Studies of the bacterial endosymbionts of "anaerobic protozoa" using fluorescently-labelled rRNA-targetted oligonucleotide probes.
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Anaerobiosis↗
Biomedical subjects
D Lloyd
Publications and source records attributed to D Lloyd.
Hydrogenosomes in trichomonads are calcium stores and have a transmembrane electrochemical potential.
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Aniline Compounds↗
Membrane potential studies of Saccharomyces cerevisiae during cider fermentation.
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Alcohols↗
Antioxidant defences in the microaerophilic protozoan Trichomonas vaginalis: comparison of metronidazole-resistant and sensitive strains.
The sensitivity of the microaerophilic protozoan Trichomonas vaginalis to oxygen and products of its reduction, and the antioxidant defences employed by this organism, were investigated. Studies revealed that this amitochondrial flagellate is sensitive to oxygen tensions above those experienced in situ in the vagina (i.e. > 60 microM) and that metronidazole-resistant strains (CDC 85 and IR78) were more sensitive to elevated oxygen levels than a metronidazole-sensitive isolate (1910). In the presence of radical scavengers, inactivation of organisms at 60 microM oxygen was significantly lessened. Investigation of the antioxidant enzymes present in this organism revealed that activities of peroxide-reducing enzymes (e.g. catalase and general peroxidase) were not detectable, but that a cyanide-insensitive, azide-sensitive superoxide dismutase was present in cell extracts. Measurement of thiol-cycling enzymes indicated that NADPH could drive the reduction of oxidized glutathione (thiol reductase); however, the corresponding peroxidase activity was not detected. Analysis of thiols in whole cells of T. vaginalis indicated that glutathione was absent, but high levels of other thiols, propanethiol, methanethiol and H2S, were present. No significant differences were detected in thiol levels or antioxidant enzyme activities on comparison of metronidazole-sensitive and resistant strains. These results indicate that the sensitivity of T. vaginalis to oxygen above physiological levels is due to the lack of adequate peroxide-reducing enzymes and radical-scavenging mechanisms.
Animals↗
Rapid estimation of bacterial antibiotic susceptibility with flow cytometry.
Bacterial antibiotic susceptibility was rapidly estimated for Escherichia coli and Staphylococcus spp. by flow cytometry. This was achieved by measuring the uptake of a negatively charged membrane potential sensitive dye bis-(1,3-dibutyl-barbituric acid) trimethine oxonol and observing changes in low-angle light scatter (excitation light scattered by up to 15 degrees). Estimations of ampicillin, gentamicin and ciprofloxacin susceptibilities were possible within 2-5 h from a plate culture, depending on the species and antibiotic used. This includes the time necessary to establish steady-state growth in liquid culture.
Anti-Bacterial Agents↗
Rapid induction of microsomal delta 12(omega 6)-desaturase activity in chilled Acanthamoeba castellanii.
The activity of microsomal delta 12-desaturase in Acanthamoeba castellanii was increased after growing cultures were chilled from the optimal growth temperature (30 degrees C) to 15 degrees C. This increase was detectable in microsomes isolated from organisms subjected to only 10 min chilling. The mechanism of induction was investigated. The increase in activity on chilling was greatly reduced when protein synthesis was blocked before the temperature shift. Thus the major mechanism for the induction of delta 12-desaturase is increased protein synthesis. delta 12-Desaturase activity was higher when assayed at 20 degrees C than when assayed at 30 degrees C, but these changes were not due to the increased solubility of O2 at 20 degrees C. The major substrate of delta 12-desaturase was found to be 1-acyl-2-oleoyl phosphatidylcholine.
Acanthamoeba↗
An inexpensive infrared growth sensor array for detection of bacterial antibiotic susceptibility.
