Biomedical subjects
Publications and source records attributed to J G McCormack.
An immunocompromised patient with an indwelling Hickman catheter developed Mycobacterium neoaurum bacteremia. This rapidly growing mycobacterium was previously isolated from soil, dust, and water but has not been described as a human pathogen. The infection responded to therapy with cefoxitin and gentamicin. It was not necessary to remove the Hickman catheter.
Human neutrophil adherence was enhanced by recombinant human tumour necrosis factor-beta (TNF beta) but suppressed by recombinant human interleukin-2 (IL-2). The opposite effects of these two lymphokines were observed over a range of concentrations consistent with their other biological activities, occurred within 15 min of incubation, and were still evident after 60 min. Pretreatment of neutrophils with both IL-2 and TNF beta resulted in adherence values intermediate between the values obtained with the individual lymphokines. IL-2 suppressed the stimulatory effects of both the chemotactic peptide formyl-methionyl-leucyl-phenyl-alanine (FMLP) and the phorbol ester phorbol myristate acetate (PMA). The combination of TNF beta with either FMLP or PMA produced enhancement of neutrophil adherence which exceeded that of either agent alone. These effects of the lymphokines were not due to endotoxin contamination since their effects were sensitive to heating and insensitive to polymyxin B treatment. These experiments provide further evidence for the critical role of these lymphokines in the regulation of acute and chronic inflammatory processes.
Three important dehydrogenases in the mitochondria of mammalian tissues are activated by Ca2+ ions: these are pyruvate dehydrogenase, NAD-isocitrate dehydrogenase, and oxoglutarate dehydrogenase. Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+ in rat hearts and livers that this results in a parallel rise in the intramitochondrial concentration of Ca2+. In this way, pyruvate oxidation and citric acid cycle flux are stimulated and there is an increase in NADH supply for the respiratory chain under conditions where there is an enhanced demand for ATP.
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The results of 2439 blood cultures that were taken in an acute children's hospital over a two-year period were reviewed. Three hundred and twenty-two organisms were cultured from 310 patients. One hundred and thirty-five (5.5%) isolates were considered to be pathogenic and 187 (7.7%) isolates were considered to be contaminants. Coagulase-positive staphylococci and enteric Gram-negative organisms were the isolates of which the significance was most difficult to determine. Community-acquired bacteraemia that affected children of less than five years of age was caused by Haemophilus influenzae in 65% of cases. Staphylococcus aureus was the major pathogen in older children. In 20% of cases, antimicrobial agents were commenced or changed after blood culture results. Delayed or inappropriate therapy was significantly more common in patients without an apparent focus of infection. The results of our study suggest that narrow spectrum antimicrobial agents can be used as appropriate empiric therapy for unlocalized infections in previously-well children. In children of between three months and five years of age, treatment should be directed against Haem. influenzae and Streptococcus pneumoniae, and in children of over five years of age, antistaphylococcal therapy should also be included.
A patient from Vanuatu with a mycetoma of the foot of 25 years' duration is described. Culture of a biopsy specimen from the foot grew a fungus which was identified as Acremonium falciforme. This agent has not been described previously in association with mycetomas in the Pacific region.
The accumulation of 2-deoxy-D-[1-3H]glucose, a non-metabolised analogue of glucose provides a quantitative measurement of the state of activation of phagocytic cells. A microassay for 3H-deoxyglucose uptake by human neutrophils and monocytes is described. Optimal conditions for the assay include the use of 5 X 10(5) cells and 0.78 microCi/ml of deoxyglucose in a final volume of 0.2 ml per microtitre well, and an incubation time of 30 min at 37 degrees C. This simple, rapid and reproducible technique may find application in experimental immunology.
Two cases of tuberculosis have been seen over the past two years in southeast Asian women who had recently emigrated to Australia and who attended a single maternity hospital in Brisbane. One patient died of tuberculous meningitis and the other had an asymptomatic tuberculous pleural effusion. Both cases illustrate some of the difficulties in the diagnosis of extrapulmonary tuberculosis, particularly in pregnant women, and the low level of awareness of this condition. Antenatal screening for tuberculosis is not performed as a routine in Australia. Pregnant women who have emigrated recently from areas of high prevalence constitute a group at increased risk for tuberculosis who should be screened selectively with tuberculin tests and chest x-ray examinations.
