Multi-enzyme inhibition assay for the detection of insecticidal organophosphates and carbamates by high-performance thin-layer chromatography applied to determine enzyme inhibition factors and residues in juice and water samples

Esterase inhibition assays provide an effect-directed tool of rapid screening for inhibitors in environmental and food samples. According to a multi-enzyme microtiter-plate assay, rabbit liver esterase (RLE), Bacillus subtilis esterase (BS2), and cutinase from Fusarium solani pisi (CUT) were used for the detection of 21 organophosphorus and carbamate pesticides by high-performance thin-layer chromatography–enzyme inhibition assays (HPTLC–EI). Staining was performed with Fast Blue Salt B coupling to α-naphthol enzymatically released from the respective acetate used as substrate. Quantitative analysis was achieved by densitometric evaluation at 533 nm. Enzyme inhibition factors derived from HPTLC–EI were calculated from the slopes of the linear calibration curves, which allowed comparisons to published inhibition constants and well correlated to sensitivity parameters. Limits of detection ranged from a few pg/zone for organophosphates as strongest inhibitors to a few ng/zone for most carbamates, when RLE and BS2 were used. Without oxidation, chlorpyrifos and parathion were directly detectable at approximately 60 and 14 ng/zone, respectively. As the enzyme of lowest sensitivity, CUT was able to detect insecticides of high and low inhibitory power from the ng to μg range per zone. Due to high selectivity of enzyme inhibition, oxon impurities of thionophosphate standards were strongly detected, although only present in low traces. The exemplary application of HPTLC–EI (RLE) to apple juice and drinking water samples spiked with paraoxon (0.001 mg/L), parathion (0.05 mg/L) and chlorpyrifos (0.5 mg/L) resulted in mean recoveries between 71 and 112% with standard deviations of 2.0–18.3%.     

Thiourea mediated regulation in the expression profile of aquaporins and its impact on water homeostasis under salinity stress in Brassica juncea roots

Bioregulatory molecules such as thiourea (TU) play an important role in imparting stress tolerance to crops. However, the molecular mechanism involved in the TU-mediated tolerance has not been elucidated. Towards this endeavour, the expression profile of various PIPs (plasma membrane intrinsic proteins) was studied under salt stress (NaCl; 700 mM) with/without thiourea (TU; 6.5 mM) at different time periods in roots of Brassica juncea. Various aquaporin isoforms demonstrated an upregulation upon salinity stress imposition, whereas they were downregulated upon TU supplementation. TU treatment also led to a decrease in the accumulation of reactive oxygen species and delimited the need for an enhanced accumulation of osmolytes. The vacuolar pH was also maintained in NaCl + TU treatment as demonstrated by in vivo ³¹P NMR of roots. In conclusion, TU supplementation to salt stressed seedlings was found to maintain the water homeostasis of roots through coordinated regulation of different PIP isoforms.     

Detrimental impacts of the dinoflagellate Karenia mikimotoi in Fujian coastal waters on typical marine organisms

Blooms of the toxic dinoflagellate Karenia mikimotoi (K. mikimotoi) have occurred frequently in the East China Sea in recent decades and were responsible for massive mortalities of abalones in Fujian coastal areas in 2012, however, little is known about the effects of these blooms on other marine organisms. In this study, the toxic effects and the possible mechanisms of toxicity of K. mikimotoi from Fujian coastal waters on typical marine organisms at different trophic levels, including zooplankton (Brachionus plicatilis, Artemia salina, Calanus sinicus, and Neomysis awatschensis) and aquaculture species (Penaeus vannamei and Scophthalmus maximus) were investigated. At a bloom density of 3 × 10⁴ cells/mL, the Fujian strain of K. mikimotoi significantly affected the tested organisms, which had mortality rates at 96 h of 100, 23, 20, 97, 33, and 53%, respectively. Moreover, the intact cell suspension was toxic to all tested species, whereas cell-free culture and the ruptured cell suspension had no significant effects on the tested organisms. Possible mechanisms for this toxic effect, including reactive oxygen species (ROS) and hemolytic toxins, were evaluated. For K. mikimotoi, 0.014 ± 0.004 OD/(10⁴ cells) superoxide (O₂⁻) and 3.00 ± 0.00 nmol/(10⁴ cells) hydrogen peroxide (H₂O₂) were measured, but hydrogen peroxide did not affect rotifers at that concentration, and rotifers were not protected from the lethal effects of K. mikimotoi when the enzymes superoxide dismutase and catalase were added to counteract the ROS. The lipophilic extract of K. mikimotoi had a hemolytic effect on rabbit erythrocytes but exhibited no significant toxicity. These results suggest that this strain of K. mikimotoi can have detrimental effects on several typical marine organisms and that its toxicity may be associated with intact cells but is not related to ROS or hemolytic toxins.     

