Temperature and nutrient conditions modify the effects of phenological shifts in predator-prey communities.

Although there is mounting evidence indicating that the relative timing of predator and prey phenologies determines the outcome of trophic interactions, we still lack a comprehensive understanding of how the environmental context (e.g., abiotic conditions) influences this relationship. Environmental conditions not only frequently drive shifts in phenologies, but they can also affect the very same processes that mediate the effects of phenological shifts on species interactions. Therefore, identifying how environmental conditions shape the effects of phenological shifts is key to predicting community dynamics across a heterogeneous landscape and how they will change with ongoing climate change in the future. Here I tested how environmental conditions shape the effects of phenological shifts by experimentally manipulating temperature, nutrient availability, and relative phenologies in two predator-prey freshwater systems (mole salamander-bronze frog vs. dragonfly larvae-leopard frog). This allowed me to (1) isolate the effects of phenological shifts and different environmental conditions; (2) determine how they interact; and (3) evaluate how consistent these patterns are across different species and environments. I found that delaying prey arrival dramatically increased predation rates, but these effects were contingent on environmental conditions and the predator system. Although nutrient addition and warming both significantly enhanced the effect of arrival time, their effect was qualitatively different across systems: Nutrient addition enhanced the positive effect of early arrival in the dragonfly-leopard frog system, whereas warming enhanced the negative effect of arriving late in the salamander-bronze frog system. Predator responses varied qualitatively across predator-prey systems. Only in the system with a strong gape limitation were predators (salamanders) significantly affected by prey arrival time and this effect varied with environmental context. Correlations between predator and prey demographic rates suggest that this was driven by shifts in initial predator-prey size ratios and a positive feedback between size-specific predation rates and predator growth rates. These results highlight the importance of accounting for temporal and spatial correlations of local environmental conditions and gape limitation when predicting the effects of phenological shifts and climate change on predator-prey systems.

Broadening the ecology of fear: non-lethal effects arise from diverse responses to predation and parasitism.

Research on the 'ecology of fear' posits that defensive prey responses to avoid predation can cause non-lethal effects across ecological scales. Parasites also elicit defensive responses in hosts with associated non-lethal effects, which raises the longstanding, yet unresolved question of how non-lethal effects of parasites compare with those of predators. We developed a framework for systematically answering this question for all types of predator-prey and host-parasite systems. Our framework reveals likely differences in non-lethal effects not only between predators and parasites, but also between different types of predators and parasites. Trait responses should be strongest towards predators, parasitoids and parasitic castrators, but more numerous and perhaps more frequent for parasites than for predators. In a case study of larval amphibians, whose trait responses to both predators and parasites have been relatively well studied, existing data indicate that individuals generally respond more strongly and proactively to short-term predation risks than to parasitism. Apart from studies using amphibians, there have been few direct comparisons of responses to predation and parasitism, and none have incorporated responses to micropredators, parasitoids or parasitic castrators, or examined their long-term consequences. Addressing these and other data gaps highlighted by our framework can advance the field towards understanding how non-lethal effects impact prey/host population dynamics and shape food webs that contain multiple predator and parasite species.

Stage structure alters how complexity affects stability of ecological networks.

Resolving how complexity affects stability of natural communities is of key importance for predicting the consequences of biodiversity loss. Central to previous stability analysis has been the assumption that the resources of a consumer are substitutable. However, during their development, most species change diets; for instance, adults often use different resources than larvae or juveniles. Here, we show that such ontogenetic niche shifts are common in real ecological networks and that consideration of these shifts can alter which species are predicted to be at risk of extinction. Furthermore, niche shifts reduce and can even reverse the otherwise stabilizing effect of complexity. This pattern arises because species with several specialized life stages appear to be generalists at the species level but act as sequential specialists that are hypersensitive to resource loss. These results suggest that natural communities are more vulnerable to biodiversity loss than indicated by previous analyses.

Basic survival needs and access to medicines--coming to grips with TRIPS: conversion + calculation.

