Biomedical subjects

P J O'Brien

Publications and source records attributed to P J O'Brien.

At least 181 records · Page 10Linked to original sources

PubMed1990

Rapid ventricular pacing of dogs to heart failure: biochemical and physiological studies.

Chronic, rapid ventricular pacing produces congestive heart failure in dogs. The objectives of this study were to determine whether or not (i) in vitro myocardial biochemical alterations reported for heart failure by volume or pressure overload also occurred with heart failure due to rate overload, and (ii) these biochemical alterations were related to relevant in vivo cardiac physiologic alterations. We compared 27 dogs that were paced to advanced heart failure with 21 sham-operated dogs. Dogs with heart failure had 55% lower left ventricular ejection fraction (22.5 +/- 7.6 vs. 50.5 +/- 5.1%) and cardiac index (81 +/- 22 vs. 178 +/- 48 mL.min-1.kg-1), 287% higher pulmonary capillary wedge pressure (27.5 +/- 6.8 vs. 7.1 +/- 3.4 mmHg; 1 mmHg = 133.3 Pa), and 64% greater left ventricular diastolic area (18.4 +/- 3.7 vs. 11.2 +/- 1.3 cm2) (all p less than 0.05). Dogs with heart failure also had (i) 69% lower norepinephrine (232 +/- 139 vs. 747 +/- 220 ng/g protein), (ii) 25-50% lower activities of myofibrillar Ca ATPase (0.188 +/- 0.026 vs. 0.253 +/- 0.051 U/mg myofibrils), sarcoplasmic reticulum Ca-transport ATPase (0.155 +/- 0.074 vs. 0.288 +/- 0.043 U/mg membrane), and the glycolytic enzyme phosphofructokinase (33.4 +/- 10.0 and 47.7 +/- 15.8 U/g), (iii) 32% higher activity of the beta-oxidation enzyme hydroxyacyl-CoA dehydrogenase (11.43 +/- 1.48 vs. 8.67 +/- 1.70 U/g), and (iv) 60% higher activity of Krebs cycle oxoglutarate dehydrogenase (2.89 +/- 0.77 vs. 1.81 +/- 0.95 U/g) (all p less than 0.05). No differences between groups were observed for isozyme patterns and ATPase activity of myosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Biochemical transformation of canine skeletal muscle for use in cardiac-assist devices.

Skeletal muscle has an inherent biochemical phenotypic plasticity that provides the possibility for it to be remodeled into a "heart-like" muscle for use in cardiac-assist devices. The purpose of this study was to chronically stimulate skeletal muscle electrically to transform the biochemical capacities of the three major subcellular systems (i.e., metabolic, calcium regulating, and contractile) to resemble those of heart muscle. The latissimus dorsi muscle (LDM) of mongrel dogs weighing 22-27 kg was stimulated via the thoracodorsal nerve at 2 Hz for 6-8 wk. This stimulation protocol reduced the phosphorylase (glycogenolytic) and phosphofructokinase (glycolytic) activities by 70%. The aerobic (citrate synthase activity) and fatty acid oxidative (3-hydroxyacyl-CoA dehydrogenase activity) capacities were not significantly increased by chronic stimulation and remained at about one-fourth those in the canine heart. The calcium-dependent sarcoplasmic reticulum adenosinetriphosphatase (ATPase) activity in the microsomal fraction, which was sixfold greater in the nonstimulated LDM than in the heart, was reduced by electrical stimulation to a level similar to that of the dog heart. The contractile capacity was evaluated by determining the percentage of types I and II fibers, the myofibrillar ATPase activity, and the proportion of myosin isoforms. The transformed muscle was comprised of 93 +/- 2% type I fibers, a myofibrillar ATPase activity similar to that in heart with primarily a slow-twitch muscle myosin isoform. In conclusion, electrical stimulation of canine LDM at 2 Hz for 6-8 wk resulted in two of the three biochemical systems, which confer physiological expression and fatigue resistance to muscle being transformed to resemble those of the myocardium.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1990

Peroxidase/hydrogen peroxide--or bone marrow homogenate/hydrogen peroxide--mediated activation of phenol and binding to protein.

