Adolescence alcohol exposure impairs fear extinction and alters medial prefrontal cortex plasticity.

After experiencing a traumatic event people often turn to alcohol to cope with symptoms. In those with post-traumatic stress disorder (PTSD) and a co-occurring alcohol use disorder (AUD), PTSD symptoms can worsen, suggesting that alcohol changes how traumatic memory is expressed. The objective of this series of experiments is to identify how alcohol drinking (EtOH), following cued fear conditioning and extinction, impacts fear expression in mice. Molecular (activity-regulated cytoskeleton-associated protein, Arc/arg3.1) and structural (dendrite and spine morphometry) markers of neuronal plasticity were measured following remote extinction retrieval. Mouse age (adolescent and adult) and sex were included as interacting variables in a full factorial design. Females drank more EtOH than males and adolescents drank more EtOH than adults. Adolescent females escalated EtOH intake across drinking days. Adolescent drinkers exhibited more conditioned freezing during extinction retrieval, an effect that persisted for at least 20 days. Heightened cued freezing in the adolescent group was associated with greater Arc/arg3.1 expression in layer (L) 2/3 prelimbic (PL) cortex, greater spine density, and reduced basal dendrite complexity. In adults, drinking was associated with reduced L2/3 infralimbic (IL) Arc expression but no behavioral differences. Few sex interactions were uncovered throughout. Overall, these data identify prolonged age-related differences in alcohol-induced fear extinction impairment and medial prefrontal cortex neuroadaptations.

Case Study Part 2: Probabilistic modelling of long-term effects of pesticides on individual breeding success in birds and mammals.

Long term exposure of skylarks to a fictitious insecticide and of wood mice to a fictitious fungicide were modelled probabilistically in a Monte Carlo simulation. Within the same simulation the consequences of exposure to pesticides on reproductive success were modelled using the toxicity-exposure-linking rules developed by R.S. Bennet et al. (2005) and the interspecies extrapolation factors suggested by R. Luttik et al. (2005). We built models to reflect a range of scenarios and as a result were able to show how exposure to pesticide might alter the number of individuals engaged in any given phase of the breeding cycle at any given time and predict the numbers of new adults at the season's end.

Case Study Part 1: How to calculate appropriate deterministic long-term toxicity to exposure ratios (TERs) for birds and mammals.

In the European Union, first-tier assessment of the long-term risk to birds and mammals from pesticides is based on calculation of a deterministic long-term toxicity/exposure ratio (TER(lt)). The ratio is developed from generic herbivores and insectivores and applied to all species. This paper describes two case studies that implement proposed improvements to the way long-term risk is assessed. These refined methods require calculation of a TER for each of five identified phases of reproduction (phase-specific TERs) and use of adjusted No Observed Effect Levels (NOELs) to incorporate variation in species sensitivity to pesticides. They also involve progressive refinement of the exposure estimate so that it applies to particular species, rather than generic indicators, and relates spraying date to onset of reproduction. The effect of using these new methods on the assessment of risk is described. Each refinement did not necessarily alter the calculated TER value in a way that was either predictable or consistent across both case studies. However, use of adjusted NOELs always reduced TERs, and relating spraying date to onset of reproduction increased most phase-specific TERs. The case studies suggested that the current first-tier TER(lt )assessment may underestimate risk in some circumstances and that phase-specific assessments can help identify appropriate risk-reduction measures. The way in which deterministic phase-specific assessments can currently be implemented to enhance first-tier assessment is outlined.

Disruption of Golgi structure and function in mammalian cells expressing a mutant dynamin.

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

Iontophoretically applied lidocaine reduces pain on propofol injection.

We have compared iontophoretically and locally applied lidocaine for relief of pain on propofol injection. Pain was assessed on insertion of a 20-gauge i.v. cannula and at 10-s intervals for 30 s after injection of propofol. Pain scores on cannulation were significantly less in the iontophoresis group (median 1.1) than in the sham (control) group (median 2.8) (P < 0.005). Pain after injection of propofol was significantly reduced at 10 (P < 0.002), 20 (P < 0.001) and 30 s (P < 0.001). We conclude that iontophoretically applied lidocaine decreased the pain of cannulation and propofol injection.

Interactions between pesticides; a review of reported effects and their implications for wildlife risk assessment.

: Reviews of pesticide usage survey data and vertebrate wildlife and honeybee poisoning incident schemes in the UK show that there is considerable potential for wildlife to be exposed to combinations of agricultural pesticides. According to the published literature the toxicity of many pesticide combinations is at least additive. In some cases pesticide mixtures, particularly those involving insecticides, have been shown to be synergistic, with reported increases in toxicity of up to 100-fold. However, these effects are species, time and dose dependent and are therefore difficult to predict routinely. It is suggested that risk assessments should routinely take additive toxicity into account and those based on synergism should be targeted at those mixtures for which a further defined increase in toxicity would result in a high-risk classification. In order to support this risk assessment approach there is a need to develop and validate a standard in vivo test in order to confirm the interaction in those cases where additive or synergistic toxicity results in a high-risk classification.

Avian serum esterases: species and temporal variations and their possible consequences.

Avian serum esterases are predominantly of the 'B' type (cholinesterases and carboxylesterases) and are inhibited by carbamates and the 'active' oxon forms of organophosphorus pesticides. Selective inhibition of mammalian serum carboxylesterase, a 'B' esterase, has shown that this enzyme may play an important role in detoxication by irreversibly binding, and thus inactivating, anticholinesterase compounds. Studies have shown differences between carnivorous and omnivorous/herbivorous avian species in the level of activity and range of forms of carboxylesterases and cholinesterases in sera. In addition, these enzymes show seasonal, diurnal and developmental variations in activity. This paper will discuss species and temporal variations in avian serum 'B' esterases in relation to their possible influence on pesticide toxicity.