A case of cutaneous infection with Mycobacterium gordonae and other reports of extrapulmonary infection due to this organism are reviewed. This case confirms the pathogenic potential of M. gordonae which must now be included among the scotochromogens capable of causing cutaneous disease. Isolates of this organism should be tested against a full range of antimicrobial agents since traditional antituberculous therapy may be of limited efficacy. Pending the results of in vitro susceptibility testing, amikacin, ethambutol, rifampicin and co-trimoxazole are suggested as empirical therapy for infections caused by this organism.
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In the heart and other mammalian tissues, there are three exclusively intramitochondrial dehydrogenases that occupy key sites in oxidative metabolism which can be activated by increases in Ca2+ in the range 0.1-10 microM. They are the pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases. Activation of these enzymes can be demonstrated within intact mitochondria (incubated with Na+ and Mg2+) when extramitochondrial Ca2+ is raised within the expected physiological range (0.05-5 microM). Evidence is presented to suggest that the increase in cytoplasmic [Ca2+] caused by positive inotropic agents in the heart result in increases in intramitochondrial [Ca2+] and activation of the dehydrogenases and hence oxidative energy metabolism. Therefore it is proposed that the main function of the mitochondrial Ca2+-transport system is to regulate matrix rather than cytoplasmic Ca2+. In this way the stimulation of energy utilisation as contraction is enhanced is balanced by a parallel stimulation of ATP producing reactions.
Three key dehydrogenases in the mitochondria of higher animals have been found to be activated by Ca2+ ions; these are pyruvate dehydrogenase and two enzymes in the citric acid cycle, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. Activation can also be demonstrated within permeabilized and intact mitochondria. Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+, then this results in an increase in the intramitochondrial concentration of Ca2+. In this way, rates of pyruvate oxidation and citric acid cycle flux are increased, and hence there is an increase in NADH supply for the respiratory chain under conditions where there is an enhanced demand for ATP. In contrast, the activation of pyruvate dehydrogenase which is observed in adipose and other tissues exposed to insulin is brought about by a Ca2+-independent mechanism.
Mitochondria were prepared by a method including a Percoll purification step after the rapid homogenization of livers of fed rats which had been perfused either under unstimulated conditions or in the presence of vasopressin and/or glucagon. The two hormones separately or together increased the total calcium content of the mitochondria. This enhancement was accompanied by parallel increases in activities of the Ca2+-sensitive intramitochondrial enzymes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The effects of the two hormones on total mitochondrial calcium and on the activities of the oxidative enzymes were additive. The persistent enhancements of mitochondrial calcium content and enzyme activities were partially reversed by the addition of Na+ ions to the mitochondrial incubations; these effects of Na+ were blocked by diltiazem, a selective inhibitor of Na+-induced Ca2+ release. Mitochondria from control livers were incubated in vitro with CaCl2 to achieve various calcium content, and mitochondrial enzyme activities and calcium content were measured. A good correlation was obtained between the total calcium content and the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase. The results obtained are consistent with the hypothesis that vasopressin and glucagon additively cause increases in intramitochondrial [Ca2+] and so bring about the activations of these key enzymes of mitochondrial oxidative metabolism.
Three important dehydrogenases in vertebrate mitochondria are activated by Ca2+ ions with half-maximal effects at about 1 microM. These are pyruvate dehydrogenase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase. Activation of these enzymes can also be demonstrated within intact mitochondria when extramitochondrial Ca2+ is increased within the range of concentrations generally considered to occur in the cytoplasm of vertebrate cells. It is argued that the main role of the calcium transport system in the inner membrane of vertebrate mitochondria is to relay changes in the cytoplasmic concentration of Ca2+ into the mitochondrial matrix. In this way, hormones and other extracellular stimuli which stimulate ATP-requiring processes such as contraction and secretion through increases in the cytoplasmic concentration of Ca2+ may also increase intramitochondrial oxidative metabolism and hence the replenishment of ATP.