1. 14C-Phenol was metabolized by rat bone marrow homogenate and H2O2. The homogenate catalyst, however, was inactivated by preincubation with H2O2, presumably due to inactivation of the enzyme(s) involved in phenol metabolism. 2. The majority of the metabolized 14C-phenol was bound to bone marrow proteins. o,o'-Biphenol and p,p'-biphenol were the principal non-protein-bound products. Ascorbate was unable to remove phenol oxidation products bound to protein, although o,o'-biphenol recovery from the reaction mixture was markedly enhanced. Prior alkylation of protein thiols with N-ethylmaleimide decreased the binding of 14C-phenol oxidation products to bone marrow proteins by only 10-20%. 3. 14C-Phenol (200 microM) metabolism by horseradish peroxidase (10 micrograms) and H2O2 (200 microM) also resulted in extensive binding to externally added bovine serum albumin. The absorption spectrum of 14C-phenol oxidation products bound to bovine serum albumin was similar to that of bound oxidation products of o,o'-biphenol but not of p,p'-biphenol. 4. Protease digestion of bovine serum albumin bound 14C-phenol oxidation products, followed by ethyl acetate extraction, extracted 75% of the 14C, indicating that most of the binding is probably non-covalent. Up to 32% of the 14C-phenol oxidation products binding to bovine serum albumin may be covalent, since derivation with dinitrofluorobenzene and extraction under acid, but not alkaline, conditions extracted the 14C. The percentage of metabolites covalently bound to bovine serum albumin was increased to 59% when horseradish peroxidase concentration was decreased to 0.2 micrograms. 5. The thiol groups of bovine serum albumin were unaffected by o,o'-biphenol oxidation products, slightly decreased by phenol oxidation products, but were completely depleted by p,p'-biphenol oxidation products. 6. These results indicate that o,o'-biphenol oxidation products are responsible for much of the 14C-phenol binding to protein.

Amino Acids↗

PubMed1990

Molecular mechanisms for bromotrichloromethane cytotoxicity in isolated rat hepatocytes.

1. Bromotrichloromethane added to isolated rat hepatocytes resulted in increased cell death as determined by trypan blue uptake. Toxicity increased in a concentration-dependent fashion between 2.0-5.0 M bromotrichloromethane. 2. Lipid peroxidation (malondialdehyde) increased in a time-dependent fashion but in contrast to toxicity reached a maximum level at 2.0 mM bromotrichloromethane. 3. Hypoxia increased the toxicity of bromotrichloromethane three-fold but only decreased the amount of lipid peroxidation to a small degree. 4. In spite of this poor correlation between toxicity and lipid peroxidation, the antioxidant butylated hydroxyanisole and the iron chelator desferal protected the cells from toxicity under both aerobic and hypoxic conditions and prevented lipid peroxidation. 5. During treatment with bromotrichloromethane, cellular glutathione levels slowly decreased and oxidized glutathione appeared in the media. The addition of cystine to the incubation media prevented the formation of extracellular oxidized glutathione, indicating that cellular glutathione had leaked from the cell during treatment and was oxidized in the incubation media. Although this suggested that glutathione does not play a protective role against bromotrichloromethane toxicity, diethyl maleate-pretreatment of the cells to decrease glutathione levels markedly increased bromotrichloromethane toxicity. 6. The addition of ascorbic acid to the incubation media increased bromotrichloromethane toxicity. This was attributed to the reductive activation of bromotrichloromethane in an iron and oxygen-dependent reaction. 7. It was concluded that peroxidation of essential phospholipids contributes to bromotrichloromethane-induced hepatocyte cytotoxicity.

Aerobiosis↗

PubMed1990

Toxicity of nitrobenzene compounds towards isolated hepatocytes: dependence on reduction potential.