Implementation of a dose–response curve for γ-radiation in the Portuguese population by use of the chromosomal aberration assay

An in vitro dose–response curve following exposure to γ-radiation was determined at the IST/ITN, by use of the chromosomal aberration assay. This is the first study of this kind carried out among the Portuguese population. Un-irradiated and γ-irradiated peripheral blood lymphocytes from 16 healthy donors were cultured. A total of 22,395 metaphases were analyzed for frequency and distribution of dicentrics and centric rings, as a function of the radiation dose. The dose–response data for dicentrics and dicentrics plus centric rings were fitted by use of a linear–quadratic model: Y_dic = (0.0011 ± 0.0006) + (0.0105 ± 0.0035)D + (0.0480 ± 0.0019)D² and Y_{dic + rings} = (0.0011 ± 0.0006) + (0.0095 ± 0.0036)D + (0.0536 ± 0.0020)D². Also, calibration curves related to age and gender were determined, but no significant differences were found. Following the establishment of the dose–response curves, a validation experiment was carried out with three individuals. Real and estimated doses, obtained with the dose–response curves, were in agreement. These results give us confidence to apply both dose–response calibration curves in future biological dosimetry requirements.     

On-line detection of low naphthalene concentrations with a bioluminescent sensor

On-line tests with Pseudomonas fluorescens HK44 were carried out using a biosensor system with a flow-through cell and sample injections to detect low concentrations of naphthalene. Kinetic experiments permitted a 19 min response time and definition of a 50 min induction period for bioluminescence assays. A mathematical model showed that 0.2 g/l is the best cell concentration to be used in the detection of low naphthalene concentrations, with the highest acceleration rate of bioluminescence sensitivity increase (0.46 nA l/(g min²)). Calibration curves with different concentrations of naphthalene showed a linear correlation up to 0.4 mg/l (3 μM) corresponding to 211 nA output signal. The lower limit of naphthalene detection by strain HK44 was in the region of 0.02 mg/l (0.16 μM), below the naphthalene health advisory limit for drinking water consumed over a lifetime suggested by EPA.     

Production of channel catfish with sperm cryopreserved by rapid non-equilibrium cooling

This report describes the feasibility of using vitrification for fish sperm. Vitrification can be used to preserve samples in the field and offers an alternative to conventional cryopreservation, although it has not been systematically studied for sperm of aquatic species. The overall goal of the project was to develop streamlined protocols that could be integrated into a standardized approach for vitrification of aquatic species germplasm. The objectives of the present study in channel catfish (Ictalurus punctatus) were to: (1) evaluate the acute toxicity of 5%, 10%, 20% and 30% methanol, N,N-dimethyl acetamide, dimethyl sulfoxide, 1,2-propanediol, and methyl glycol; (2) evaluate a range of devices commonly used for cryopreservation and vitrification of mammalian sperm; (3) compare vitrification with and without cryoprotectants; (4) evaluate the post-thaw membrane integrity of sperm vitrified in different cryoprotectant solutions, and (5) evaluate the ability of vitrified sperm to fertilize eggs. Cryoprotectant concentrations of higher than 20% were found to be toxic to sperm. Methanol and methyl glycol were the least toxic at a concentration of 20% with an exposure time of less than 5 min. We evaluated a method reported for human sperm, using small volumes in loops (15 μl) or cut standard straws (20 μl) with and without cryoprotectants plunged into liquid nitrogen. Cryoprotectant-free vitrification using loops did not yield fertilization (assessed by neurulation), and the fertilization rates observed in two trials using the cut standard straws were low (∼2%). In general, fertilization values for vitrification experiments were low and the use of low concentrations of cryoprotectants yielded lower fertilization (<10%) than the use of vitrification solutions containing high cryoprotectant concentrations (as high as 25%). The highest neurulation obtained was from a mixture of three cryoprotectants (20% methanol + 10% methyl glycol + 10% propanediol) with a single-step addition. This was reflected in the flow cytometry data from which the highest membrane integrity using loops was for 20% methanol + 10% methyl glycol + 10% propanediol (∼50%). We report the first successful sperm vitrification in fish and production of offspring from vitrified sperm in channel catfish. Although the fertilization values were low, at present this technique could nevertheless be used to reconstitute lines (especially in small aquarium fishes), but it would require improvement and scaling up before being useful as a production method for large-bodied fishes such as catfish.     

