Merle S. Olson

[25] The MBEC assay system: Multiple equivalent biofilms for antibiotic and biocide susceptibility testing

Publisher Summary A number of technologies have been developed to study biofilm growth. Although these technologies produce reproducible biofilms for the study of biofilm growth, structure, and physiology, they have not been amenable for the routine study of biofilm susceptibility to antibiotics and biocides. For this reason, virtually every antibiotic and biocide available has been selected for activity against planktonic organisms. These drugs often have been found to lack activity against microbial biofilms. The MBEC (minimum biofilm eradication concentration) Assay System using the Calgary Biofilm Device provides, for the first time, an assay easily applicable to screening antibiotics and biocides for activity against microbial biofilms. The MBEC Assay System is ideally suited either for screening new putative antibiotics and/or biocides, or for the determination of both the MIC (minimal inhibitory concentration) and MBEC values in clinical situations for the treatment of chronic, recurrent, or device-related infections. The MBEC Assay System produces 96 equivalent biofilms formed under flow conditions, without the need for pumps. Further, as it is based on the standard 96 well platform, it conforms to existing technology available in most laboratories. The MBEC Assay System consists of a two-piece disposable plastic apparatus used for biofilm formation.

Studies of mitochondrial calcium movements using chlorotetracycline

1. The association of calcium with isolated rat liver mitochondrial membranes under various metabolic conditions was monitored using the fluorescent chelate probe, chlorotetracycline. Chlorotetracycline fluorescence increased markedly during energized calcium uptake in the absence of a permeant anion. Uncoupler and a respiratory chain inhibitor caused a rapid decrease in chlorotetracycline fluorescence when added either before or after calcium. During calcium uptake experiments concentrations of calcium exceeding 100 muM caused a transient fluorescence increase followed by an extensive decrease in fluorescence. 2. Changes in the chlorotetracycline-associated fluorescence of the mitochondrial suspensions were correlated with the uptake of exogenous 45Ca. A positive correlation was observed between fluorescence and energized 45Ca uptake in the absence of permeant anions. Addition of the permeant anion, phosphate, caused an extensive decrease in chloretetracycline fluorescence but an enhanced uptake of exogenous 45Ca. 3. The interaction of endogenous mitochondrial calcium with the fluorescent chelate probe was studied under a number of experimental conditions using mitochondria labeled during preparation with 45Ca. Endogenous 45Ca was lost rapidly from the mitochondria upon treatment with uncoupler, antimycin A, and A23187. Potassium phosphate and EGTA had no effect on the endogenous calcium as measured by either the 45Ca content of the mitochondria or the fluorescence of the probe. 4. Mitochondria treated with antimycin A lost most of their endogenous 45Ca within 3 min; subsequent energization of the mitochondria resulted in a partial uptake of the released 45Ca but caused nearly a complete return of the chlorotetracycline fluorescence to the original level. Addition of phosphate did not change the fluorescence level but resulted in an almost complete accumulation of the 45Ca previously released. 5. Following this energized uptake of 45Ca, EGTA, p-trifluoromethoxyphenyl hydrazone of carbonyl cyanide, A23187 and calcium chloride all caused a nearly complete loss of the 45Ca from the mitochondria and, with the exception of calcium chloride, caused an extensive decrease in the fluorescence level. Hence, the apparent location and/or properties of the endogenous calcium in this rat liver mitochondrial system were altered significantly by manipulation of the energetic state of the mitochondrial membrane.

The inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

Abstract The influence of fatty acid on the interconversion of the pyruvate dehydrogenase complex (PDH) between its active (dephospho-) and inactive (phospho-) forms and on the intramitochondrial ATP ADP , NADH NAD + and acetyl-CoA CoASH ratios was studied in isolated rat liver mitochondria. Conditions were found in which the PDH activity was inversely correlated only with the NADH NAD + ratio. Under other conditions the PDH activity was inversely correlated solely with the acetyl-CoA CoASH ratio. These experiments suggest that the activity of the regulatory enzymes involved in the inactivation and reactivation of the pyruvate dehydrogenase multienzyme complex may be controlled by both the intramitochondrial NADH NAD + and acetyl-CoA CoASH ratios.

