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1. Room-temperature (18 degrees C) CO difference spectra of mitochondrial fractions from the amoeba Acanthamoeba castellanii reveal the presence of at least four CO-reacting haemoproteins. As well as cytochrome a3, other components reacting with CO are: (i) a c-type cytochrome; (ii) a b-type cytochrome; and (iii) another a-type cytochrome. 2. The same components can be identified in low-temperature photodissociation experiments with intact cells or mitochondria. 3. The time of exposure to CO and the nature of the reductant are both important in identifying all the components present, in that the b-type cytochrome is more readily distinguished after longer exposure to CO and more of the c-type cytochrome is detectable when NADH is the reductant 4. Treatment of mitochondria with ultrasound releases two components, identifiable in low-temperature difference spectra as a c-type and a b-type cytochrome; only the latter appears to have any reaction with CO, and the CO-reacting c-type cytochrome is retained in submitochondrial particles. 5. The complexity of the CO-reacting haemoproteins in this organism is compared with the simpler systems found in other eukaryotic organisms.  相似文献   

3.
DNA was prepared from isolated mitochondria of Acanthamoeba castellanii and was shown to behave as a single component in density gradients, on ;melting' and on renaturation. From measurements of renaturation kinetics, sedimentation coefficient and electron micrographs the genome size of the mitochondrial DNA was calculated to be about 3.4x10(7) daltons. A small proportion of the preparations could be isolated as relaxed circular molecules of mean contour length 16.2mum.  相似文献   

4.
Although 5S rRNA is a highly conserved and universal component of eubacterial, archaeal, chloroplast, and eukaryotic cytoplasmic ribosomes, a mitochondrial DNA-encoded 5S rRNA has so far been identified only in land plants and certain protists. This raises the question of whether 5S rRNA is actually required for and used in mitochondrial translation. In the protist Acanthamoeba castellanii, BLAST searches fail to reveal a 5S rRNA gene in the complete mitochondrial genome sequence, nor is a 5S-sized RNA species detectable in ethidium bromide-stained gels of highly purified mitochondrial RNA preparations. Here we show that an alternative visualization technique, UV shadowing, readily detects a novel, mitochondrion-specific small RNA in A. castellanii mitochondrial RNA preparations, and that this RNA species is, in fact, a 5S rRNA encoded by the A. castellanii mitochondrial genome. These results emphasize the need for caution when interpreting negative results that suggest the absence of 5S rRNA and/or a mitochondrial DNA-encoded 5S rRNA sequence in other (particularly protist) mitochondrial systems.  相似文献   

5.
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.  相似文献   

6.
Acanthamoeba castellanii is a free-living amoeba found in soil, freshwater, and marine environments and an important predator of bacteria. Acanthamoeba castellanii is also an opportunistic pathogen of clinical interest, responsible for several distinct diseases in humans. In order to provide a genomic platform for the study of this ubiquitous and important protist, we generated a sequence survey of approximately 0.5 x coverage of the genome. The data predict that A. castellanii exhibits a greater biosynthetic capacity than the free-living Dictyostelium discoideum and the parasite Entamoeba histolytica, providing an explanation for the ability of A. castellanii to inhabit a diversity of environments. Alginate lyase may provide access to bacteria within biofilms by breaking down the biofilm matrix, and polyhydroxybutyrate depolymerase may facilitate utilization of the bacterial storage compound polyhydroxybutyrate as a food source. Enzymes for the synthesis and breakdown of cellulose were identified, and they likely participate in encystation and excystation as in D. discoideum. Trehalose-6-phosphate synthase is present, suggesting that trehalose plays a role in stress adaptation. Detection and response to a number of stress conditions is likely accomplished with a large set of signal transduction histidine kinases and a set of putative receptor serine/threonine kinases similar to those found in E. histolytica. Serine, cysteine and metalloproteases were identified, some of which are likely involved in pathogenicity.  相似文献   

7.
During development of Acanthamoeba castellanii in a non-nutrient medium, the pattern of synthesis of proteins changes. Comparison of in vivo and in vitro patterns of protein synthesis reveals concomitant relative increases of five proteins, indicating a control of synthesis of these proteins at the level of the RNA content. The decrease in the overall rate of protein synthesis and relative decreases in the synthesis of actin and ribosomal proteins during development, not accompanied by equivalent changes in the content of mRNA, suggest control mechanisms also at the level of translation. Patterns of ribosomal proteins do not change qualitatively during encystation, indicating that the inhibition in the overall rate of protein synthesis and the formation of inactive monosomes is not controlled by this mechanism; however, phosphorylation of one ribosomal protein, S 3, is observed occasionally during encystation. Phosphorylation of S 3 is also detected after transfer of stationary phase cells into fresh nutrient medium. It was found that only such cells having RNA of aberrant properties are able to phosphorylate S 3 after transfer into fresh nutrient medium. Since these changes in the property of RNA are never observed in cysts, in which phosphorylation of S 3 sometimes occurs, it is concluded that either other alterations in the properties of RNA than those detected or other parameters are responsible for changes in phosphorylation of S 3.  相似文献   