Evaluating agro-industrial by-products as dietary roughage source on growth performance of fattening steers

Silages from pineapple peel, sweet corn husk and cob mixed with bagasse and vinasse were evaluated to determine their chemical composition and fermentation characteristics as well as feeding performance in fattening steers. The experiment, which lasted 90 days, involved 48 fattening steers (264 ± 37.4 kg BW) randomly allocated to three diets. Treatments included: a control diet containing rice straw and molasses (T1); diet containing bagasse–vinasse mixture including sweet corn husk and cob silage (BS; T2); and diet containing bagasse–vinasse mixture including pineapple peel silage (BP; T3). All treatments included a commercial concentrate feed (13% CP) and ad libitum rice straw throughout the experiment. Results from chemical analysis showed that dry matter (DM) of BS was higher than BP (P < 0.05), whereas the protein content of BS and BP was similar (P > 0.05). For fermentation characteristics, pH in BP was lower than BS (P < 0.05); in addition, acetic and butyric acids in BS were higher than BP (P < 0.05). Findings from growth trial showed that total DM intake in steers fed T1 was higher compared to the other dietary treatments (P < 0.05), whereas the average BW gain was found to be grater in T3 steers (P < 0.05). As result from our findings, bagasse–vinasse mixture with pineapple peel silage appeared to be a viable feed ingredient in fattening steer diet and moreover it could become an economically feasible agro-industrial by-product for farmers.     

Immune responses,ROS generation and the haemocyte damage of scallop Chlamys farreri exposed to Aroclor 1254

The toxic effects of Aroclor 1254 (0.05, 0.5, 5 and 50 μg l^?1) on scallop (Chlamys farreri) immune system in vivo were studied. The results showed that Aroclor 1254 had significant toxic effect on the parameters tested in this paper (P < 0.05). The total number of haemocytes, the proportion of granulocytes, phagocytosis in all groups as well as the lysosomal membrane stability (LMS) in 5, 50 μg l^?1 and bacteriolytic activity 0.5, 5, 50 μg l^?1 treatments decreased significantly, while the proportion of hyalinocytes and the production of O₂^- in all treatments remarkably increased during the sampling time and tended to be stable gradually after 6–15 d. The bacteriolytic activity in 0.05 μg l^?1 treatments, LMS in 0.05, 0.5 μg l^?1 groups and the DNA damage (comet ratios and arbitrary values) in all treatments increased at the beginning of exposure and reached their peaks on day 1, day 1, day 6 and day 3, following that they all decreased gradually and became stable after 9–15 d. When the indices reached stability, except for DNA damage was higher than controls, the others were all significantly lower than those of controls (P < 0.05). Thus, Aroclor 1254 has evident toxic effects on scallop immune system, which supports the view that a relationship exists between pollution and immunomodulation in aquatic organisms. Also it supports the speculation that the PCBs pollution is one of the important reasons of the mass mortality of the C. farreri.     

