Biomedical subjects
Publications and source records attributed to D Lloyd.
Iodine-123-labeled 4-(3-dimethylpropylamino)-7-iodoquinoline was evaluated in nine patients. By using a specially designed dual-pinhole ocular collimator, it was possible to obtain positive images at 2-6 hr for only 70% of the cases with subsequently proven ocular melanomas.
The respiratory burst of polymorphonuclear leukocytes, induced by the addition of chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine) and cytochalasin B was found to consist of two phases. The first phase of very rapid oxygen uptake lasted 1-3 min. and was followed by a second more prolonged phase of lower magnitude. The apparent Km for oxygen of unstimulated cells was 9.6 +/- 0.67 microM, while that of the second phase of stimulation was 3.7 +/- 1.6 microM oxygen. The possibility that lowered oxygen concentrations may regulate polymorphonuclear leukocyte activity in some pathological conditions is discussed.
Apparent Km values for O2 for the soil amoeba Acanthamoeba castellanii determined polarographically and by bioluminescence gave similar values (0.37 and 0.41 microM respectively). Mitochondria oxidizing succinate or NADH in the presence or absence of ADP gave values in the range 0.21-0.36 microM-O2. Oxidation of respiratory-chain components to 50% of the aerobic steady states in intact cells was observed at the following O2 concentrations: cytochrome aa3, 0.1-0.25 microM; cytochrome c, 0.3-0.6 microM; cytochrome b, 0.35-0.45 microM; flavoprotein, 2 microM. In isolated mitochondria corresponding values for a-, c- and b-type cytochromes were 0.007, 0.035-0.05 and 0.06-0.09 microM-O2. It is concluded that an O2 gradient exists between plasma membrane and mitochondria in A. castellanii.
Simultaneous and continuous measurements of changes in CO2 and O2 concentrations in glucose-metabolizing yeast suspensions by mass spectrometry enabled a study of the Pasteur effect (aerobic inhibition of glycolysis) in Saccharomyces uvarum and Schizosaccharomyces pombe. A different control mechanism operates in Candida utilis to give a damped oscillation after the anaerobic-aerobic transition. The apparent Km values for respiration of the three yeasts were in the range 1.3-1.8 microM-O2. The apparent Km values for O2 of the Pasteur effect were 5 and 13 microM for catabolite-repressed and derepressed S. uvarum respectively and 7 microM for Sch. pombe. These results are discussed with respect to currently accepted mechanisms for the control of glycolysis.
1. Room-temperature CO-reduced minus reduced difference spectra of intact cells of Acanthamoeba castellanii show the presence of CO-reacting haemoproteins in cells from the early-exponential, late-exponential and stationary phases of growth. 2. The relative rates of reaction with CO of the two haemoproteins differ; that of cytochrome a/a3 with CO is complete within 1 min of bubbling with CO, whereas that of cytochrome b takes longer than 90 min. 3. Photochemical action spectra reveal cytochrome a/a3 as the predominant haemoprotein oxidase at all stages of growth. 4. It is concluded that the alternative oxidases known to be present in these organisms are not cytochromes.
A method of obtaining photochemical action spectra for the relief of CO inhibition of respiration is described. Continuous readout from a membrane-covered oxygen electrode of dissolved oxygen in a stirred suspension of microorganisms under CO-oxygen gas mixtures in an open reactor enables measurement of increased respiration on illumination. Advantages presented by the use of a liquid dye laser include high intensity of emission and narrow spectral bandwidth; just two dyes (rhodamine 6G and rhodamine 110) are required to match the alpha-absorption maxima of the CO complexes of all known bacterial and mitochondrial terminal oxidases.
1. Both Isotricha intestinalis and I. prostoma possess microbody-like organelles, with a highly granular appearance. 2. These organelles, which are sedimentable at 10(5) g-min, bear no morphological similarity to mitochondria, but are enzymatically similar to organelles possessed by certain other anaerobic protozoa and termed hydrogenosomes. 3. The hydrogenosomes isolated from a preparation of mixed isotrichs bear a closer similarity to those isolated from the other rumen holotrich. Dasytricha ruminantium, than those recently identified in a mixed entodiniomorph preparation, or the trichomonads, in that the enzyme malate dehydrogenase (decarboxylating) is non-sedimentable and phosphoacetyl transferase together with acetate kinase are involved in the transformation of acetyl CoA to acetate. 4. The results enable a scheme of acetate, CO2 and H2 formation from carbohydrates to be proposed and extends the number of protozoa known to possess this organelle.
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The endogenous respiration of the rumen ciliate Dasytricha ruminantium maintained under an O2 tension of 2kPa (approximately 0.02 atm) was partially inhibited by KCN (40% inhibition) and NaN3 (58% inhibition). The organisms lack cytochromes, and sensitivity of respiration to KCN, NaN3, chloroquine and quercetin suggest that the operation of flavoprotein-iron-sulphur-mediated electron transport. As in Tritrichomonas foetus, hydrogenosomal respiration can be stimulated by the addition of CoA in the presence of 0.025% Triton X-100; stimulation by ADP was not detected. Stimulation of pyruvate-supported O2 uptake by Pi suggests that acetate is produced via acetyl phosphate.
