were also found to act as antidotes against NAPQI- and 2,6-diMeNAPQI-induced cytotoxicity in isolated rat hepatocytes if added within 2 min of the addition of the quinoneimines. However, the addition of DEDC or DTT 10 min after either quinoneimine did not prevent subsequent cytotoxicity or restore GSH levels, indicating that the alkylation of GSH and of protein thiols was irreversible at that time. Fast atom bombardment mass spectrometry was used to show that DEDC formed conjugates with both NAPQI and 2,6-diMeNAPQI. Furthermore, these conjugates were found to be nontoxic. This suggests that DEDC acts as a trap for the toxic quinoneimines, thus preventing alkylation of essential macromolecules. In contrast, DTT reduced the quinoneimines to their respective nontoxic parent compounds and presumably also reduced mixed-protein disulfides and GSSG, thereby regenerating protein thiols and GSH. Therefore, this study suggests that DEDC and DTT act as antidotes by two different mechanisms.
Acetaminophen↗
Effects of mild cardiac hypertrophy, induced by volume overload in turkeys, on myocardial sarcoplasmic reticulum calcium-pump and calcium-channel activities and on the creatine kinase system.
Recent studies indicate that in animals with marked cardiac hypertrophy, there is depressed function of Ca2+ sequestration by myocardial sarcoplasmic reticulum (SR) because of down regulation of the Ca(2+)-ATPase gene. However, in several animal models we have observed enhancement of myocardial Ca2+ sequestration in response to chronic cardiac stimulation. We tested the hypothesis that in animals with mild cardiac hypertrophy, there is enhanced Ca(2+)-cycling activity by the SR Ca2+ pump and Ca(2+)-release channel. Because creatine kinase activity is consistently decreased in cardiomyopathy, we also determined whether enhanced Ca2+ cycling was accompanied by down regulation or inhibition of the creatine kinase system. Mild cardiac hypertrophy was induced by volume overload; 2% salt was added to the diet of 2-week-old turkey poults for 4 weeks. Compared with age-matched controls, volume overload resulted in 14.3% increase in heart weight and 21.5% increase in heart-to-body weight ratios. The hypertrophied heart had approximately 20% increased activities of the SR Ca2+ pump and the SR Ca2+ channel. Net Ca2+ transport was increased by 16.5%. Compared with controls and in contrast to several other myocardial enzymes, creatine kinase activity was diminished in the hypertrophied hearts by 23% and creatine content was decreased by 8%. Differences between groups were not detected for lactate dehydrogenase, aspartate transaminase, and alanine transaminase. We concluded that an early adaptation of the myocardium undergoing hypertrophy in compensatory response to functional overload is an enhancement of Ca2+ cycling activity by the Ca2+ pump and Ca2+ channel of the SR.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Lymphocyte calcium extrusion: kinetic and thermodynamic measurements using ratiometric dual-emission spectrofluorometry.
A method is described for monitoring intracellular ionized calcium (Ca2+) and determining kinetic and thermodynamic parameters of Ca2(+)-extrusion from intact lymphocytes. The method uses ratiometric spectrofluorometry and the fluorescent Ca2+ dye indo-1. Lymphocytes were loaded with calcium and placed in a low calcium medium. A novel formula for calculation of intracellular Ca2+ that corrects for background fluorescence and fluorescence quenching was used. Calcium extrusion resulted in exponential decrease in cytoplasmic Ca2+ with a rate constant of 0.031 +/- 0.003 sec-1, maximal rate of 23 +/- 7 nM/sec, dissociation constant of 366 +/- 63 nM, Hill coefficient of 2.3 +/- 0.4, Q10 of 2.58 +/- 0.28, and activation energy of 18.3 Kcal/mol. This method should allow for characterization of the Ca2(+)-extrusion system of lymphocytes and may be applicable to other blood cell types.
Animals↗
Calcium sequestration by isolated sarcoplasmic reticulum: real-time monitoring using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1.
