Aberrant energy-linked reactions in mitochondria isolated from the livers of rats infected with the liver fluke Fasciola hepatica.

The respiratory properties of mitochondria isolated from the livers of rats infected with the parasite Fasciola hepatica were examined. Oligomycin-sensitive ATPase activity was also examined during the acute stage (2-4 weeks post-infection). At 2,4 and 6 weeks post-infection, mitochondrial respiration in vitro (supported by site I and site II substrates) was completely uncoupled. Limited respiratory control had returned by 11 weeks post-infection, but complete recovery was not observed even at 21 weeks post-infection. At 4 weeks post-infection, uncoupled respiration (from all three energy-conserving sites) was also markedly attenuated (to the greatest extent with NADH-linked substrate). Except for pyruvate-supported respiration, this attenuation was not apparent at any other stage of the infection. The attenuation of pyruvate-supported respiration declined, but was still present, at 6 weeks post-infection. In addition to these perturbations in mitochondrial respiratory properties, mitochondrial ATPase activity at 4 weeks post-infection was insensitive to oligomycin, indicating a change in the structural integrity of the ATPase complex.     

Comparison of the effect of hypoxia on the secretion of the atrial natriuretic factor in spontaneously hypertensive and normotensive rats.

The effect of short lasting hypoxia on blood pressure, plasma atrial natriuretic peptide level and number of specific atrial granules were studied in 26 male spontaneously hypertensive and 24 normotensive Wistar rats. A great difference occurred in ANP secretion between hypertensive and normotensive rats. In the hypertensive animals elevated plasma ANP concentration (130 +/- 27 pg/ml) and decreased granularity in the right atria (73 +/- 2) were found on the first day of hypoxia with a slight elevation in urinary sodium content versus normotensive controls. The blood pressure also decreased although not significantly (190 +/- 14 mm Hg). In Wistar rats increased plasma ANP (130 +/- 34 pg/ml) and decreased atrial granularity versus normotensive controls (72 +/- 10 in the left and 113 +/- 16 in the right atrium) were observed only on the third day of hypoxia without changes in blood pressure and natriuresis. In SHR the rapid but short timed ANP release might be of right atrial origin and probably the consequence of a continuous and perhaps increased secretion of the peptide in normoxic conditions too. In Wistar rats the plasma ANP elevation could be secondary due to the increased plasma level of different vasoactive hormones to hypoxia. In the altered effect of ANP in hypertensive and normotensive hypoxic animals, structural and functional changes in the vascular bed may play a role.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

Molecular phylogeny and divergence times of drosophilid species

The phylogenetic relationships and divergence times of 39 drosophilid species were studied by using the coding region of the Adh gene. Four genera--Scaptodrosophila, Zaprionus, Drosophila, and Scaptomyza (from Hawaii)--and three Drosophila subgenera--Drosophila, Engiscaptomyza, and Sophophora--were included. After conducting statistical analyses of the nucleotide sequences of the Adh, Adhr (Adh-related gene), and nuclear rRNA genes and a 905-bp segment of mitochondrial DNA, we used Scaptodrosophila as the outgroup. The phylogenetic tree obtained showed that the first major division of drosophilid species occurs between subgenus Sophophora (genus Drosophila) and the group including subgenera Drosophila and Engiscaptomyza plus the genera Zaprionus and Scaptomyza. Subgenus Sophophora is then divided into D. willistoni and the clade of D. obscura and D. melanogaster species groups. In the other major drosophilid group, Zaprionus first separates from the other species, and then D. immigrans leaves the remaining group of species. This remaining group then splits into the D. repleta group and the Hawaiian drosophilid cluster (Hawaiian Drosophila, Engiscaptomyza, and Scaptomyza). Engiscaptomyza and Scaptomyza are tightly clustered. Each of the D. repleta, D. obscura, and D. melanogaster groups is monophyletic. The splitting of subgenera Drosophila and Sophophora apparently occurred about 40 Mya, whereas the D. repleta group and the Hawaiian drosophilid cluster separated about 32 Mya. By contrast, the splitting of Engiscaptomyza and Scaptomyza occurred only about 11 Mya, suggesting that Scaptomyza experienced a rapid morphological evolution. The D. obscura and D. melanogaster groups apparently diverged about 25 Mya. Many of the D. repleta group species studied here have two functional Adh genes (Adh-1 and Adh-2), and these duplicated genes can be explained by two duplication events.      