"Access to medicines" is a broad concept. After a review of three authoritative frameworks that help to identify its constitutive components, this essay summarizes the actual situation on the ground in low- and middle-income countries on the basis of recent empirical work. An analysis of survey data from 36 countries concluded that developing countries should promote generic medicines as a key policy option for improving access to medicines. Taking an international perspective to that recommendation, this essay reviews the World Trade Organization's Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS) and, particularly, how this agreement has been applied in practice. As shown by the experience of Thailand, Brazil, and the Philippines, in order to deal effectively with international pressures for an excessive application of the TRIPS Agreement, some sort of conversion experience appears to be required, which then leads to a switch from a private enterprise, supply-driven approach to a public health vision that insists on universal and affordable access. But moral conviction is not sufficient. In order to muster and sustain the political will to face down international forces, civil society and government offices must be able and ready to show the costs and other adverse consequences of the TRIPS-based model for medicines. This calculation needs to reach beyond the health sector and calls for new alliances, nationally as well as internationally.

Phenology, ontogeny and the effects of climate change on the timing of species interactions.

Climate change is altering the phenology of many species and the timing of their interactions with other species, but the impacts of these phenological shifts on species interactions remain unclear. Classical approaches to the study of phenology have typically documented changes in the timing of single life-history events, while phenological shifts affect many interactions over entire life histories. In this study, we suggest an approach that integrates the phenology and ontogeny of species interactions with a fitness landscape to provide a common mechanistic framework for investigating phenological shifts. We suggest that this ontogeny-phenology landscape provides a flexible method to document changes in the relative phenologies of interacting species, examine the causes of these phenological shifts, and estimate their consequences for interacting species.

Evolution of sexual size monomorphism: the influence of passive mate guarding.

Some species have potential for intense mate competition yet exhibit little or no sexual size dimorphism, despite predictions from sexual selection theory. Using a conceptual model, we show the conditions for which passive mate guarding with copulatory plugs can be an alternative and more successful strategy to active (direct) guarding, reducing selection pressure on large male size. The model predicts that copulatory plugs in mammals should be favoured in species for which females have short sexual receptivity periods. Using data on 62 primate species and a phylogenetic regression approach, we show that, as predicted, copulatory plugs are negatively associated with degree of sexual dimorphism and females' sexual receptivity length. Penile spines are also significantly associated with plug use and short receptivity periods suggesting a possible offensive role in sperm competition. Results highlight that life-history characteristics, such as sexual receptivity lengths, may alter the costs and benefits of alternative male strategies and thus alter the strength of sexual selection.

Serum paraoxonase activity in dairy cows during pregnancy.

Preparturient dairy cows are at high risk of metabolic and reproductive disorders and oxidative stress is considered to be involved in these events. We investigated the serum paraoxonase activity in dairy cows during pregnancy and alterations in lipid and lipoprotein patterns in this period. The relation between paraoxonase activity and HDL-cholesterol concentration was also compared. The study was carried out on 76 pregnant lactating and 26 pregnant dry Holstein dairy cows. The serum paraoxonase activity was determined by the method of hydrolysing of paraoxon, while triglyceride, cholesterol and HDL-cholesterol concentrations were measured by the enzymatic kit methods. A significantly higher serum triglyceride concentration (P<0.001) was observed in dry cows compared to lactating cows. The total cholesterol and HDL-cholesterol concentrations were significantly lower (P<0.001) in dry cows than in lactating ones. In dry cows, paraoxonase activity was significantly lower than in those lactating (P<0.001). There was no significant difference in paraoxonase/HDL-cholesterol ratio between the investigated groups. It seems that the lower HDL concentration could be one of the causes of reduced paraoxonase activity considering the role of HDL as a carrier of most paraoxonase molecules in the blood. A decreased serum paraoxonase activity could diminish the effectiveness and total capacity of the whole antioxidative system during prepartum period in dairy cattle.

Serum paraoxonase activity and lipid parameters in the early postpartum period of dairy cows.