This study demonstrates a simple, rapid, and reproducible microassay for real-time monitoring of Ca2(+)-sequestration by isolated sarcoplasmic reticulum (SR) using ratiometric dual-emission spectrofluorometry and the fluorescent calcium-binding dye indo-1. The SR membranes were isolated by differential centrifugation and suspended in a medium including Ca2+, indo-1, ATP and oxalate. As Ca2+ was sequestered by SR, Ca2(+)-bound indo-1 fluorescence decreased equivalently but reciprocally to the increase in Ca2(+)-free indo-1 fluorescence. The kinetic and thermodynamic properties of Ca2(+)-transport measured fluorometrically were similar to those measured radiometrically by 45Ca2+, with the exception that the former monitors changes in free Ca2+ whereas the latter monitors total Ca2+. An estimate of the maximal rate of change in total Ca2+ could be made by multiplying the maximal rate of change in free Ca2+ by the ratio of initial total Ca2+ to free Ca2+ concentration.
Animals↗
Microassay for malignant hyperthermia susceptibility: hypersensitive ligand-gating of the Ca channel in muscle sarcoplasmic reticulum causes increased amounts and rates of Ca-release.
A crude preparation of heavy sarcoplasmic reticulum (HSR) was isolated using 1 gram of muscle obtained from swine susceptible to malignant hyperthermia (MH) and from control swine. The caffeine and ATP concentration-dependency of Ca-release was determined using suction filtration with radioisotopic 45Ca as a tracer. Rates of release were determined using a rapid filtration system. Caffeine and ATP-induced Ca-release from MH-susceptible (MHS) HSR occurred at one-tenth the concentration of agonist that was required for control muscle HSR. No differences in rates and amounts of release were observed when agonist concentrations were used that caused maximum release for controls. However, at the threshold concentration of caffeine causing release for control HSR, the MHS HSR released 4-times as much Ca and at 3-times the rate of controls. These findings indicate that increased rates and amounts of Ca-release are due to the hypersensitivity of the Ca-release channel of HSR and that this abnormality can be detected using 1 gram of muscle.
Adenosine Triphosphate↗
Differential cytotoxicity of diaziquone toward Chinese hamster ovary cells under hypoxic and aerobic exposure conditions.
Diaziquone [AZQ, 2,5-bis(carboethoxyamino)-3,6-diaziridinyl-1,4-benzoquinone] has been investigated for its toxicity toward Chinese hamster ovary cells AA8-4 under both aerobic and hypoxic conditions. Under acute (1-5 h) exposures to 2.5-10 microM AZQ in alpha-medium plus 10% fetal calf serum, AZQ showed an approximately linear concentration x time dependency for cell killing which was 3-4 times less under hypoxic compared to aerobic conditions. This selective toxicity toward hypoxic cells was prevented by low levels of oxygen. Under aerobic exposure conditions the toxicity of 2.5 microM AZQ was greatly increased by addition of 1-2 mM ascorbate. This ascorbate mediated toxicity of AZQ, presumably extracellular, could be prevented by the simultaneous addition of catalase. Under hypoxic exposure conditions there was no enhancement of AZQ toxicity by ascorbate or protection by catalase. The present results are consistent with two mechanisms for AZQ toxicity proposed earlier by others: toxicity due to (a) redox cycling and increased levels of oxidative stress and (b) reduction of the quinone leading to enhanced reactivity of the aziridines. The relative potency of AZQ as a hypoxic or aerobic cell selective toxin is determined by the balance between these two mechanisms.
Aerobiosis↗
Modulation of benzoquinone-induced cytotoxicity by diethyldithiocarbamate in isolated hepatocytes.
The copper-chelating thiol drug diethyldithiocarbamate protected isolated hepatocytes from benzoquinone-induced alkylation cytotoxicity by reacting with benzoquinone and forming a conjugate which was identified by fast atom bombardment mass spectrometry as 2-(diethyldithiocarbamate-S-yl) hydroquinone. In contrast to benzoquinone, the conjugate was not cytotoxic to isolated hepatocytes. The thiol reductant dithiothreitol had no effect on benzoquinone-induced alkylation cytotoxicity. However, inactivation of catalase in the hepatocytes with azide and addition of the reducing agent ascorbate markedly enhanced the cytotoxicity of the conjugate but did not affect benzoquinone-induced cytotoxicity. Furthermore, inactivation of glutathione reductase and catalase in hepatocytes greatly enhanced the cytotoxicity of the conjugate and caused oxidation of GSH to GSSG. The conjugate also stimulated cyanide-resistant respiration, which suggests that the conjugate undergoes futile redox cycling resulting in the formation of hydrogen peroxide which causes cytotoxicity in isolated hepatocytes only if the peroxide detoxifying enzymes are inactivated. Diethyldithiocarbamate does, however, protect uncompromised isolated hepatocytes from benzoquinone cytotoxicity by conjugating benzoquinone, thereby preventing the electrophile from alkylating essential macromolecules. Diethyldithiocarbamate therefore changed the initiating cytotoxic mechanism of benzoquinone from alkylation to oxidative stress, which was less toxic.
