Copper toxicity to earthworms: A comprehensive review and meta-analysis.

Copper can accumulate in agricultural topsoil through the use of Cu-based fungicides, which may harm soil organisms such as earthworms. This study aimed at reviewing the effects of copper on earthworms at different levels of biological organization, and to determine critical values of copper toxicity to earthworms using a meta-analysis and accounting for lethal and sub-lethal effects and different earthworm species and exposure conditions. Endpoints at the sub-individual level were more sensitive than at higher levels of organization. At the individual level, the most sensitive endpoints were reproduction and growth (hatching success, hatchling growth). Hormetic growth was clearly recognized at copper concentrations less than 80 mg kg-1 in dry soil. However, effects at the sub-individual level already occurred at lower concentrations. Considering all the exposure conditions, the calculated weighted means were 113 mg Cu kg-1 dry soil (95% CI -356; 582) for the LC50 (lethal concentration for 50% of the exposed individuals), 94.6 mg Cu kg-1 dry soil (95% CI 14.0; 175) for the EC50 reproduction, and 144 mg Cu kg-1 dry soil (95% CI -12.6; 301) for the EC50 growth or weight change. When accounting for the origin of the soil, earthworms were five times more sensitive to copper (LC50) in natural than in artificial soils. The different factors affecting Cu toxicity to earthworms explain the high variability of these values, making it difficult to derive thresholds. However, considering the potential negative effects of copper on earthworms, attention should be given to the more sustainable use of human-contributed copper in agricultural soils.

Avoidance behaviour response and esterase inhibition in the earthworm, Lumbricus terrestris, after exposure to chlorpyrifos.

The avoidance response of earthworms to polluted soils has been standardised using a simple and low-cost test, which facilitates soil toxicity screening. In this study, the avoidance response of Lumbricus terrestris was quantified in chlorpyrifos-spiked soils, depending on the pesticide concentration and exposure duration. The inhibition of acetylcholinesterase (AChE) and carboxylesterase (CbE) activities was also determined as indirect measures of pesticide bioavailability. The effects of different chlorpyrifos concentrations were examined in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyrifos. A modification of the test involved a pre-exposure step (24, 48 or 72 h) in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to avoid the contaminated soils. However, the esterase activities showed that all earthworms were exposed to chlorpyrifos. Acetylcholinesterase activity did not change in earthworms in the standardised behavioural test (0.58 ± 0.20 U/mg protein, mean ± SD; n = 72), whereas the CbE activity was significantly inhibited (62-87 % inhibition) in earthworms exposed to 3 and 15 mg/kg. In the modified test, earthworms had greatly inhibited AChE activity (0.088 ± 0.034 U/mg protein, n = 72), which was supported by reactivation of the inhibited enzyme activity in the presence of pralidoxime (2-PAM). Similarly, the CbE activity was significantly inhibited in earthworms with all treatments. This study suggests that the avoidance behaviour test for organophosphorus-contaminated soils could be supported by specific biomarkers to facilitate a better understanding of pesticide exposure and toxicity during this test.

Inhibition, recovery and oxime-induced reactivation of muscle esterases following chlorpyrifos exposure in the earthworm Lumbricus terrestris.

Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg(-1) chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (< or = 1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field.

Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase.

We have expressed the DNA of the fusion of CS1 to MDH1 in Escherichia coli gltA-. The fusion protein (CS1/MDH1) is the C-terminus of CS1 linked in-frame to the N-terminus of MDH1 with a short linker of glycyl-seryl-glycyl. The fusion protein produced was isolated and purified. Gel filtration studies indicated that CS1/MDH1 had a M(r) of approximately 170,000. Western blotting analysis with SDS gel indicated a M(r) of approximately 90,000-95,000 (theoretical M(r) = 87,000). This is the expected M(r) for the fusion protein subunit. The kinetics of CS1 and MDH1 activities of the fusion protein were compared to those of the free enzymes. In addition, the effect of AAT reaction, as a competitor for the intermediate OAA of the coupled MDH-CS reaction, was examined. It was observed that AAT was a less effective competitor for OAA when the CS1/MDH1 fusion protein is used than when the separate enzymes are employed. In addition, the transient time for the coupled reaction sequence was less for the fusion protein than for the free enzymes.

Structural and functional properties of a multi-enzyme complex from spinach chloroplasts. 1. Stoichiometry of the polypeptide chains.