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The regulatory properties of the Ca2+-sensitive intramitochondrial enzymes (pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in extracts of rat liver mitochondria appeared to be essentially similar to those described previously for other mammalian tissues. In particular, the enzymes were activated severalfold by Ca2+, with half-maximal effects at about 1 microM-Ca2+ (K0.5 value). In intact rat liver mitochondria incubated in a KCl-based medium containing 2-oxoglutarate and malate, the amount of active, non-phosphorylated, pyruvate dehydrogenase could be increased severalfold by increasing extramitochondrial [Ca2+], provided that some degree of inhibition of pyruvate dehydrogenase kinase (e.g. by pyruvate) was achieved. The rates of 14CO2 production from 2-oxo-[1-14C]glutarate at non-saturating, but not at saturating, concentrations of 2-oxoglutarate by the liver mitochondria (incubated without ADP) were similarly enhanced by increasing extramitochondrial [Ca2+]. The rates and extents of NAD(P)H formation in the liver mitochondria induced by non-saturating concentrations of 2-oxoglutarate, glutamate, threo-DS-isocitrate or citrate were also increased in a similar manner by Ca2+ under several different incubation conditions, including an apparent 'State 3.5' respiration condition. Ca2+ had no effect on NAD(P)H formation induced by beta-hydroxybutyrate or malate. In intact, fully coupled, rat liver mitochondria incubated with 10 mM-NaCl and 1 mM-MgCl2, the apparent K0.5 values for extramitochondrial Ca2+ were about 0.5 microM, and the effective concentrations were within the expected physiological range, 0.05-5 microM. In the absence of Na+, Mg2+ or both, the K0.5 values were about 400, 200 and 100 nM respectively. These effects of increasing extramitochondrial [Ca2+] were all inhibited by Ruthenium Red. When extramitochondrial [Ca2+] was increased above the effective ranges for the enzymes, a time-dependent deterioration of mitochondrial function and ATP content was observed. The implications of these results on the role of the Ca2+-transport system of the liver mitochondrial inner membrane are discussed.
The administration in vivo of either adrenaline or glucagon alone resulted in increases of about 2-fold in the amounts of active, non-phosphorylated, pyruvate dehydrogenase in the livers of fed male or female rats, whereas when administered together increases of about 4-fold were obtained. Ca2+-dependent increases in the amount of active enzyme of up to about 5-fold could be achieved in isolated rat liver mitochondria by incubating them with increasing extramitochondrial [Ca2+]; from this, two conditions of Ca loading were chosen which caused increases in active enzyme similar to those with the hormone treatments given above. The increases in enzyme activity owing to these Ca loads persisted through the 're-isolation' of mitochondria and their incubation in Na+-free KCl-based media containing EGTA. Differences from values obtained with unloaded controls could be diminished by adding Na+ ions to cause the egress of Ca2+ from the mitochondria, or enough extramitochondrial Ca2+ to saturate the enzyme in its Ca2+-dependent activation; the effects of Na+ could be blocked by diltiazem, an inhibitor of mitochondrial Na+/Ca2+ exchange. The re-isolated, Ca-preloaded, mitochondria also exhibited enhanced activities of 2-oxoglutarate dehydrogenase when assayed at non-saturating [2-oxoglutarate] by two different methods; effects of Na+, Ca2+ or diltiazem on the persistent activations of this enzyme were similar to those for pyruvate dehydrogenase. Na+ caused a marked depletion, which could be blocked by diltiazem, of the 45Ca content of re-isolated mitochondria which had pre-loaded with Ca, containing 45Ca, to the same degrees as above. The activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase in incubated liver mitochondria prepared from rats subjected to the hormone treatments given above were found to behave in a very similar manner to those exhibited in the re-isolated, Ca-preloaded, mitochondria. It is concluded that these hormones each bring about the activations of these rat liver enzymes by causing increases in intramitochondrial [Ca2+], and that their effects, as such, are additive.