1. The cytotoxicity of p-substituted nitrobenzenes towards isolated hepatocytes under aerobic or hypoxic conditions has been determined. The nitrobenzene concentration required to cause 50% cytoxicity in 2 h was a function of the one-electron reduction potential of the nitrobenzene, with the more cytotoxic compounds having the strongest electron-withdrawing substituents. 2. The effectiveness of the nitrobenzenes at causing cytotoxicity under aerobic but not hypoxic conditions was markedly increased if hepatocyte catalase was inhibited with azide. 3. Nitrobenzenes at cytotoxic concentrations induced cyanide-resistant respiration in isolated hepatocytes. Their effectiveness correlated with their cytotoxicity. 4. The rate of oxygen activation of these nitrobenzenes by ascorbate was also a function of the one-electron reduction potential. The nitro compounds with the strongest electron-withdrawing substituents were the most rapidly reduced. 5. Most nitrobenzenes were more cytotoxic under aerobic than hypoxic conditions. Ascorbate enhanced hypoxic, but not aerobic, cytotoxicity. 6. It was concluded that the cytotoxicity of different nitrobenzenes is related to their ease of reduction to nitro radical anions and nitrosobenzenes. Aerobic cytotoxicity is probably initiated by redox cycling and oxygen activation by the nitro radical anions whereas hypoxic cytotoxicity is probably initiated by the alkylation of macromolecules by nitrosobenzene metabolites.

Alkylation↗

PubMed1990

Malignant hyperthermia susceptibility: biochemical basis for pathogenesis and diagnosis.

Malignant hyperthermia (MH) is a hypermetabolic and hypercontractile syndrome triggered by anesthesia or various stressors that cause a sustained increase in sarcoplasmic ionized Ca. Susceptibility is apparently inherited in an autosomal dominant pattern. The primary molecular defect results in hypersensitive ligand-gating of the Ca-release channel of sarcoplasmic reticulum (SR) in skeletal muscle: channel opening is stimulated by abnormally low concentrations of agonist. We attribute MH to a mutation in the gene for the fast twitch muscle isoform of the Ca-channel, resulting in the expression of a cardiac-like isoform in fast muscle. Syndromes with some resemblance to MH can occur due to other genetic or acquired imbalances in Ca-flux across SR that favor net release of Ca. Either defective uptake or release can be detected as increased sensitivity of muscle to the contracture-producing effects of caffeine and halothane. Thus, caffeine and/or halothane contracture tests for MH-susceptibility may give false positives when there is decreased Ca-uptake, such as in muscular dystrophies. Ca-channel hypersensitivity and decreased Ca-uptake activity can be detected by assays using isolated SR. Functional assays using lymphocytes are being assessed as potential replacements for muscle contracture tests. Polymorphism analysis of proteins or nucleic acids for the MH or closely-linked genes has been used to trace the inheritance of MH-susceptibility.

Animals↗

PubMed1990

Update on trauma care in Canada. 3. Pelvic ring fractures.

Correct assessment and treatment of pelvic ring fractures in the multiply injured patient is important because it can result in fewer deaths and less long-term disability. There is ample evidence to show that surgical stabilization of unstable pelvic ring fractures is a life-saving intervention that should be done on the day of injury. Anterior frame external fixation is the standard form of treatment and is life-saving. However, it is not ideal biomechanically, and over the next decade open reduction and internal fixation of pelvic ring fractures will likely become the treatment of choice.

Clinical Protocols↗

PubMed1990

Canine stress syndrome/malignant hyperthermia susceptibility: calcium-homeostasis defect in muscle and lymphocytes.

This study provides the first comprehensive characterisation of the calcium (Ca) homeostasis defects found in muscle and lymphocytes of a malignant hyperthermia (MH)-susceptible dog. Novel findings regarding this dog are reported, compared to controls. First, a canine stress syndrome occurs, analogous to the porcine stress syndrome; susceptibility can be identified by exercise challenge testing. Secondly, caffeine causes Ca release from muscle sarcoplasmic reticulum in a greater amount and at a greater rate. Thirdly, there is a compensatory increase in Ca sequestration by sarcoplasmic reticulum. Fourthly, lymphocytes have lower cytosolic-free Ca and a greater ability to prevent Ca increase. Halothane increases Ca by a greater amount and rate. Fifthly, muscle is more resistant to the contracture-producing effects of caffeine, as occurs in the non-rigid variant of MH susceptibility in man. This resistance, despite increased caffeine-induced release through the Ca channel, may be attributable to increased Ca sequestration by sarcoplasmic reticulum. Finally, erythrocyte osmotic fragility and creatine kinase tests fail to distinguish between the MH-susceptible dog and controls.