CHLOROTETRACYCLINE-ASSOCIATED FLUORESCENCE CHANGES DURING CALCIUM UPTAKE AND RELEASE BY RAT BRAIN SYNAPTOSOMES

Abstract– The fluorescent divalent metal chelate‐probe, chlorotetracycline (CTC), was used as a dynamic monitor of calcium association with rat brain snynaptosomes. The determined fluorescence excitation and emission maxima, 412 nm and 522 nm respectively, were used to monitor membrane‐calcium interactions as a function of various parameters. Positive correlations were observed between increased or decreased fluorescence quantum yield and the uptake of both CTC and 45Ca by synpatosomes. The divalent metal ionophore A23187 enhanced fluorescence as well as probe and 45Ca uptake. Whereas, the polar chelator, EGTA, markedly reduced fluorescence, and the synaptosomal bound CTC and 45Ca. The CTC fluorescence changes also demonstrated the saturable manner in which 45Ca bound synaptosomes. At concentrations greater than 100μg/ml, CTC bound to the synaptosomes in a manner which quenched fluorescence at 522 nm. Also, CTC, at concentrations above 15 μg/ml, enhanced the uptake of 45Ca. At CTC concentrations between 10 and 15 μg/ml the quenching and iono‐phoretic properties of the probe were minimized without affecting the capability of using the probe to visualize calcium interactions with synaptosomal membranes. Also, at a low CTC concentration (12.5 μg/ml) the inhibition of calcium uptake by increasing monovalent ion concentrations was clearly demonstrated.

The effects of cyclopropane carboxylic acid on hepatic pyruvate metabolism

The effects of the hypoglycemic agent, cyclopropane carboxylate, on the metabolism of various substrates that enter the mitochondrion via the mitochondrial monocarboxylate transporter were investigated in perfused rat livers. Metabolism of pyruvate, branched-chain alpha-keto acids, acetoacetate and, to a lesser extent beta-hydroxybutyrate, were all inhibited by cyclopropane carboxylate. In addition, the stimulation of pyruvate decarboxylation by beta-hydroxybutyrate at low pyruvate concentrations because of exchange of extramitochondrial pyruvate with intramitochondrially generated acetoacetate, was abolished by cyclopropane carboxylate. Gluconeogenesis from alanine, which is not transported via the monocarboxylate translocator, was not inhibited by low concentrations of cyclopropane carboxylate. Cyclopropane carboxylate also inhibited fatty acid oxidation, as measured by ketone body production. These results support previous findings that the hypoglycemic agent, cyclopropane carboxylate, exerts at least some of its metabolic effects at the level of the mitochondrial monocarboxylate transporter.

The inhibition of l(—)-palmitylcarnitine oxidation by α-ketoglutarate in rat liver mitochondria

Abstract 1. 1. The effect of α-ketoglutarate addition to uncoupled rat liver mitochondria oxidizing L(—)-palmitylcarnitine plus L-malate was studied with regard to various metabolic parameters. 2. 2. L(—)-Palmitylcarnitine plus L-malate oxidation was strongly inhibited by α-ketoglutarate in uncoupled rat liver mitochondria. This inhibition was dependent upon the concentration of α-ketoglutarate. 3. 3. Citrate production was markedly inhibited following α-ketoglutarate addition but ketogenesis was not stimulated. The inhibition of citrate production was not correlated with a high ATP level in the mitochondrial suspension but followed closely the inhibition of oxygen consumption. 4. 4. ADP addition prevented the establishment of the α-ketoglutarate induced inhibitory state. The prevention of the inhibitory state by ADP addition was atractyloside sensitive but oligomycin insensitive. 5. 5. The inhibitory state induced by α-ketoglutarate oxidation was not specific for L(—)-palmitylcarnitine plus L-malate oxidation but caused strong inhibition of the oxidation of all NAD-linked substrates. Succinate oxidation was not affected. 6. 6. It is postulated that this inhibitory effect induced by α-ketoglutarate was due to an elevated intramitochondrial level of GTP which inhibited NAD-linked substrate oxidation.