Rapid phosphorylation of MAP kinase-like proteins in two species of Arctic kelps in response to temperature and UV radiation stress

Mitogen-activated protein kinases (MAPKs) are a group of cytoplasmic phosphoproteins that constitute the central core of the signalling network to respond to stress in most organisms. Their role in stress responses has been extensively studied in organisms from yeast to humans, and recently, their presence has also been described in higher plants as well as in micro- and macroalgae. In this study, we demonstrate via short experiments (1 h in duration), the rapid activation of two MAPKs similar to p38 and JNK of mammalian cells, in the Arctic kelps Laminaria solidungula and Saccharina latissima exposed to temperature and UV stress. The molecular mass of p38 is 40 kDa in L. solidungula and 42 kDa in S. latissima, while two JNKs were detected in both species, of 36 and 42 kDa in L. solidungula, and 36 and 40 kDa in S. latissima. These MAPKs are highly phosphorylated in response to temperature and UV light. In S. latissima, both p38 and the JNK showed higher phosphorylation at 2 °C than at 7 °C, while the reverse response was shown for L. solidungula. In addition, a significant increase in phosphorylation of both kinases was found following exposure to UV radiation (UVR). Exposure to PAR + UVA + UVB induced higher phosphorylation than PAR + UVA in L. solidungula, especially at 7 °C. In S. latissima, this response occurred only with JNK, and no differences in p38 phosphorylation between PAR + UVA and PAR + UVA + UVB at any temperature were observed. These results indicate the possible participation of MAPK-like proteins in response to stress in Arctic kelps, and that their activation is species-specific.     

Time-course correlation of early toxic events in three models of striatal damage: Modulation by proteases inhibition

Metabolic alterations in the nervous system can be produced at early stages of toxicity and are linked with oxidative stress, energy depletion and death signaling. Proteases activation is responsible for triggering deadly cascades during cell damage in toxic models. In this study we evaluated the early time-course of toxic events (oxidative damage to lipids, mitochondrial dysfunction and LDH leakage, all at 1, 3 and 6 h) in rat striatal slices exposed to quinolinic acid (QUIN, 100 μM) as an excitotoxic/pro-oxidant model, 3-nitropropionic acid (3-NP, 1 mM) as an inhibitor of mitochondrial succinate dehydrogenase, and a combined model produced by the co-administration of these two toxins at subtoxic concentrations (21 and 166 μM for QUIN and 3-NP, respectively). In order to further characterize a possible causality of caspases or calpains on the toxic mechanisms produced in these models, the broad calpain inhibitor IC1 (50 μM), and the pan-caspase inhibitor Z-VAD (100 μM) were tested. Lipid peroxidation (LP) was increased at all times and in all models evaluated. Both IC1 and Z-VAD exerted significant protection against LP in all models and at all times evaluated. Mitochondrial dysfunction (MD) was consistently affected by all toxic models at 3 and 6 h, but was mostly affected by 3-NP and QUIN at 1 h. IC1 differentially protected the slices against 3-NP and QUIN at 1 h and against QUIN at 3 h, while Z-VAD exhibited positive actions against QUIN and 3-NP at all times tested, and against their combination at 3 and 6 h. LDH leakage was enhanced at 1 and 3 h in all toxic models, but this effect was evident only for 3-NP + QUIN and 3-NP at 6 h. IC1 protected against LDH leakage at 1 h in 3-NP + QUIN and 3-NP models, at 3 h in all toxic models, and at 6 h in 3-NP + QUIN and 3-NP models. In turn, Z-VAD protected at 1 and 6 h in all models tested, and at 3 h in the combined and QUIN models. Our results suggest differential chronologic and mechanistic patterns, depending on the toxic insult. Although LP, MD and membrane cell rupture are shared by the three models, the occurrence of each event seems to obey to a selective recruitment of damaging signals, including a differential activation of proteases in time. Proteases activation is likely to be an up-stream event influencing oxidative stress and mitochondrial dysfunction in these toxic models.     