Phosphoenolpyruvate carboxykinase from Fasciola hepatica

Behm C. A. and Bryant C. 1982. Phosphoenolpyruvate carboxykinase from Fasciola hepatica. International Journal for Parasitology12: 271–278. The kinetic properties of a partially purified preparation of phosphoenolpyruvate carboxykinase (PEPCK) from F. hepatica were examined. The pH optimum for the carboxylation reaction is 5.8–6.2. The enzyme is more active with Mn²⁺ than Mg²⁺ and the Mn²⁺ saturation curve was sigmoid. Apparent K_m values for the substrates GDP, IDP, PEP and HCO₃^? were determined and found to be in the same range as those reported for other helminths except that the enzyme is less sensitive to low PEP concentrations. GTP and ATP at 0.5 and 1.0 mM inhibit the enzyme; the GTP inhibition was greater in the presence of Mg²⁺ than Mn²⁺ and was competitive with GDP. It was concluded that the activity of PEPCK from F. hepatica is controlled by the concentration of reactants and the ambient pH, that the accumulation of GTP is a sensitive mechanism for inhibiting the carboxylation reaction and that PEPCK activity in the cytosol is likely to be favoured over that of pyruvate kinase except when pH is high and PEP concentration low.     

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

Regulatory properties of a partially purified preparation of pyruvate kinase from Fasciola hepatica

It possesses sigmoid kinetics with PEP; FBP activation changes the relationship to a rectangular hyperbola. The enzyme is inhibited by malate, which competes with PEP; FBP relieves the inhibition slightly. ATP and bicarbonate ions are also inhibitory at high concentrations. ATP inhibition is mixed-competitive with PEP; bicarbonate inhibition is non-competitive. It is suggested that pyruvate kinase may regulate both lactate and acetate production by moderating the size of the cytosolic pyruvate pool.     

ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria

Köhler P. B.,Ryant C. and Behm Carolyn A. 1978. ATP synthesis in a succinate decarboxylase system from Fasciola hepatica mitochondria. International Journal for Parasitology8: 399–404. Succinate decarboxylation was measured by the formation of ¹⁴CO₂ from 1,4-¹⁴C-succinate in a particle free, dialysed mitochondrial extract from liver fluke. It has an absolute requirement for Mg²⁺ and CoA. ATP, ADP and inorganic phosphate are essential for optimal activity. Ap₅A, an inhibitor of adenylate kinase, and glutathione are also necessary. GTP supports decarboxylation as well as ATP, provided ADP is also present. The formation of CO₂ and propionate greatly exceeds the amount of ATP and CoA initially present in the reaction mixture. A net, substrate-level phosphorylation of ADP occurs, the amount of ATP formed being equivalent to the production of CO₂ or propionate. This system is inhibited in flukes incubated in vitro with mebendazole.It is concluded that ATP is required to spark the fermentation system when succinate is the initial substrate and intermediate substrates are absent; that the terminal step in propionate formation is catalysed by a transferase which transfers CoA from propionyl CoA to succinate; and that ATP formation is coupled to the decarboxylation of methylmalonyl-CoA. A reaction scheme is presented.     

Studies of regulatory metabolism in Moniezia expansa: the role of phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase (PEPCK) from M. expansa has been partially purified and its behaviour in a range of different assay conditions has been determined. Different PEPCK's were found in the cytosol and mitochondria. Some kinetic parameters for each are presented. Both enzymes are activated by Mn²⁺; cytosolic PEPCK is also activated by Mg²⁺. The enzymes have pH optima in the range 6·4–7·0. They do not differ with respect to their apparent affinities for inosine and guanosine diphosphates, but the latter allows higher maximal activity. Little activity is observed with adenosine diphosphate. Adenosine and inosine triphosphates exert weak inhibitory effects on the Mn²⁺ activated enzymes; a much strongsr inhibition is exerted on the cytosolic enzyme when activated by Mg²⁺. A number of non-nucleotide compounds were tested for possible inhibitory effects with no success. The forward and back reactions catalyzed by PEPCK proceed at similar rates, suggesting that the enzyme may be readily raversible in vivo.