1. Mitochondria-enriched fractions of the ciliate protozoan Tetrahymena pyriformis ST contained CO-reacting cytochromes b560 and a620. 2. A non-photodissociable oxygen-containing compound of cytochrome a620 was formed in whole cell suspensions at -114 degrees C after photolysis of CO in the presence of 200 microM-O2. 3. Electron transport, indicated by the oxidation of cytochrome a620 and cytochrome c, occurred at temperatures higher than -72 degrees C. 4. Photochemical action spectra for the relief of respiratory inhibition of whole cells by CO obtained by using a liquid dye laser indicate that the only CO-reacting terminal oxidase detectable was cytochrome a620. 5. It is concluded that the alternative electron transport chains in this organism utilize non-cytochrome terminal oxidases.
1. The mitochondrial ATPase of Acanthamoeba castellanii accumulated discontinuously in synchronous cultures prepared by a minimally perturbing size-selection technique. 2. Enzyme activity per ml of culture doubled overall during one cell cycle time of 8 h, but oscillated to give seven maxima during this period. Similar oscillations were observed in the specific activities of ATPase and of the naturally occurring inhibitor protein. 3. These variations in enzyme activity reflected changes in amount of enzyme protein as assayed by an immunological technique. 4. Large variations in I50 values (micrograms of inhibitor/mg of protein necessary for 50% inhibition of inhibitor-sensitive activity) for inhibition of ATPase activity by seven different inhibitors of energy conservation were observed. Activity was more sensitive to inhibition by oligomycin, efrapeptin, citreoviridin and quercetin when values were highest. 5. The results are discussed in relation to the phased organization of biosynthesis and degradation of cellular components known to occur during the cell cycle of this organization.
1. The mitochondrial adenosine triphosphatase (ATPase) of Acanthamoeba castellanii is Mg2+-requiring (optimum cation: ATP ratio of 1.5) and has two pH optima of activity (at pH 6.6 and 8.1). 2. ATPase activity of submitochondrial particles is effectively inhibited by twelve different inhibitors of energy conservation suggesting similarities in inhibitor-binding sites to other previously characterized complexes. 3. Gel filtration by passage through Sephadex G-50 increases ATPase activity of submitochondrial particles between 1.5 and 3.5 fold indicating the presence of a low molecular weight inhibitor protein. 4. After removal of the inhibitor protein, sensitivity to inhibitors of energy conservation decreases by between 1.5 and 14 fold. Crude F1-inhibitor preparations from A. castellanii, Schizosaccharomyces pombe, Tetrahymena pyriformis and bovine heart also inhibit ATPase activity. 5. Large variations in ATPase activity, F1-inhibitor protein activity, and amounts of immunologically-determined ATPase protein were observed during exponential growth, and the correlation between changes in these measurements is discussed. 6. The results are also discussed highlighting the similarities between the mitochondrial ATPase of A. castellanii and other mitochondrial ATPases.
Synchronous cultures of the soil amoeba Acanthamoeba castellanii, established by a selection procedure, show significant oscillations of respiration and total cell protein. There was little difference between the period of these oscillations, which averaged 76 min, although the five incubation temperatures used varied between 20 degrees C and 30 degrees C and the cell division time increased from 7.8 to 16 hr. The phase of these oscillations also corresponded approximately at all incubation temperatures. Similar observations made over the whole division cycle at three temperatures indicated that similar oscillations occurred, with a constant period of 65 min, although these data were too variable to show this unequivocally. Control (asynchronous) cultures show that the oscillations are not a consequence of metabolic perturbation produced by the centrifugal selection procedure. It is suggested that these temperature-compensated epigenetic oscillations serve a dual role in cell cycle and circadian timekeeping and that cell cycle time is quantized.
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Oxygen-dependent bioluminescence of Photobacterium (Vibrio) fischeri was used to measure oxygen affinities of four protozoa. The aerobic organisms Acanthamoeba castellanii and Tetrahymena pyriformis showed apparent Km values for O2 of 0.42 and 2.43 microM respectively. The aerotolerant anaerobe Tritrichomonas foetus, and the more strictly anaerobic rumen ciliate Dasytricha ruminantium, both of which have hydrogenosomes, respired with apparent Km values of 1.08 and 1.70 microM-O2. We conclude that mitochondrial respiration is not the only process conferring on organisms a high affinity for O2.
A family is described in which 2 siblings born to healthy parents presented with abnormal facies, persistent diarrhoea, and early death. Exhaustive pathological and biochemical investigations failed to find a cause. The scalp hair of both babies had an abnormal amino-acid composition, and presented an appearance that was unique on scanning electron microscopical examination; this fact and the clinical picture probably represents a new syndrome.
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1. Mitochondria-enriched fractions of the soil amoeba Acanthamoeba castellanii contained four haemoproteins that in their reduced forms reacted with CO to give photodissociable CO complexes; these were cytochromes a 3, a 614, b- and c-type cytochromes. 2. Non-photodissociable oxygen-containing compounds were formed at temperatures between -130 and -150 degrees C after photodissociation of CO in the presence of 200 microM-O2, 3. Electron transport, indicated by the oxidation of cytochromes a + a3 and cytochrome c, did not occur until the temperature was raised to -80 degrees C.