Insecticidal and developmental inhibitory properties of monoterpenes on Culex pipiens L. (Diptera: Culicidae)

Twelve monoterpenes were evaluated for larvicidal and adulticidal activities towards Culex pipiens. Geraniol and cuminaldehyde were the most toxic monoterpenes to larvae, with LC₅₀ values of 38.6 and 38.9 mg/l after 24 h of treatment, respectively, whereas cuminaldehyde was the most potent compound after 48 h of treatment, followed by geraniol and thymol. In fumigant toxicity experiments, (R)-carvone and geraniol were the most toxic monoterpenes against the adults at all three tested concentrations and after both 24 and 48 h. When tested at sublethal concentrations (0.5 LC₅₀), (R)-carvone, (S)-limonene and cuminaldehyde decreased hatchability, pupation and adult emergence and induced high larval mortality. Our results suggest that geraniol, cuminaldehyde and (R)-carvone are promising toxicants against Culex pipiens and could be useful in the search for new natural insecticides.     

Evaluation of the adverse effects of two commonly used fertilizers,DAP and urea,on motility and orientation of the green flagellate Euglena gracilis

The effect of two commonly used fertilizers, DAP (diammonium phosphate) and urea was studied on the freshwater flagellate Euglena gracilis using the automatic biotest ECOTOX. NOEC and EC₅₀ values for various parameters like motility, velocity, cell shape and gravitaxis were calculated. The NOEC and EC₅₀ values obtained for DAP were much lower than those for urea; i.e. DAP showed a stronger inhibitory effect as compared to urea. The inhibition caused by DAP increased with increasing exposure time over 24 h but urea showed no augmentation with increasing exposure time. Application of DAP resulted in an increased pH and high concentrations of ammonia but urea did neither affect the pH nor affect the ammonia concentration. Recovery experiments in deionized water after urea application showed a reconstitution of motility after 72 h. After an application of 1.35 g L⁻¹ (24 h EC₅₀ for motility) DAP motility recovered after 72 h but motility did not recover when the concentration was doubled (2.7 g L⁻¹). The EC₅₀ values obtained were compared with the EC₅₀/LC₅₀ values reported for other aquatic organisms and were found to be comparable with the reported values.     

Toxic strains of the Alexandrium ostenfeldii complex in southern South America (Beagle Channel,Argentina)

During phytoplankton monitoring in the Beagle Channel (≈54°52′ S, 67°32′ W) a previously undetected Alexandrium species was observed in coincidence with mouse bioassay toxicity. Detailed thecal plates analysis using epifluorescence and scanning electron microscopy revealed the presence of the Alexandrium ostenfeldii species complex, showing a mixture of the diagnostic features usually used to discriminate between the morphospecies A. ostenfeldii and A. peruvianum. Cells of the A. ostenfeldii complex were commonly observed during spring after the main annual diatom bloom, when temperatures and salinities were respectively around 7.5–10 °C and 30–30.5 psu, and nutrients showed a seasonal decrease. Toxin analysis by liquid chromatography–mass spectrometry revealed the production of 13-desmethyl spirolide C and 20-methyl spirolide G in cell cultures. The cellular contain of spirolides during exponential phase growth was 0.5906 ± 0.0032 and 0.1577 ± 0.0023 pg cell⁻¹ for 13-desMe-C and 20-Me-G, respectively. A third unknown compound, with a structure resembling that of spirolides was also detected in culture. Moreover, an additional compound with a similar m/z (692) than that of 13-desMe-C but presenting a higher retention time (Rt = 40.5 min) was found in high proportions in mussel samples. PSP toxins were present at low concentration in mussels but were not detected in cultures. These results extend the world-wide distribution of toxic strains of the A. ostenfeldii complex to the Beagle Channel (southern South America), where toxic events have been traditionally linked to the presence of Alexandrium catenella. This is the first confirmed occurrence of spirolides in mussels and plankton from Argentina, which highlights the importance of monitoring these toxins and their producing organisms to protect public health and improve the management of shellfish resources.     

Rational design and synthesis of dihydropyrimidine based dual binding site acetylcholinesterase inhibitors

Based on the pharmacological importance of dihydropyrimidine (DHPM) scaffold, substituted DHPMs linked with acetamide linker to substituted aromatic anilines were synthesized and evaluated for their potency as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The good AChE inhibitory activity of 4-dihydropyrimidine-2-thione (4a–h) and 2-amino-1,4-dihyropyrimidines (5a–h) series was observed with compound 4a and 4d identified as the most potent compounds with IC₅₀ values of 0.17 ± 0.01 and 0.39 ± 0.04 μM respectively. The inhibition of BChE was found in a broader range of concentrations (2.37–56.32 μM). To explore the binding insights into the enzyme, molecular docking study was carried out using GOLD software. The binding mode analysis indicated that all of these inhibitors are well accommodated in the active site and interact with the key amino acid residues of Catalytic anionic site (CAS) and peripheral anionic site (PAS). Furthermore, in silico ADMET predictions suggest that these compounds are non-AMES toxic with good blood brain barrier (BBB) penetration, human intestinal absorption.     