Animals↗

PubMed1990

External fixation and delayed intramedullary nailing of open fractures of the tibial shaft. A sequential protocol.

Between 1983 and 1989, forty-one open fractures of the tibial shaft were treated with débridement and provisional external fixation, followed by delayed soft-tissue closure and subsequent intramedullary nailing with reaming. The average duration of external fixation was seventeen days (range, six to fifty-two days). The average time between removal of the fixator and intramedullary nailing was nine days (range, zero to twenty-four days). Of thirty-nine patients who had adequate follow-up, two (5 per cent) subsequently had a deep infection. Both infections healed, with retention of the nail and without chronic osteomyelitis. There were two nonunions and one delayed union. Satisfactory alignment was achieved in thirty-seven patients (95 per cent). This sequential protocol for treatment, which involved a short period of external fixation and thus minimized colonization of the pin tracks, yielded excellent results and a low rate of infection.

Adolescent↗

PubMed1990

Compensatory increase in calcium extrusion activity of untreated lymphocytes from swine susceptible to malignant hyperthermia.

We tested the hypothesis that lymphocytes from swine with susceptibility to malignant hyperthermia (MH) had calcium extrusion activity higher than unaffected swine. Cytoplasmic concentration of ionized calicum was determined by use of dual emission spectrofluorometry and measurement of the ratio of free to calcium-bound form of the fluorescent calcium dye indo-1. Net calcium accumulation and unidirectional calcium extrusion rate were dependent on intracellular calcium concentration. Calcium extrusion from calcium-loaded lymphocytes was monitored while calcium influx was inhibited by suspending the cells in calcium-free medium with a calcium chelator. Net calcium accumulation of untreated lymphocytes was monitored in calcium-replete medium. A novel method of calculation of ionized calcium was used. This method confirmed our previous findings of lower ionized calcium concentration (86 +/- 40 and 370 +/- 216 nmol/L; P less than 0.01) and slower rates of calcium accumulation 39 +/- 16 and 127 +/- 52 nmol/L/min) in untreated lymphocytes from MH-susceptible swine compared with controls. These changes were attributable to calcium extrusion activity two- to three-fold higher in lymphocytes of MH-susceptible swine (154 +/- 36 and 408 +/- 47 nmol/L/min at 175 nmol/L; 972 +/- 111 and 1,690 +/- 505 nmol/L/min at 425 nmol/L). These data were compatible with our model of higher calcium extrusion activity being a compensatory adaptation of MH-susceptible swine lymphocytes to their hypersensitivity to stimuli that increase cytoplasmic calcium concentration.

Animals↗

PubMed1989

Diaziquone-induced cytotoxicity in isolated rat hepatocytes.

2,5-Diaziridinyl-3,6-bis(carboethoxyamine)-1,4-benzoquinone (AZQ) is a lipid-soluble antitumor agent. The following evidence using isolated rat hepatocytes as a model for cytotoxicity studies suggests that, under aerobic conditions, AZQ participates in futile oxidation-reduction cycling and oxygen activation. The H2O2 formed can mediate oxidative stress cytotoxicity in compromised cells. (a) Addition of AZQ to hepatocytes causes the stoichiometric oxidation of glutathione (GSH) to oxidized glutathione. No subsequent reduction back to GSH occurred. This was found to be the result of reversible inactivation of glutathione reductase by AZQ. The extent of GSH oxidation increased with AZQ concentration. (b) Cytotoxicity occurred when AZQ concentrations were sufficient to completely deplete GSH. (c) Addition of AZQ to hepatocytes enhanced cyanide-resistant respiration. (d) If the hepatocytes were compromised with azide or cyanamide to inhibit catalase, cytotoxicity was increased 10-fold or 100-fold if ascorbate was also present. (e) AZQ readily induced Ca2+ release by energized mitochondria. Ascorbate markedly enhanced the effectiveness of AZQ, and catalase delayed Ca2+ release. H2O2 formed by aerobic oxidation-reduction cycling of AZQ may therefore cause mitochondrial Ca2+ release.

Animals↗

PubMed1989

Allyl alcohol- and acrolein-induced toxicity in isolated rat hepatocytes.