8.
Benzodiazepine binding sites were studied in mitochondria of unicellular eukaryotes, the amoeba Acathamoeba castellanii and the yeast Saccharomyces cerevisiae, and also in rat liver mitochondria as a control. For that purpose we applied Ro5-4864, a well-known ligand of the mitochondrial benzodiazepine receptor (MBR) present in mammalian mitochondria. The levels of specific [(3)H]Ro5-4864 binding, the dissociation constant (K(D)) and the number of [(3)H]Ro5-4864 binding sites (B(max)) determined for fractions of the studied mitochondria indicate the presence of specific [(3)H]Ro5-4864 binding sites in the outer membrane of yeast and amoeba mitochondria as well as in yeast mitoplasts. Thus, A. castellanii and S. cerevisiae mitochondria, like rat liver mitochondria, contain proteins able to bind specifically [(3)H]Ro5-4864. Labeling of amoeba, yeast and rat liver mitochondria with [(3)H]Ro5-4864 revealed proteins identified as the voltage dependent anion selective channel (VDAC) in the outer membrane and adenine nucleotide translocase (ANT) in the inner membrane. Therefore, the specific MBR ligand binding is not confined only to mammalian mitochondria and is more widespread within the eukaryotic world. However, it can not be excluded that MBR ligand binding sites are exploited efficiently only by higher multicellular eukaryotes. Nevertheless, the MBR ligand binding sites in mitochondria of lower eukaryotes can be applied as useful models in studies on mammalian MBR.  相似文献   

9.
Light from 350 to 680 nm at intensities up to 1.62 × 105 ergs per sec per cm2 slowed exponential growth and lowered the maximum yield in axenic cultures of Acanthamoeba castellanii. Photoinhibition was a linear function of light intensity up to 1.25 × 105 ergs per sec per cm2. At higher intensities, growth was too slow to be measured accurately. A photochemical change occurring in the growth medium on irradiation was a function of light dosage and not intensity per se. Light in dosages which appreciably changed the growth-supporting properties of the medium exceeded the dosages received by exponentially growing cultures during irradiation. Consequently, photoinhibition of growth was attributed to a direct effect of light on the amoebae, not to photodegradation of the medium. The growth-supporting properties of irradiated media could be restored by the addition of yeast extract and Proteose peptone. The reduced growth rate in the light was not due to cyst formation or induction of multinuclearity. Light affected the amoebae either through absorption by intracellular pigment(s) or through binding to the amoebae of a photosensitizing compound in the medium.  相似文献   

10.
Fifteen aminoacyl-transfer ribonucleic acids (tRNA's) from vegetative cells (trophozoites) and mature cysts of Acanthamoeba castellanii were compared by reversed-phase 5 chromatography. Little or no differences were detected in reversed-phase 5 chromatography elution profiles of alanyl-, arginyl-, isoleucyl-, phenylalanyl-, prolyl-, seryl-, threonyl-, tryptophanyl- and valyl-tRNA's. Significant differences in the relative proportions of isoaccepting species of leucyl-, lysyl-, methionyl-, aspartyl-, histidyl-, and tyrosyl-tRNA's were observed. Based upon the criterion of cyanogen bromide reactivity with the modified nucleoside queuosine, the content of queuosine in aspartyl-tRNA of A, castellanii is significantly greater in mature cysts than in trophozoites. The similarity of change in reversed-phase 5 chromatography elution profiles of aspartyl-, histidyl-, and tyrosyl-tRNA suggests that a common mechanism is responsible for alterations in the chromatographic patterns.  相似文献   

11.
Mitochondria of Acanthamoeba castellanii possess a cyanide-resistant GMP-stimulated ubiquinol alternative oxidase in addition to the cytochrome pathway. In a previous work it has been observed that an interaction between the two ubiquinol-oxidizing pathways exists in intact A. castellanii mitochondria and that this interaction may be due to a high sensitivity of the alternative oxidase to matrix pH. In this study we have shown that the alternative oxidase activity reveals a pH-dependence with a pH optimum at 6.8 whatever the reducing substrate may be. The GMP stimulation of alternative oxidase is also strongly dependent on pH implicating probably protonation/deprotonation processes at the level of ligand and protein with an optimum pH at 6.8. The ubiquinone redox state-dependence of alternative oxidase activity is modified by pH in such a way that the highest activity for a given ubiquinone redox state is observed at pH 6.8. Thus pH, binding of GMP, and redox state of ubiquinone collaborate to set the activity of the GMP-stimulated alternative oxidase in isolated A. castellanii mitochondria. The high pH sensitivity of the alternative oxidase could link inactivation of the cytochrome pathway proton pumps to activation of the alternative oxidase with acceleration of redox free energy dissipation as a consequence.  相似文献   

12.
Vibrio parahaemolyticus is a food-borne pathogen that naturally inhabits both marine and estuarine environments. Free-living protozoa exist in similar aquatic environments and function to control bacterial numbers by grazing on free-living bacteria. Protozoa also play an important role in the survival and spread of some pathogenic species of bacteria. We investigated the interaction between the protozoan Acanthamoeba castellanii and the bacterium Vibrio parahaemolyticus. We found that Acanthamoeba castellanii does not prey on Vibrio parahaemolyticus but instead secretes a factor that promotes the survival of Vibrio parahaemolyticus in coculture. These studies suggest that protozoa may provide a survival advantage to an extracellular pathogen in the environment.  相似文献   