The effect of early lactation on serum paraoxonase activity was studied on 21 postpartum dairy cows and 19 non-pregnant late lactating dairy cows. A significant decrease of the paraoxonase activity was found in the early postpartum period compared to the late non-pregnant lactation. The serum triglyceride, cholesterol and LDL-cholesterol concentration were also markedly reduced during the postpartum period, while the serum HDL-cholesterol concentration showed no significant change. The results indicate that lower serum paraoxonase activity is associated with lipid metabolic disorders in the early postpartum period. A decreased serum paraoxonase activity may lead to the reduction of the antioxidative capacity and antioxidative protection during the early postpartum period.

Faraday Ripples, Parametric Resonance, and the Marangoni Effect.

In general, the combined actions of two destabilizing mechanisms do not simply add to each other. Here we show that there is a subtle interplay between parametric excitation and thermal gradients leading to interfacial instability, overstability, and generation of surface waves. The case studied refers to the stability of a liquid layer with an open free surface subjected to a transverse temperature gradient (with the Marangoni effect) and also subjected to the simultaneous action of periodic vibrations normal to the layer. Stability is examined in the weak viscosity approximation by applying a multiscale method. To a first approximation, whatever the imposed thermal gradient, vibrations with fairly large amplitude are responsible for excitation of ripples with half the imposed vibration frequency, but their amplitude depends on the Marangoni number. However, as the Marangoni number increases, the critical amplitude decreases from the excitation threshold of Faraday ripples, and after passing through a minimum it monotonically increases with increasing thermal gradient. Another salient finding is that the threshold of the Marangoni overstability is found to be independent of the imposed vibration frequency and amplitude. Copyright 2001 Academic Press.

Serum paraoxonase activities in hemodialyzed uremic patients: cohort study.

AIM: To determine whether paraoxonase activity, paraoxonase phenotypes, and lipid status are altered in uremic patients on long-term hemodialysis treatment as compared to healthy population. METHODS: Patients (n = 69) and control subjects (n = 145) were from the area of Slavonski Brod, Croatia. Paraoxon was used as a substrate for measuring basal or sodium chloride-stimulated (NaCl-stimulated) paraoxonase activity, and phenylacetate for measuring arylesterase activity. The double substrate method was used to assign phenotypes. Cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-cholesterol) were determined by methods routinely used in medical-biochemical laboratories. Enzyme activities are expressed as international units per liter of serum or per mmol of HDL-cholesterol (HDL-standardized activities). RESULTS: Basal and NaCl-stimulated paraoxonase activity, as well as arylesterase activity expressed per serum volume, were significantly lower in the hemodialyzed uremic patients compared to the controls; 69% (p < 0.001), 73% (p < 0.001) and 49%, (p < 0.001), respectively. However, basal and NaCl-stimulated paraoxonase activity standardized for HDL-cholesterol concentrations were not significantly reduced in the hemodialyzed uremic patients as compared to controls (86%, p = 0.614 and 87%, p = 0.720, respectively), contrary to arylesterase activity, which remained significantly lower (72%, p < 0.001). The distribution of paraoxonase phenotypes in hemodialyzed uremic patients and controls was as follows: AA 45% and 39%, AB 37% and 48%, BB 18%, and 13%, respectively. CONCLUSION: Patients on long-term hemodialysis have decreased paraoxonase/arylesterase activity, which might indicate a greater risk of premature atherogenesis.

Interlaboratory study into the proficiency of serum cholinesterase activity measurement.

The measurement of cholinesterase activity is an important function of a clinical laboratory. Participation in appropriate quality assurance schemes is essential in ensuring a high analytical standard. Samples of human serum were distributed to thirty-five laboratories for the measurement of cholinesterase activity. Because of methodological differences between the participants, findings of each laboratory were compared either by the use of duplicate samples or by analysis of six mixtures of two samples, one having a high and one a low activity. Of 4,964 distributed samples 95% were analysed and findings reported in 596 reports. Thirty-four percent of all reports were considered very good (less than 5% within-run error) and 38% less than satisfactory (within-run error over 10%). Access to a proficiency programme such as this enables laboratories to evaluate the quality of their analytical service.

Comparison of protocols for measuring activities of human blood cholinesterases by the Ellman method.