Development of Complement-Fixing 19S, Anti-Heart Mitochondria Autoantibody, Following Myocardial Infarction in Dogs

The development of anti-heart autoantibody following experimental myocardial infarction in dogs was studied with respect to (1) the subcellular localization of the heart autoantigen(s), (2) the incidence of autoantibody following myocardial infarction, and (3) the temporal development of the autoantibody and its relation to serum enzyme elevations and electrocardiographic changes. Complement-fixing anti-heart autoantibody developed following myocardial infarction induced by either two-stage coronary artery ligation or by the injection of microspheres directly into the coronary circulation. The autoantibody was directed toward an autoantigen residing in the mitochondrial fraction of myocardial tissue and was found to be associated with the 19S class of immunoglobulin. Eighteen of 20 dogs with infarction produced anti-heart mitochondria autoantibody 10 days following induction of myocardial damage; the autoantibody persisted 4 to 6 weeks thereafter. In these dogs the presence of anti-heart mitochondria autoantibody was diagnostic of myocardial infarction at times when the serum enzymes and electrocardiograms were within normal limits. Our studies indicate that a serologic test, based on the detection of complement-fixing anti-heart mitochondria autoantibody, may be a valuable adjunct to tests currently available for diagnosing recent myocardial infarction.

Characterization of anti-heart mitochondria autoantibodies produced in dogs following myocardial infarction.

A complement-fixing, heart-reactive serum component that develops in dogs following experimental myocardial infarction was characterized with respect to its immunoglobulin nature and its subcellular membrane and organ specificities. The immunoglobulin nature of the heart-reactive serum factor was established by the following evidence: (1) the factor was not removed from the serum following absorption of up to 90% of the first component of dog complement with bovine serum albumin (BSA)-anti-BSA immune precipitates, (2) the serum factor sedimented in the 19S region of sucrose density gradients, and (3) affinity chromatography using a Sepharose column coupled with rabbit anti-dog μ-chain-specific antibody resulted in the removal of nearly all of the heart-reactive substance. The major subcellular autoantigenic locus of the dog heart was determined to be the outer mitochondrial membrane. Serum containing the autoantibody demonstrated a six-fold greater autoantibody titer when outer mitochondrial membrane was used as the test antigen than it did when inner mitochondrial membrane was used. Absorption experiments suggested that the autoantigenic determinants residing on inner and outer mitochondrial membranes were unique to the individual membranes. Significant cross-reaction of the anti-heart autoantibody between heart and skeletal muscle mitochondria was observed, but no cross-reaction was seen with dog liver mitochondrial membranes.

Characterization of autoantigenic sites on isolated dog heart mitochondria

1. Anti-heart mitochondria autoantibodies were developed in serum from dogs following experimental myocardial infarction. 2. Heart mitochondria frozen and thawed repeatedly in a sucrose/Tris-chloride buffer retained both their functional integrity as measured by the respiratory control ratio and their ability to serve as an antigen in a complement fixation test. Mitochondria frozen and thawed in a potassium chloride/Tris-chloride buffer lost both their functional integrity and their autoantigenic activity after one freeze-thaw cycle. 3. Extraction of the heart mitochondria with acetone/water mixtures to remove phospholipids from the membrane led to a complete loss of the ability of the mitochondria to react in the complement fixation test but did not affect the ability of the membranes to bind autoantibody in absorption experiments. 4. Treatment of the mitochondrial membranes with increasing concentrations of trypsin caused a loss of up to approximately 50% of the membrane protein with a gradual decrease in the autoantigenic activity of the membrane without impairment of the ability of the membrane to bind autoantibody. 5. Removal of up to 90% of the sialic acid of the mitochondrial membrane with neuraminidase resulted in a considerable increase in the complement-fixing autoantigenic activity of the membrane without changing the apparent ability of the membrane to bind autoantibody in absorption experiments. 6. Exposure of mitochondrial membranes to autoantibody and complement caused an inhibition of both an inner mitochondrial membrane enzyme, i.e. cytochrome oxidase (48%) and an outer mitochondrial membrane enzyme, i.e. NADH cytochrome c reductase (rotenone insensitive) (37%).

[60] Measurement of branched-chain α-keto acid dehydrogenase flux rates in perfused heart and liver

Abstract The methods described here represent a flexible set of procedures for investigating the metabolism of the branched-chain α-keto acids and other substances in perfused organs, notably the rat heart and liver. These procedures have been used to investigate many aspects of the metabolism of the branched-chain α-keto acids not discussed here, such as the effects on branched-chain α-keto acid metabolism by exposure to α-adrenergic agents,28 by inhibition of the monocarboxylate translocator,29 and by the coinfusion of other metabolites.30,31