An inexpensive infrared sensor was constructed and used for the rapid testing of bacterial antibiotic susceptibility by detection of changes in absorbance at 950 nm. By comparing cultures of clinical isolates together with control strains (Escherichia coli NCTC 10418, Staphylococcus aureus NCTC 6571 or Pseudomonas aeruginosa NCTC 10662) after addition of an antibiotic, results on susceptibility were obtained within 3-5 h from the original plate culture. Representative strains of E. coli, P. aeruginosa, and S. aureus were tested successfully against ampicillin, penicillin, gentamicin or ciprofloxacin.
Ampicillin↗
Electron transport components of the parasitic protozoon Giardia lamblia.
The energy metabolism of the intestinal parasite, Giardia lamblia, involves the iron-sulphur protein, pyruvate:ferredoxin oxidoreductase. Cell fractionation studies showed that this enzyme is associated with the membranes. NADH and NADPH dehydrogenases were found in both the membrane and cytosolic fractions. EPR spectroscopic studies showed the presence of iron-sulphur clusters in the membrane fraction and in the cytosolic fraction, non-sedimentable at 6 x 10(6) g.min. An acidic, soluble protein fraction was separated from the cytosol. It had an EPR spectrum in the reduced state, characteristic of the 2[4Fe-4S] type of ferredoxin, with g-factors at 2.04. 1.93 and 1.89, and the midpoint redox potential was estimated to be -360 mV. This species is probably a ferredoxin, like those of anaerobic bacteria such as Clostridium and Desulfovibrio spp. and also that of Entamoeba histolytica. The protein was readily and irreversibly oxidized to give [3Fe-4S] clusters.
Animals↗
The need for involvement--an interview with David Lloyd. Interview by Sharon Beesley.
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General Practice, Dental↗
Temperature-induced membrane-lipid adaptation in Acanthamoeba castellanii.
A method has been developed for the separation of the major membrane fractions of Acanthamoeba castellanii after growth at different temperatures. The acyl-lipid compositions of individual membrane fractions, microsomal membranes, plasma membrane and mitochondria were analysed after a shift in culture temperature from 30 degrees C to 15 degrees C. The major change in lipid composition observed was an alteration in the relative proportions of oleate and linoleate. This reciprocal change was seen in all the membrane fractions, but occurred most rapidly in the phosphatidylcholine of the microsomal fraction. Thus, there appears to be a rapid induction of delta 12-desaturase activity in A. castellanii after a downward shift in growth temperature. Changes were also seen in the proportions of the n-6 C20 fatty acids, with a decrease in the proportions of icosadienoate and increases of icosatrienoate and arachidonate. However, unlike the alteration in oleate/linoleate ratios, this change was not seen in all the individual lipids of each membrane fraction.
Acanthamoeba↗
Effects of growth with ethanol on fermentation and membrane fluidity of Saccharomyces cerevisiae.
Saccharomyces cerevisiae HSc was grown with ethanol at concentrations up to 10% (v/v). The immediate effects of additions of externally added ethanol on CO2 production and O2 consumption of washed organisms were studied by stopped-flow membrane inlet quadrupole mass spectrometry. Fermentative activities of organisms grown with ethanol (0-5% v/v) showed similar sensitivities to inhibition by ethanol, whereas those grown with 10% (v/v) ethanol had become protected and were markedly less sensitive. The fluidity of subcellular membrane fractions was measured by determination of the temperature dependence of the rotational order parameter of the spin label 5-doxyl stearic acid (free radical) by electron spin resonance. Mitochondria prepared from yeasts grown with 0, 7, and 9% (v/v) ethanol showed similar overall fluidity, although differences in temperature-dependent behaviour indicate altered lipid composition or lateral phase separations. On the other hand the microsomal fraction from organisms grown with 9% ethanol showed a remarkable increase in fluidity. These data suggest that the protective effects of growth with ethanol near the limit of tolerance on fermentative activities may arise from altered plasma membrane fluidity properties.
Adaptation, Physiological↗
Intracellular coordination by the ultradian clock.