Toxic and essential elements in children's blood (<6 years) from Kinshasa,DRC (the Democratic Republic of Congo)

In this study we determined the concentration of 9 trace elements (As, Cd, Cu, Hg, Mn, Mo, Pb, Se and Zn) in whole blood of children (n = 100, 64 girls, 36 boys and median age: 36 months) using inductively coupled plasma mass spectrometry (ICP-MS). The proportion of children potentially deficient in essential elements or poisoned by toxic elements was evaluated. The aging effects on the concentration of these elements were also investigated. The median values were 3.17 μg/L (As), 0.15 μg/L (Cd), 1.1 mg/L (Cu), 2.1 μg/L (Hg), 10.4 μg/L (Mn), 17.7 μg/L (Mo), 8.7 μg/dL (Pb), 10.7 μg/L (Se) and 5.0 mg/L (Zn). The concentration of many elements (As, Cd, Hg, Mn, Pb and Zn) showed significant age variations but not sex influence. Regarding levels of the essential elements (Cu, Mn, Mo, Se and Zn), B-Cu, B-Mn, B-Se and B-Zn were in the normal range, whereas exceeded levels were observed for B-Mo. None of these children was deficient in essential elements. Except B-Cd, all toxic elements showed exceeded blood levels. The proportion of children potentially poisoned by toxic elements varies from 10% (n = 10) to 95% (n = 95) and depends on toxic element: 95% for As, 10% for Hg and 35% for Pb. The main health concerns emerging from this study are the high As, Hg and Pb exposures of the Kinshasan children requiring further documentation, corrective actions and the implementation of appropriate regulations.     

Effects of dichromate on growth and root system architecture of Arabidopsis thaliana seedlings

The hexavalent form of chromium [Cr(VI)] is toxic for most organisms; however, very little information is available regarding the effects of this metal on plant morphogenesis. In this work, we investigated the effects of Cr(VI) on the growth and development of Arabidopsis thaliana, a species widely used as a model for studying the diverse physiological and cellular processes in plants. Elongation of root hairs and biomass production were stimulated by relatively low concentrations (100 μM) of Cr(VI) as potassium dichromate. Concentrations of Cr(VI) greater than 200 μM were toxic to plants as revealed both by arrested growth of roots and shoots and the development of chlorosis in leaves. At 200 μM the primary root growth was totally inhibited but the plants continued their growth manifesting different alterations in root development. These alterations correlated with changes in mitotic activity and in cellular expansion. The analyses of A. thaliana transgenic plants that express the auxin-inducible marker DR5:uidA, and the response of the auxin-resistant mutants axr2 and aux1–7 to dichromate suggest that auxins do not participate as mediators in the cellular and physiological responses to this metal. The primary root growth inhibition by 200 μM dichromate was alleviated by more than 70% by increasing the sulfate, phosphate or nitrate concentration in the media, which suggests a relation of dichromate with these mineral nutrients.     

Trace amounts of nickel in belowground rhizomes of Vaccinium myrtillus L. decrease anthocyanin concentrations in aerial shoots without water stress