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We studied FFA (free fatty acid)-induced uncoupling activity in Acanthamoeba castellanii mitochondria in the non-phosphorylating state. Either succinate or external NADH was used as a respiratory substrate to determine the proton conductance curves and the relationships between respiratory rate and the quinone reduction level. Our determinations of the membranous quinone reduction level in non-phosphorylating mitochondria show that activation of UCP (uncoupling protein) activity leads to a PN (purine nucleotide)-sensitive decrease in the quinone redox state. The gradual decrease in the rate of quinone-reducing pathways (using titration of dehydrogenase activities) progressively leads to a full inhibitory effect of GDP on LA (linoleic acid) induced proton conductance. This inhibition cannot be attributed to changes in the membrane potential. Indeed, the lack of GDP inhibitory effect observed when the decrease in respiratory rate is accompanied by an increase in the quinone reduction level (using titration of the quinol-oxidizing pathway) proves that the inhibition by nucleotides can be revealed only for a low quinone redox state. It must be underlined that, in A. castellanii non-phosphorylating mitochondria, the transition of the inhibitory effect of GDP on LA-induced UCP-mediated uncoupling is observed for the same range of quinone reduction levels (between 50% and 40%) as that observed previously for phosphorylating conditions. This observation, drawn from the two different metabolic states of mitochondria, indicates that quinone could affect UCP activity through sensitivity to PNs.  相似文献   

15.
Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii . Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function.  相似文献   

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In this study we report observations on the structural mechanisms of the cytopathic effect of Acanthamoeba castellanii trophozoites on cultured MDCK cell monolayers. Co-incubations were carried out for a maximum of 24h. The first evidence of damage to the cell monolayer was detected by measuring the transepithelial resistance of cell monolayers that interacted with the amoebae. At 6h, transepithelial resistance diminished to 51% and amoebae required 5-6h to produce evidence of structural injury at the light microscopy level. Following 12h of incubation, the cell monolayer was severely damaged. After making intimate contact with the surface of target cells, trophozoites detached cells from the substrate, lysed and by means of food-cups ingested the damaged cells. There was no morphological evidence of modifications in MDCK cell membranes, membrane fusion or junction formation between the amoeba and host plasma membrane. The lytic capacity of the amoebas appears to be the result of cytotoxic factors secreted by the amoebae since, when monolayers were incubated with conditioned medium, there was also a decrease in the transepithelial resistance. Besides, mechanical injury produced by the attachment and movement of the trophozoites may contribute to the disruption of the cell monolayer. As in other pathogenic amoebae, the cytopathic action of A. castellanii on the cell monolayers can subjectively be separated into four stages: adhesion, cytolysis, phagocytosis, and intracellular degradation.  相似文献   

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When E. coli cells are subjected to energy source downshift, the accumulation of RNA (and overall cell growth) is drastically restricted within 1 to 2 min. However, the identity of the primary metabolic signal for this adjustment is a mystery. Earlier studies, and further evidence presented here, show that there is no satisfactory correlation between the sudden adjustment of RNA accumulation and the kinetics of changes in the levels of prospective signalling compounds, such as glycolytic intermediates, ppGpp, ATP, or the three adenylate nucleotides. We have discovered an unusual nucleotide, which we call the phantom spot, whose level decreases dramatically within a minute of downshift, correlating well with the adjustment of RNA accumulation. Preliminary characterization of the phantom spot indicates that it is a triphosphate derived from the guanylate pathway, and suggests that it is a form of GTP with a modification of the imidazole portion of the purine ring. We postulate that this nucleotide serves as a regulatory facsimile of ATP, linking the rate of RNA accumulation and other anabolic processes to the overall rate of phosphorylation.  相似文献   

20.
We have studied the effects of 1 mM solutions of L-amino acids on the X-ray- and heat-induced generation of hydrogen peroxide and hydroxyl radicals in phosphate buffer (5 mM, pH 7.4). Hydrogen peroxide was estimated by enhanced chemiluminescence in the luminol/p-iodophenol/peroxidase system; hydroxyl radicals were detected with a fluorescent probe coumarin-3-carboxylic acid. We demonstrate that amino acids can be grouped into three categories by their effect on X-ray-induced H2O2 production: those that reduce, increase, and have no influence on H2O2 yield. Similar amino acid effects were observed upon heating; however, the composition of respective amino acid groups was different. All amino acids lowered the X-ray-induced hydroxyl radical production, and the most effective were Cys > His > Phe = Met = Trp > > Tyr (in descending order). Hydroxyl radical generation induced by heating was inhibited by Met, His, and Phe; enhanced by Ser; and not affected by Tyr and Pro. Thus, amino acids have different effects on the production of reactive oxygen species by X-rays and heating, and some amino acids appear to be effective natural antioxidants.  相似文献   

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