The time structure of a biological system is at least as intricate as its spatial structure. Whereas we have detailed information about the latter, our understanding of the former is still rudimentary. As techniques for monitoring intracellular processes continuously in single cells become more refined, it becomes increasingly evident that periodic behaviour abounds in all time domains. Timekeeping is essential for synchronization and coordination of intracellular processes. The presence of a temperature-compensated oscillator provides such a timer. The coupled outputs (epigenetic oscillations) of this ultradian clock constitute a special class of ultradian rhythm. These are undamped and endogenously driven by a device which shows biochemical properties characteristic of transcriptional and translational elements. Energy-yielding processes, protein turnover, motility, and the timing of the cell division cycle processes, are all controlled by the ultradian clock. Different periods 30 min-4h characterize different species.
Activity Cycles↗
Oxygen affinities of metronidazole-resistant and -sensitive stocks of Giardia intestinalis.
The common protozoon, Giardia intestinalis, parasitizes the upper small intestine of man, and is often refractory to treatment by metronidazole. Defective oxygen-scavenging mechanisms have been implicated as a cause of metronidazole resistance of another flagellate Trichomonas vaginalis, where metronidazole is also the most common drug treatment. Oxygen consumption of six clinical isolates of G. intestinalis and one line selected for resistance to metronidazole was measured over 0-50 microM-O2 using an oxygen electrode open for gas exchange. At > 30 microM-O2, inhibition of respiration was demonstrated in all seven stocks. Apparent oxygen affinities (KmO2) were found to range from 0.5 to 5.2 microM-O2; however, isolates from patients who failed to respond to treatment with metronidazole did not have measurably defective O2-scavenging capabilities compared with metronidazole-sensitive isolates. These strains did, however, show elevated NADPH-oxidase activities compared with metronidazole-sensitive strains. Results indicate that biochemical mechanisms of drug resistance in G. intestinalis may be quite different from those operating in T. vaginalis.
Animals↗
The effects of oxygen on fermentation in Giardia lamblia.
Detailed study of the effects of oxygen on the carbohydrate metabolism of Giardia lamblia revealed that low concentrations of oxygen (< 0.25 microM) produced profound alterations in the carbon balance of this organism. Although this concentration of oxygen could not be detected by mass spectrometry, a marked stimulation of ethanol production was observed. Associated with this was an inhibition of alanine production and oxidation of the intracellular NAD(P)H pool. Higher concentrations of oxygen inhibited ethanol production and further reduced levels of alanine. These results suggest that this stimulation is due to changes in carbon flux. Analysis of cell and medium hydrolysates after the growth of trophozoites in [U-14C]glucose suggests that G. lamblia does not synthesise detectable levels of labelled amino acids, except alanine and to a lesser extent valine, from this sugar. Trophozoites of G. lamblia have both glutamate dehydrogenase and alanine aminotransferase activity. As glutamate is taken up from the medium, it is suggested that glutamate dehydrogenase and alanine aminotransferase cooperate to convert pyruvate to alanine, with the concomitant oxidation of NAD(P)H.
Alanine↗
Hypothesis: the central oscillator of the circadian clock is a controlled chaotic attractor.
Controlled chaos may be important for the generation of rhythmic behaviour in living systems. A model is proposed in which the central circadian oscillator is a chaotic attractor. Whereas a limit cycle mechanism (previously invoked to explain circadian clocks as well as ultradian clocks and cell division cycles) can provide only a single stable periodic orbit, a chaotic attractor can generate rich dynamic behaviour. Control by feedback makes accessible a selected stabilized orbit; this can be chosen so as to optimize system performance. Such a system can accommodate a wide variety of requirements, e.g. that a single clock mutation can affect both period and temperature compensation, and the generation of higher periods from an ultradian oscillator. Simultaneous operation of more than one clock (with differing periods) may require a high-dimension chaotic attractor. Attractive features of such a model include versatility of period selection (e.g. as in the per mutants of Drosophila) and the use of control elements of the type already well known in metabolic circuitry.
Animals↗
Biological rhythms as organization and information.