Antibodies have been raised specifically against chloroplast phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase and ribulose 1,5-bisphosphate carboxylase-oxygenase. Each of these antibodies recognizes the same macromolecular entity isolated and purified from chloroplasts. This entity is a multi-enzyme complex, previously isolated and made up of ribose-phosphate isomerase, phosphoribulokinase, ribulose 1,5-bisphosphate carboxylase-oxygenase, phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase. Under denaturing conditions the multi-enzyme complex contains two polypeptides of 54 kDa and 15 kDa corresponding to the large and the small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase, the two polypeptides of the glyceraldehyde-3-phosphate dehydrogenase of 39 kDa and 37 kDa, one polypeptide of 40 kDa pertaining to phosphoribulokinase and one polypeptide of 30 kDa very likely pertaining to ribose-phosphate isomerase. The combined use of immunochemical and densitometric techniques allows one to determine the number and the stoichiometry of the various types of polypeptide chains and to compare them with the quaternary structure of the corresponding isolated enzymes. Ribulose 1,5-bisphosphate carboxylase-oxygenase of higher plants consists of eight large and eight small subunits. Glyceraldehyde-3-phosphate dehydrogenase is made up of two types of polypeptide chains called A and B and its simplest quaternary structure is A2B2. Finally, phosphoribulokinase is a dimer made up of two identical subunits. Therefore, for the three isolated enzymes, the stoichiometry of the polypeptide chains is always 1:1. Within this multi-enzyme complex, there are two subunits of phosphoribulokinase, two A and B subunits of glyceraldehyde-3-phosphate dehydrogenase and two large and four small subunits of ribulose 1,5-bisphosphate carboxylase-oxygenase. Therefore the number and the stoichiometry of the polypeptide chains of phosphoribulokinase and glyceraldehyde-3-phosphate dehydrogenase are the same in the multi-enzyme complex and in the free enzymes, but those of ribulose 1,5-bisphosphate carboxylase-oxygenase are completely different. This conclusion that the multi-enzyme complex contains two active sites for ribulose 1,5-bisphosphate may be confirmed independently by kinetic inhibition studies using 6-phosphogluconate.

Structural and functional properties of a multi-enzyme complex from spinach chloroplasts. 2. Modulation of the kinetic properties of enzymes in the aggregated state.

The carboxylase activity of free ribulose 1,5-bisphosphate carboxylase-oxygenase has been compared to that of the five-enzyme complex present in chloroplasts. Kinetic results have shown that the V/active site is lower for the free enzyme than for the complex. Conversely the Km is smaller for the complex than for the free enzyme. This implies that the catalytic activity of the enzyme is enhanced when it is embedded in the complex. Under reducing conditions and in the presence of reduced thioredoxin, inactive oxidized phosphoribulokinase, free in solution or inserted in the multi-enzyme complex, becomes active. The kinetics of this activation process has been studied and shown to be exponential. The time constant of this exponential decreases, for the free enzyme, as thioredoxin concentration is increased. Alternatively, for the enzyme embedded in the complex, this time constant increases with thioredoxin concentration almost in a linear fashion. This implies that the complex is much more rapidly activated by reduced thioredoxin than is the free phosphoribulokinase. The variation of the amplitude of this activation process as a function of thioredoxin concentration is a hyperbola. The concentration of thioredoxin which results in half the asymptotic value of this hyperbola is smaller for the complex than for the free enzyme. A kinetic model has been proposed and the dynamic equations resulting from this model have been derived. They fit the experimental results exactly. From the variation of the amplitude of the activation process one may derive the binding constants of thioredoxin on either the oxidized enzyme or on a partly dithiothreitol-reduced enzyme (both of them free or inserted in the complex). In either case, the affinity of reduced thioredoxin is larger for the complex than for the free enzyme. The individual values of some of the rate constants have also been estimated from the variation of the time constants as a function of thioredoxin concentration. Taken together, these results show that at least two enzymes, ribulose 1,5-bisphosphate carboxylase-oxygenase and phosphoribulokinase, have quite different kinetic properties depending on whether they are in free solution or embedded in the multi-enzyme complex.

Thioredoxin activation of phosphoribulokinase in a chloroplast multi-enzyme complex.

The activation process of spinach phosphoribulokinase by thioredoxin f has been studied with the enzyme in a free, isolated state, or integrated in a multi-enzyme complex. The time periods required for enzyme activation are always smaller and the maximal enzyme velocities are always greater when chloroplast phosphoribulokinase is included in the multi-enzyme complex than when it is in the isolated state. Comparative kinetic studies show that phosphoribulokinase extracted from the complex behaves exactly as in the isolated state. The reduced form of the kinase, whatever it has been included in the complex or isolated from the chloroplasts, are deactivated by oxidized thioredoxins. In the absence of thioredoxin f however, the reduced form of the isolated enzyme undergoes spontaneous oxidation whereas the reduced kinase included in the multi-enzyme complex is stable. 'Unspecific' proteins such as bovine serum albumin do not provide any protection of the kinase against autooxidation, whereas 'homologous' specific proteins such as ribulose-1,5-bisphosphate carboxylase/oxygenase dramatically decrease the rate of this autooxidation process. These results therefore support the view that interactions between phosphoribulokinase and the other components of the multi-enzyme complex play an important role in the modulation of the activity of this enzyme. The possible part of these interactions in the control of the Calvin cycle is discussed.

Diabetes mellitus 14 years after a subtotal pancreatectomy for neonatal hyperinsulinism.

A 14-year-old girl developed diabetes mellitus 14 years after subtotal pancreatectomy for neonatal hyperinsulinism. Diabetes mellitus seemed to be due to pancreatectomy and not to the usual juvenile type 1. To the best of our knowledge, this type of complication had never been reported such a long time after a pancreatectomy.

Familial hypermethioninemia partially responsive to dietary restriction.

Hypermethioninemia and absolute methionine intolerance were observed in three siblings. These patients had several peculiar clinical features comprising failure to thrive, mental and motor retardation, facial dysmorphy with abnormal hair and teeth, and myocardiopathy. Hepatic S-adenosylhomocysteine hydrolase activity was decreased by 80% in the three children. These clinical and biochemical features differ from those of hypermethioninemias previously described, and thus represent a new form of inherited disorder of methionine metabolism. Whether S-adenosylhomocysteine hydrolase deficiency is primary or secondary to an unknown metabolic defect remains to be determined.