Animals↗
Myeloperoxidase catalysed cooxidative metabolism of methimazole: oxidation of glutathione and NADH by free radical intermediates.
The myeloperoxidase catalysed oxidation of methimazole in the presence of NADH or GSH resulted in oxygen uptake suggesting that metabolism proceeded via a one electron mechanism. The GSH was oxidised to GSSG and the thiyl radical could be trapped with DMPO while NADH was oxidized to NAD+. Metabolism proceeded without the inactivation of the enzyme myeloperoxidase. Myeloperoxidase catalyzed oxidation of other substrates which proceed via one electron intermediates; 2,6-dimethylphenol, N,N,N',N'-tetramethyl-phenylenediamine and luminol, were all stimulated by methimazole providing further evidence for a methimazole free radical. The presence of iodide stimulated the oxidation of methimazole but inhibited the oxygen uptake in the presence of GSH or NADH suggesting that metabolism in this case proceeded by a two electron mechanism. In contrast, another S-thioureylene drug, thiourea; did not cause oxygen uptake when oxidised in the presence of GSH or NADH indicating that the myeloperoxidase oxidation of thiourea proceeded primarily by a two electron mechanism. The horseradish peroxidase catalysed one electron oxidation of p'p'-biphenol, and 3,3',5,5'-tetramethylbenzidine was reversibly inhibited by methimazole and thiourea by preventing the accumulation of oxidation products via reductive mechanisms whereas the reversible inhibition of guaiacol and luminol oxidation was the result of competitive inhibition. With p,p'-biphenol, and 3,3',5,5'-tetramethylbenzidine unstable adduct formation could be demonstrated.
Free Radicals↗
Reversible drug effects on the metabolic activation of polymorphonuclear leukocytes.
Polymorphonuclear leukocytes (PMNs) stimulated by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP) were effectively inhibited by chlorpromazine (10 microM) and azelastine (20 microM) in terms of superoxide generation, and restored by the addition of dodecylbenzenesulfonic acid (DBS) in a range of concentrations from 20 to 40 microM. The stimulation of superoxide generation by DBS was also inactivated by dodecylamine (DA) but was restored by the subsequent addition of DBS. A dose dependent competitive inhibition and activation of leukocytes was observed between azelastine (10 microM) and DBS (20 microM). The release of arachidonic acid from leukocytes activated by the chemotactic peptide was decreased by DA or chlorpromazine, but could be restored by DBS. The changes in membrane potential of leukocytes as monitored by cyanine dye were also decreased by DA, chlorpromazine or azelastine. These observations indicate that some cationic drugs reversibly inhibit membrane bound enzymes or receptors. The physiological responses of these inhibited PMNs can then be restored by appropriate anionic amphiphiles.
Amines↗
Metabolic labeling of rod outer segment phospholipids in miniature poodles with progressive rod-cone degeneration (prcd).
The recessive genetic defect in miniature poodles which results in progressive rod-cone degeneration (prcd) has been investigated in an attempt to determine the biochemical abnormality involved. In the present study, the rod outer segments of young prcd affected miniature poodles and normal dogs have been compared with respect to the incorporation of intravitreally injected [3H]palmitic acid. [14C]linolenic acid, and [14C]docosahexaenoic acid into neutral lipids and phospholipids as well as [3H]palmitate and [14C]leucine into rhodopsin. In addition, 3 mm trephined punches of retinas were incubated with [3H]palmitic acid, [3H]arachidonic acid, [14C]linolenic acid, [3H]serine, [14C]glycerol and [14C]leucine. No difference in incorporation of labeled precursors into lipids or rhodospin was noted between prcd affected and normal retinas. Phosphatidyl choline appeared to function as a carrier of fatty acids to the rod outer segment where they were redistributed to other phospholipids. An interesting lack of conversion of the essential fatty acid linolenic acid to docosahexaenoic acid was noted in both normal and affected retinas. This conversion involves elongation and desaturation of linolenic acid and may take place primarily in extraretinal tissues such as the liver. This finding, in conjunction with a parallel study of plasma fatty acids which has shown significantly lower levels of docosahexaenoic acid in prcd affected poodles, points to a possible systemic defect in the metabolism or transport of docosahexaenoic acid, a fatty acid uniquely enriched in the photoreceptor outer segments.