This paper presents a protocol for routine assays of human blood cholinesterase activities which separates erythrocytes from plasma by centrifugation and measures acetylcholinesterase activity in unwashed erythrocytes and butyrylcholinesterase activity in the plasma. The recommended substrate for both enzymes is 1.0 mM acetylthiocholine. The protocol is compared with other two recommended protocols for the activity measurements of the two enzymes using the Ellman method. The paper discusses the advantages and disadvantages of each and concludes with a proposal for an international agreement between laboratories for the evaluation of a standardized protocol.

Cholinesterase: substrate inhibition and substrate activation.

The relationship between activities and substrate concentrations (pS-curves) was analysed for reactions of acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8). Catalytic constants Km, Kss, Vm, n and b were calculated from the Michaelis, Haldane, Hill and Webb equations in order to assess whether a given substrate also acts as an inhibitor or activator. It is suggested that the term substrate inhibition should only be attributed to substrates revealing bell-shaped pS-curves, while the terms apparent substrate inhibition or apparent substrate activation should relate to calculated values of the catalytic constants.

Amino acid residues involved in the interaction of acetylcholinesterase and butyrylcholinesterase with the carbamates Ro 02-0683 and bambuterol, and with terbutaline.

In order to identify amino acids involved in the interaction of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) with carbamates, the time course of inhibition of the recombinant mouse enzymes BChE wild-type (w.t.), AChE w.t. and of 11 site-directed AChE mutants by Ro 02-0683 and bambuterol was studied. In addition, the reversible inhibition of cholinesterases by terbutaline, the leaving group of bambuterol, was studied. The bimolecular rate constant of AChE w.t. inhibition was 6.8 times smaller by Ro 02-0683 and 16000 times smaller by bambuterol than that of BChE w.t. The two carbamates were equipotent BChE inhibitors. Replacement of tyrosine-337 in AChE with alanine (resembling the choline binding site of BChE) resulted in 630 times faster inhibition by bambuterol. The same replacement decreased the inhibition by Ro 02-0683 ten times. The difference in size of the choline binding site in the two w.t. enzymes appeared critical for the selectivity of bambuterol and terbutaline binding. Removal of the charge with the mutation D74N caused a reduction in the reaction rate constants for Ro 02-0683 and bambuterol. Substitution of tyrosine-124 with glutamine in the AChE peripheral site significantly increased the inhibition rate for both carbamates. Substitution of phenylalanine-297 with alanine in the AChE acyl pocket decreased the inhibition rate by Ro 02-0683. Computational docking of carbamates provided plausible orientations of the inhibitors inside the active site gorge of mouse AChE and human BChE, thus substantiating involvement of amino acid residues in the enzyme active sites critical for the carbamate binding as derived from kinetic studies.

Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by 4,4'-bipyridine and by a coumarin derivative.

Inhibition of recombinant mouse wild type AChE (EC 3.1.1.7) and BChE (EC 3.1.1.8), and AChE peripheral site-directed mutants and human serum BChE variants by 4,4'-bipyridine (4,4'-BP) and the coumarin derivative 3-chloro-7-hydroxy-4-methylcoumarin (CHMC) was studied. The enzyme activity was measured with acetylthiocholine as substrate. Enzyme-inhibitor dissociation constants for the catalytic and peripheral sites were evaluated from the apparent dissociation constants as a function of the substrate concentration. Inhibition by 4,4'-BP of AChE, BChE and the AChE mutant Y72N/Y124Q/W286A, was consistent with inhibitor binding to both catalytic and peripheral sites. The dissociation constants for the peripheral site were about 3.5-times higher than for the catalytic site. The competition between CHMC and substrate displayed two binding sites on the AChE mutants Y72N, Y124Q, W286A and W286R, and on the atypical and fluoride-resistant BChE variants. The dissociation constants for the peripheral site were on average two-times higher than for the catalytic site. CHMC displayed binding only to the catalytic site of Y72N/Y124Q/W286A mutant and only to the peripheral site of w.t. AChE and the human usual BChE. Modelling of the 4,4'-BP and CHMC binding to wild type mouse AChE substantiated the difference between the inhibitors in their mode of binding which was revealed in the kinetic studies.