Incubation of isolated hepatocytes with allyl alcohol results in GSH depletion and subsequent cytotoxicity which is prevented by pyrazole, an inhibitor of alcohol dehydrogenase. Both GSH depletion and cytotoxicity were much more rapid when hepatocytes were incubated with acrolein, the reactive metabolite, and were not affected by pyrazole. However, cytotoxicity of both allyl alcohol and acrolein was enhanced by the aldehyde dehydrogenase inhibitors cyanamide and disulfiram. Malondialdehyde, a lipid peroxidation product, was also formed when hepatocytes were incubated with either agent, and treatment of the hepatocytes with a ferric ion chelator, desferrioxamine, or an antioxidant delayed the cytotoxicity without affecting GSH depletion. Although no GSSG was formed and addition of disulfide reductant dithiothreitol did not restore GSH levels, cytotoxicity was prevented if dithiothreitol was added some time after either agent.

1-Propanol↗

PubMed1989

Addition of the chromophore to rat rhodopsin is an early post-translational event.

Rat retinas were labeled either by intravitreal injection of [14C]leucine or by incubation with [3H]-leucine or [35S]-methionine. Subcellular fractions were prepared on linear sucrose gradients and rhodopsin was extracted with detergent and purified by chromatography on ConA-Sepharose. A fraction enriched in rough endoplasmic reticulum (RER) and substantially free of rod outer segments (ROS) was found to contain a light-sensitive protein exhibiting the properties of rhodopsin on ConA-Sepharose or Agarose chromatography and on SDS-polyacrylamide gel electrophoresis, as well as immunologically. Intravitreal injection of [3H]-retinol also labeled the rhodopsin in the RER under conditions in which the rhodopsin in the ROS was not heavily labeled. Thus the chromophore appears to be attached to opsin shortly after the apoprotein is translated in the RER.

Animals↗

PubMed1989

Metabolic labeling of normal canine rod outer segment phospholipids in vivo and in vitro.

Twenty-four hours after the intravitreal injection of [3H]palmitate and [14C]docosahexaenoate in dogs, the rod outer segment phospholipids are highly labeled. Palmitate is found predominantly in phosphatidylcholine (PC), with lesser amounts in phosphatidylethanolamine (PE) and very little in either phosphatidylserine (PS) or phosphatidylinositol (PI). Docosahexaenoate most heavily labeled PE followed by PC, with lesser amounts in PS and very little in PI. Two-hour incubations of 3 mm trephine buttons removed from dog retinas produced very similar patterns of labeling with palmitate and docosahexaenoate. In vitro incubation of retina buttons with [3H]arachidonate produced heavy labeling of PI, with much less in PC and very little in either PS or PE. [3H]Glycerol labeled in PC, PI and PE in descending order but PS almost not at all. [3H]Serine labeled PS predominantly, but small amounts were found in PC, PE and PI. The trephine retina buttons can be utilized for multiple-precursor incubations and studies of differential metabolism in retinal regions, particularly when studying scarce tissue from mutant animals or humans with inherited retinal degenerations.

Animals↗

PubMed1989

Diethyldithiocarbamate (DEDC) enhances quinone mediated oxidative stress cytotoxicity in isolated hepatocytes by forming toxic quinone conjugates.

The copper-chelating thiol drug, diethyldithiocarbamate (DEDC) had previously been used to inhibit superoxide dismutase (SOD) and enhance oxidative stress mediated cytotoxicity. Using isolated rat hepatocytes, it was confirmed that DEDC enhances oxidative stress cytotoxicity induced by 1,4-naphthoquinone (1,4-NQ) and 1,4-naphthoquinone-2-sulphonate (1,4-NQ-2S). However, equimolar concentrations of DEDC also enhances cytotoxicity induced by benzoquinone, previously shown to cause cytotoxicity as a result of alkylation and not oxidative stress. Higher DEDC concentrations on the other hand protected against benzoquinone-induced cytotoxicity. Finally, the susceptibility of hepatocytes to quinone mediated oxidative stress cytotoxicity was not enhanced if the DEDC was removed before incubating the hepatocytes with naphthoquinone or benzoquinone. Enhanced oxidative stress cytotoxicity was only observed if the DEDC was present when hepatocytes were treated with quinones. It was concluded that DEDC forms conjugates with quinones which undergo futile redox cycling in the hepatocyte and form H2O2 as well as increase the susceptibility of hepatocytes to H2O2.