Animals↗
The localization and timing of post-translational modifications of rat rhodopsin.
Rat retinas were labeled by incubation with, or intravitreal injection of, [14C]leucine along with tritiated palmitic acid, glucosamine or galactose. At selected intervals, subcellular fractions were prepared on linear sucrose gradients and rhodopsin was extracted and purified by affinity chromatography and gel electrophoresis. Time courses revealed that leucine rapidly and transiently labeled the rhodopsin in the rough endoplasmic reticulum (RER), with a maximum at 1.5 hr post-injection. Subsequently, the rod outer segments (ROS) contained the labeled rhodopsin, with the ROS labeling maximally at 6-12 hr. Palmitate labeling followed the same pattern but was subject to a delay, presumably because of a large intracellular pool of the fatty acid. With palmitate the RER rhodopsin was not maximally labeled until 12 hr. The acylation of rhodopsin takes place in the RER sometime after the polypeptide has been translated but before transport to the Golgi. Glucosamine labeling was also delayed because of intracellular pools of the sugar or its metabolic derivatives. But because of secondary glycosylation in the Golgi, the rhodopsin in the ROS also labeled maximally with glucosamine at about 6 hr. Administration of [3H]galactose resulted in the labeling of rhodopsin both in vivo and in vitro, in part possibly because of its conversion to mannose and subsequent insertion into the core oligosaccharide on the RER. However, in the ROS the ratio of tritium, derived from [3H]galactose, to [14C]leucine decreased by a factor of 2 between 6 and 24 hr post-injection. Moreover, between 6 and 12 hr post-injection, labeled rhodopsin molecules in the ROS underwent a shift in mobility on gels indicative of trimming to a lower molecular weight. Thus some sugar residues may be added to the rhodopsin in the inner segment and removed in the ROS.
Animals↗
Myocardial biochemical changes in furazolidone-induced cardiomyopathy of turkeys.
This study tested the hypothesis that membrane transport is the major biochemical system of the myocardium altered in furazolidone-induced cardiomyopathy (round heart disease), before the development of myocardial failure, and that metabolic enzymes and contractile proteins are less affected. Compared with controls, maximal percentage depression of activities of myocardium from furazolidone-treated birds were 40 for creatine kinase, 30 for glycolysis, 30 for glycogen, 20 for myofibrils, 20 for Krebs's cycle enzymes, 15 for fatty acid oxidation and 10 for total soluble protein. Sodium and potassium transport, antioxidant system activity, myosin, myosin isoenzyme patterns and amino acid aminotransferases were unaffected. In marked contrast, the calcium-transport ATPase activity of the sarcoplasmic reticulum had undergone a 60 per cent compensatory increase in activity. The pattern of biochemical changes observed is consistent with a role of ischaemia in the pathogenesis of round heart disease and indicates that calcium transport by the sarcoplasmic reticulum is the major biochemical system affected.
Animals↗
Genes that can be mutated to unmask hidden antigenic determinants in the cuticle of the nematode Caenorhabditis elegans.
Rabbit antisera directed against a mixture of proteins solubilized from the wild-type adult Caenorhabditis elegans cuticle were used to isolate mutants, induced by ethyl methanesulfonate treatment, that exhibit alterations in surface antigenicity by immunofluorescence. Genetic mapping and complementation data for four such mutations define two genes, srf-2(I) and srf-3(IV). The mutant phenotypes observed by immunofluorescence appear to result from unmasking of antigenic determinants that are normally hidden in the wild-type cuticle. In support of this hypothesis, surface radioiodination experiments indicate that components labeled on the wild-type surface are missing or less readily labeled on the surface of srf-2 and srf-3 mutants.
Animals↗