Species differences in avian serum B esterases revealed by chromatofocusing and possible relationships of esterase activity to pesticide toxicity.

Serum cholinesterase (BChE) and carboxylesterase (CbE) activities were investigated in ten species of birds. Multiple forms of serum BChE and CbE were also separated by chromatofocusing. Higher CbE activity and a wider range of CbE and BChE forms were present in the sera of omnivorous/herbivorous birds than carnivores. Omnivores/herbivores studied were the starling, house sparrow, tree sparrow, pigeon, partridge and magpie. Serum CbE activities of these species ranged from 0.46 to 2.93 mumol/min/mL with 2-6 forms separated by chromatofocusing. 0-6 forms of BChE were separated by the same method. The serum CbE activities of the little owl, tawny owl, barn owl and razorbill ranged from 0.19 to 0.58 mumoles/min/mL with 0-2 forms separated by chromatofocusing. No ChE forms were present within the pH gradient. These results may be significant in contributing to the understanding of the selective toxicity of organophosphorus and carbamate pesticides.

Distinction between 'A'-esterases and arylesterases. Implications for esterase classification.

'A'-esterase activities (substrates paraoxon and pirimiphos-methyloxon) and arylesterase activities (substrate phenyl acetate) were assayed in the sera of 14 species of birds representing seven different orders and 11 species of mammal representing five different orders. Ten species of birds had no detectable 'A'-esterase, and the remaining four species only low activity, yet all birds showed considerable arylesterase activity (16.8-99.3 mumol/min per ml of serum). Ten species of mammal showed both 'A'- and 'aryl'-esterase activities. In humans, gel filtration of serum completely separated peaks representing paraoxonase and arylesterase activities. Thus, in both birds and humans, serum enzymes exist that express arylesterase activity but not 'A'-esterase activity. These findings suggest that a distinction should be made between these two types of esterase in future classifications.

Resonance Raman studies of dioxygen and carbon monoxide binding to imidazole-appended hemes.

Resonance Raman spectroscopy has been employed to probe the effects of proximal base strain on the bonding of O2 and CO in three synthetic hemins with covalently linked imidazole ligands. The strain is introduced by varying the length of the imidazole-containing side chain and by restricting the side chain flexibility with a phenyl ring. These hemins are abbreviated as "long," "short," and "stiff" hemins, respectively. In the deoxy state, the iron-imidazole stretching frequencies [nu(Fe--N epsilon)] for long, short, and stiff hemins are detected at 200, 207, and 204 cm-1, respectively. The strain induced in the iron-imidazole bond by the short hemin results in a higher nu(Fe--N epsilon) frequency, in contrast to the strain induced by sterically hindered 2-methylimidazole or 1,2-dimethylimidazole complexes in which the Fe--N epsilon bond is tilted and lengthened, but the imidazole ring remains perpendicular to the heme plane. However, in the short hemin, the plane of the imidazole ring may not be perpendicular to the plane of the porphyrin, altering the amount of pi-interaction (hence the strength of Fe--N epsilon bond) and the nature of normal mode containing Fe--N epsilon bond stretching. Upon CO binding, we have observed the nu(Fe--CO) stretching frequencies at 497 (long), 499 (short), and 496 cm-1 (stiff), somewhat lower than those reported by Mitchell et al. (Inorg. Chem., 1985, 24:967) for the chelated-heme X CO complexes (i.e., 501-506 cm-1). This is the first report of an iron-oxygen-associated vibration observed in solution for an unprotected heme. The oxy complexes were formed by introducing dioxygen to the deoxy complexes at -700C. The isotope-sensitive line was detected at 576 cm- (1602) in oxy stiff hemin, which was shifted to 545 cm-1 upon 1802 substitution. This is perhaps the largest isotope shift (31 cm-') observed to date, compared with the usual 22-24 cm-'.For the long and short hemins, the iron-oxygen-associated vibration was detected at 574 and 573 cm-', respectively.These values are very similar to those observed(N-methylimidazole) and myoglobin/hemoglobin.

Resonance Raman evidence for the mechanism of the allosteric control of O2-binding in a cobalt-substituted monomeric insect hemoglobin.

The substitution of iron for cobalt in the monomeric insect hemoglobin CTT (Chironomus thummi thummi) III does not alter the Bohr effect for O2-binding. The cobalt substitution in this hemoglobin allows us to identify not only the O-O and Co-O2 stretching mode but also the Co-O-O bending mode by resonance Raman spectroscopy. The assignments were made via 16O2/18O2 isotope exchange. The modes associated with the Co-O-O moiety are pH-dependent. These pH-induced changes of the resonance Raman spectra are correlated with the t = r conformation transition. At high pH (high-affinity state) two unperturbed O-O stretching modes are observed at 1,068 cm-1 (major component) and 1,093 cm-1 (minor component) for the 18O2 complex. These frequencies correspond to split modes at 1,107 cm-1 and 1,136 cm-1 and an unperturbed mode at approximately 1,153 cm-1 for the 16O2 complex. At low pH (low-affinity state) the minor component becomes the major component and vice versa. The Co-O2 stretching frequency varies for approximately 520 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) indicating a stronger (hence shorter) Co-O2 bond in the high-affinity state. On the other hand, the O-O bond is weakened upon the conversion of the low- to the high-affinity state. The Co-O-O bending mode changes from 390 cm-1 (pH 9.5) to 374 cm-1 (pH 5.5). In the deoxy form the resonance Raman spectra are essentially pH-insensitive except for a vinyl mode at 414 cm-1 (pH 5.5), which is shifted to 416 cm-1 (pH 5.5).