Nickel (Ni) may impair plant water balance through detrimental effects on the belowground level. Bilberry (Vaccinium myrtillus L.) plants were grown in a mesic heath forest-type soil and subjected to Ni sulphate (NiSO₄·6H₂O) concentrations of 0, 10, 50, 100 and 500 mg m⁻² during an entire growing season in northern Finland (65°N). Biomass of belowground rhizomes, and tissue water content (TWC) and anthocyanin concentrations of aerial shoots were determined from mature plants in order to study rhizospheric Ni stress, and its possible long-distance effects on aerial shoots. As the major proportion of biomass of bilberry is invested in belowground parts, it was hypothesised that Ni-induced rhizospheric disturbance causes water stress in aerial shoots and increases their anthocyanin concentrations for osmotic regulation. Uptake of Ni from the soil to the rhizome and aerial shoots was measured with X-ray fluorescence spectrometry. Ni concentrations in the soil and rhizome exhibited a dose–response relationship, but the concentrations in the rhizome were about 10-fold lower (<3 mg Ni kg⁻¹) than those in the soil (<30 mg Ni kg⁻¹). Translocation of Ni from the rhizome to aerial shoots did not occur, as Ni concentrations in shoots remained at 1 mg Ni kg⁻¹. Although Ni concentrations in the rhizome were below the threshold values of Ni toxicity (i.e. 10–50 mg Ni kg⁻¹), Ni decreased the rhizome biomass. Anthocyanins decreased in aerial shoots along with the Ni accumulation in the rhizome, while TWC was unaffected. The result suggests that anthocyanins are not involved in osmotic regulation under Ni stress, since anthocyanins in aerial shoots responded to the Ni concentrations in the rhizome despite the lack of water stress.     

The effects of accelerometer placement on mechanomyographic amplitude and mean power frequency during cycle ergometry

The purposes of this study were threefold: (1) to compare the power output related patterns of absolute and normalized MMG amplitude and MPF responses for proximal and distal accelerometer placements on the vastus lateralis (VL) muscle during incremental cycle ergometry; (2) to examine the influence of accelerometer placements on mean absolute MMG amplitude and MPF values; and (3) to determine the effects of normalization on mean MMG amplitude and MPF values from proximal and distal accelerometer placements. Fifteen adults (10 men and 5 women; mean ± SD age = 23.9 ± 3.1 years) performed incremental cycle ergometry tests to exhaustion. Two accelerometers were placed proximal and distal on the VL muscle. Paired t-tests indicated that absolute MMG amplitude values for the proximal accelerometer were greater (p < 0.05) than the distal accelerometer at all power outputs. The normalized MMG amplitude also had greater values for the proximal accelerometer at all power outputs, except 50 W. There were no differences, however, between proximal and distal accelerometers for absolute MMG MPF, except at 75 W, and normalization eliminated this difference. Twenty-seven percent of the subjects exhibited different power output related patterns of responses between accelerometer placements for MMG amplitude and 47% exhibited different patterns for MPF. These findings indicated that normalization did not eliminate the influence of accelerometer placement on MMG amplitude and highlighted the importance of standardizing accelerometer placements to compare MMG values during cycle ergometry.     

Gelation of high methoxy pectin by acidification with d-glucono-δ-lactone (GDL) at room temperature