Animals↗

PubMed1989

The Adriamycin (doxorubicin)-induced inactivation of cytochrome c oxidase depends on the presence of iron or copper.

1. It was confirmed that Adriamycin (doxorubicin) inactivates cytochrome c oxidase upon incubation. However, further investigation shows that this inactivation is strongly dependent upon the presence of Fe3+ and Cu2+. Trace amounts of these transition metal ions, present in phosphate and Tris buffers, bind strongly to the Adriamycin and the complex formed is responsible for the inactivation of cytochrome c oxidase. No Adriamycin-induced inactivation of cytochrome c oxidase occurred in the presence of EDTA or in phosphate buffers purified on a cation exchange column to remove trace metals. 2. The metal ion-induced inactivation of cytochrome c oxidase by Adriamycin results in significant decreases in both the maximum velocity and the Michaelis constant. The degree of inactivation is strongly dependent on the Fe3+ concentration. 3. Cardiolipin partially protects against cytochrome c oxidase inactivation, presumably by binding to the cytochrome c oxidase, whereas catalase or superoxide dismutase partially protect by scavenging damaging reactive oxygen species generated within a Fe3+-Adriamycin-enzyme complex.

Animals↗

PubMed1989

Fatty acid metabolism in normal miniature poodles and those affected with progressive rod-cone degeneration (prcd).

It is possible that a genetic defect observed in the prcd poodle involves the abnormality of an enzyme which functions in phospholipid or lipoprotein metabolism. In our studies thus far, we have been unable to detect any defect in retinal phospholipid biosynthesis, but we have noted a decrease in plasma levels of 22:6w3 which may be a result of an enzyme defect in liver biosynthesis of 22:6w3 from its dietary precursor, linolenic acid, or some defect in the blood lipoprotein transport of this essential fatty acid. If 22:6w3 is essential to the normal elaboration and functioning of the photoreceptor outer segment, it is possible that decreased access to this fatty acid due to lower blood levels of 22:6w3 could cause photoreceptor abnormalities. Further studies are needed to confirm the possible defect in delta-4 desaturase activity and possible dietary modification of the course of this prcd retinal degeneration.

Animals↗

PubMed1989

Porcine malignant hyperthermia susceptibility: halothane-induced increase in cytoplasmic free calcium in lymphocytes.

We tested the hypothesis that lymphocytes from swine with susceptibility to malignant hyperthermia (MH) had increased sensitivity to the membrane-perturbing effects of halothane that increase cytoplasmic calcium. Cytoplasmic concentration of ionized calcium in lymphocytes isolated from blood was determined in the presence and absence of halothane for 10 Pietrain x Poland China swine that were susceptible to MH and 20 Yorkshire swine that were resistant to MH. Calcium was determined by dual-emission spectrofluorometry and by measuring the ratio of free to calcium-bound form of the fluorescent calcium dye Indo-1. Mean values for calcium concentrations in lymphocytes from MH-susceptible (MHS) swine were 80% less than control values (40.5 +/- 38.8 and 185.3 +/- 91.6 nmol/L; P less than 0.01). Untreated lymphocytes from MHS swine accumulated calcium at half the rate observed for controls. Exposure to 1 mmol/L halothane resulted in a 3-fold increase of free calcium concentration to 127.9 +/- 81.3 nmol/L in the lymphocytes of MHS swine, but had no significant effect on lymphocytes from control swine (225.0 +/- 91.4; P less than 0.01). Exposure to 2 mmol/L halothane resulted in a 6-fold increase of free calcium concentration to 255.9 +/- 91.4 nmol/L in lymphocytes from MHS swine and a 63% increase in lymphocytes from controls (303.8 +/- 116). The rate of halothane-induced increase in cytosolic calcium was 13 times greater in lymphocytes from MHS swine, compared with controls. These data indicated that the molecular defect that results in halothane-hypersensitivity and is characteristic of muscle of MHS swine also occurs in lymphocytes from MHS swine.

Animals↗