While it is generally acknowledged that modern science began with the quantification of time in the measurement of linear physical processes in space by Galileo and Newton, the biological sciences have only recently developed appropriate experimental and mathematical methods for the description of living systems in terms of processes of non-linear, recursive dynamics. We now recognize that living organisms have patterns of exquisitely timed processes that are as intricate as their spatial structure and organization. Self-similarities of life processes in time and space have evolved to generate an ensemble of oscillators within which analogous functions may be discerned on many different time scales. The increasing complexity of periodic relationships on and between the many levels of biological organization are uncovered by current research. Recent efforts to reformulate the foundation of physics from the quantum to the cosmological level by using the concept of information as the common denominator integrating time, structure and energy remind us of an apparently analogous suggestion in the chronobiological literature which also describes the periodic dynamics of living systems as information processing. In this paper we review the periodic processes of living systems on all levels from the molecular, genetic and cellular to the neuroendocrinological, behavioural and social domains. Biological rhythms may be conceptualized as the evolution of ever more complex dynamics of information transduction that optimize the temporal integrity, development, and survival of the organism.
Animals↗
Mutations in phosphofructokinases alter the control characteristics of glycolysis in vivo in Saccharomyces cerevisiae.
Ethanol and CO2 production from glucose by non-proliferating suspensions of aerobically-grown, glucose-derepressed wild-type Saccharomyces cerevisiae is inhibited by O2; monitoring by mass spectrometry provides a direct method for measurement of the Pasteur effect. Under aerobic conditions, that part of the CO2 evolved equivalent to the O2 consumed, is produced by respiration: subtraction of this respiratory CO2 from the total gives CO2 produced by aerobic glycolysis. Pasteur quotients (anaerobic CO2/aerobic glycolytic CO2) were within the range 1.2 to 3.0. The Pasteur effect was not observed in the presence of carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of mitochondrial energy metabolism, or in a rho degree cytoplasmic petite mutant. A 'non-allosteric' mutant with an altered regulatory subunit of phosphofructokinase showed no Pasteur effect. Strains bearing a nonsense mutation pfk1 in the catalytic subunit of soluble phosphofructokinase (PFKI) also showed no Pasteur effect; the residual fermentative activity of this strain was dependent on PFKII, the particulate phosphofructokinase. A double mutant lacking both PFKI and glucose-6-phosphate dehydrogenase showed similar characteristics to those of the single pfk1 mutant; this indicates that the hexose monophosphate shunt is not acting to bypass the phosphofructokinase block. A 'hyper-allosteric' mutant altered in the regulatory subunit encoded by the gene PFK2 showed characteristics of glucose fermentation and ethanol oxidation very similar to those of wild-type organisms. These results indicate that either of the two phosphofructokinases can carry out glycolysis.
Aerobiosis↗
A general method for preparation of peptides biotinylated at the carboxy terminus.
A method for the preparation of a biotinylated resin that can be elongated by standard methods of solid-phase peptide synthesis to give peptides biotinylated at the carboxy terminus is described. This methodology is particularly important for the preparation of biotinylated peptides in which a free amino terminus is required. Coupling of N epsilon-9-fluorenylmethoxycarbonyl-(Fmoc)-N alpha-tert-butyloxycarbonyl(Boc)-L- lysine to p-methylbenzhydrylamine resin, followed by removal of the Fmoc protecting group and reaction with (+)-biotin-4-nitrophenyl ester yielded N alpha-Boc-biocytin-p-methyl-benzhydrylamine resin. The utility of this resin was tested by the synthesis of a biotinylated peptide, Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg-biocytin-NH2, for use as an in vitro substrate for myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation. Analysis of the peptide derivative by HPLC and mass spectrometry revealed a single major product of the expected mass, indicating that the biotin group survived cleavage and deprotection with HF. The biotinylated peptide served as a substrate for NMT, and the resulting myristoylated peptide could be quantitatively recovered by adsorption to immobilized avidin.
Amino Acid Sequence↗