Rheological comparisons have been made between preparations of high methoxy pectin (DE ≈ 70%) gelled by acidification with d-glucono-δ-lactone (GDL) on holding for 16 h at 25 °C in the presence of 60 wt% sucrose, and otherwise identical preparations gelled by acidification with citric acid at high temperature and cooling from 90 to 25 °C at 1 °C/min. Two series of experiments were carried out for both methods of acidification. In the first series, the concentration of pectin (c) was held constant at 1.0 wt% and the final pH attained after holding (with GDL) or cooling (with citric acid) was varied from 3.75 to 2.25. In the second series, the final pH was held constant at 3.0 and c was varied from 0.25 to 2.00 wt%. All samples were then heated (1 °C/min) from 25 to 90 °C. Rheological changes on cooling/holding and heating were characterised by low-amplitude oscillatory measurements of storage modulus (G′) and loss modulus (G′′) at 1 rad s^?1 and 0.5% strain, and mechanical spectra were recorded at 25 °C. Selected samples, gelled with GDL, were also characterised by compression testing (at 25 °C), and a direct linear relationship was found between the logarithm of yield stress and log G′.The concentration-dependence of moduli for the samples acidified to pH 3.0 with GDL had the form typical of biopolymer gels, with log G′ versus log c approaching a limiting slope of 2 as c was raised above the minimum critical gelling concentration (c_o ≈ 0.3 wt%). Under all conditions of pH and pectin concentration studied, the values of G′′ (at 25 °C) for the samples acidified with citric acid were higher than those of the corresponding GDL-induced networks. The values of G′ were also higher, except at very low pH (below ～2.7 at c = 1.0 wt%) or very high concentrations of pectin. At pectin concentrations above ～1.5 wt%, the moduli of the samples gelled with citric acid (at pH 3.0) levelled out, or decreased slightly, with the values of G′ dropping below those of the GDL-induced networks towards the end of the concentration range studied (at c ≈ 2 wt%). All samples acidified with citric acid showed gel-like response (G′ > G′′) at 90 °C, attributed to hydrophobic association. The downturn in moduli at 25 °C for high concentrations of pectin is attributed to formation and disruption of strong networks during mixing with citric acid at high temperature (“pregelation”). It is suggested, however, that “weak gels” formed at lower concentrations or at pH values above ～2.7 may enhance gel properties by preserving a continuous network as hydrophobic junctions dissociate on cooling and are replaced by hydrogen-bonded junctions, in contrast to random percolation during gelation with GDL at 25 °C. On re-heating from 25 to 90 °C, the reverse processes (dissociation of hydrogen-bonded structures and formation of hydrophobic associations) were evident in an initial reduction and subsequent increase in moduli, as observed in previous studies. Similar heating traces were obtained for samples acidified with GDL to pH values above ～3.0 (at c = 1.0 wt%) or with pectin concentrations below ～1.0 wt% (at pH 3.0). However, at higher concentrations or lower values of pH (i.e. conditions favourable to extensive intermolecular association) an abrupt decrease in G′, with an accompanying maximum in G′′, was observed on heating through the temperature range ～60–80 °C. This is attributed to excessive hydrophobic association, causing collapse of network structure. It is further suggested that, for samples acidified with citric acid, there is preferential association of chain sequences of high ester content into hydrophobic junctions at 90 °C, leaving sequences with a high content of unesterified carboxyl groups available to form long hydrogen-bonded junctions during cooling, and thus giving gels that are stronger and more resistant to network collapse.     

Imagerie moléculaire du transporteur vésiculaire de l’acétylcholine (VACh) par le [123I]-5-iodobenzovézamicol dans la démence de type Alzheimer (DTA)

Alzheimer's disease (AD) is characterized by a premature decline of cholinergic neurons. The 5-IBVM is an analogue of the vesamicol that binds to the presynaptic vesicular acetylcholine transporter (VAChT). The exploration of this target should be useful to make an early diagnosis of AD. Our first aim was to propose a method of non invasive VAChT quantification according to 5-IBVM kinetic. 5-IBVM was injected to four AD patients (age = 77 ± 3.9 years and MMSE = 24.5 ± 1.02) were included in this methodological study. The single-photon emission computed tomography (SPECT) images were obtained at five, 20, 35 and 50 minutes, at then at three, five and 22 hours after intravenous injection of 5-IBVM (185MBq). The time activity curves were obtained after SPECT images coregistration on a MRI masque. Specific volume of interest (SPE = SPEcific) were manually drawn on striatum, pons, thalamus and para-hippocampic gyrus including hippocampus; reference volumes of interest (REF = REFerence) were drawn on frontal and occipital cerebral cortex. On the basis of uptake kinetic, two modelisation approaches were considered: transient equilibrium model for reversible ligand (binding potential (BP) = (SPE ? REF)/REF) and Patlak graphical analysis for irreversible tracers (slope given by K_i/DV_ref where K_i is the influx contant and DV_ref is the distribution volume of the reference region). We observed an inflection or a stady state of the activity curves in the different regions studied between 250 and 1400 minutes, what seems to confirm that the tracer is little reversible. BP values obtained at 21 hours with occipital areas as reference and K_i/DV_ref values were respectively 4.62 ± 0.42 and 0.07 ± 0.01. The SPE classification according to BP and K_i/DV_ref values were similar to the classification according to the compartmental analysis (Kuhl 1994).The transient equilibrium model with late acquisition seems the more suitable because IBVM kinetic and practical simplicity of performing in clinical routine.