An explanation for the different inhibitory characteristics of human serum butyrylcholinesterase phenotypes deriving from inhibition of atypical heterozygotes.

The time course of inhibition of butyrylcholinesterase (EC 3.1.1.8) by the dimethylcarbamate Ro 02-0683 in sera taken from patients heterozygous for the usual (U), atypical (A), K or J variants was followed using propionylthiocholine as substrate. Data obtained were used to determine rate constants of inhibition together with the contribution made by each variant to total enzyme activity. The findings substantiate earlier reports that J and K mutations lead to quantitative changes in the concentration of usual enzyme in contrast to the qualitative changes of the atypical variant. The contribution of the atypical enzyme to the total activity in serum from UA, AK and AJ heterozygotes was respectively 17-20, 24-31 and 34-53%. The altered ratios of atypical to usual, K or J enzyme in UA, AK and AJ together with the constants on the usual enzyme alone, explain the differences in observed inhibitor numbers which enable these heterozygotes to be identified.

Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase variants.

Catalysed hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluoride-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinesterase (EC 3.1.1.8) variants was measured in phosphate buffer pH 7.4 at 25 degrees C. pS-curves for all phenotypes were S-shaped; the activities rose to a plateau with increasing substrate concentration except at 100 mM where there was a small decrease. To obtain the catalytic constants, three equations were applied: Michaelis-Menten equation (Eq. 1), Hill equation (Eq. 2) and an equation which assumes simultaneous binding of the substrate to the catalytic site and to a peripheral site on the enzyme (Eq. 3). Over a range from 0.01 to 50 mM BTCh, the activity versus substrate concentration relationship deviated from Michaelis-Menten kinetics (Eq. 1) while data fitted well with Eqs. 2 and 3. The Michaelis-Menten equation was applied separately to two BTCh concentration ranges: the corresponding Km constants for the UU, FS, AK, AJ and AA phenotypes ranged from 0.1 to 0.2 mM (at 0.01-1.0 mM BTCh) and from 0.3 to 2.0 mM (at 1.0-50 mM BTCh). Hill coefficients (nH) calculated from Eq. 2 were similar for all phenotypes (nH approximately 0.5). The dissociation constants K1 and K2 calculated from Eq. 3 for two sites on the enzyme fell between 0.02 and 0.12 mM (K1) and 0.89 and 4.9 mM (K2) for the five phenotypes. Experimental data support the assumption that the phenotypes studied have two substrate binding sites.

3-Hydroxyquinuclidinium derivatives: synthesis of compounds and inhibition of acetylcholinesterase.

Four compounds were prepared: 3-hydroxy-1-methylquinuclidinium iodide (I), 3-(N,N-dimethylcarbamoyloxy)-1-methylquinuclidinum iodide (II), and two conjugates of I and II with 2-hydroxyiminomethyl-3-methylimidazole in which two parts of the molecule were linked by -CH2-O-CH2- (III and IV). III and IV are new compounds and their synthesis and physical data were given. All compounds were tested as inhibitors of human erythrocyte acetylcholinesterase (EC 3.1.1.7, AChE). The enzyme activity was measured in 0.1 M phosphate buffer (pH 7.4) at 10 and 37 degrees C with acetylthiocholine (ATCh) as the substrate. The obtained enzyme/inhibitor dissociation constants were between 0.05 and 0.5 mM at 10 degrees C and between 0.2 and 0.6 mM at 37 degrees C. At both temperatures compounds III and IV had higher affinities for the enzyme than compounds I and II and this difference was more pronounced at 10 than at 37 degrees C. The carbamates II and IV were also progressive AChE inhibitors. For compound II the rate constants of inhibition were 6300 and 2020 M(-1) min(-1) at 37 and 10 degrees C, respectively. Compound IV was a very weak carbamoylating agent with rate constants of inhibition of 100 and 63 M(-1) min(-1) at 37 and 10 degrees C, respectively. The oxime group in compounds III and IV hydrolyzed ATCh at rates of 23 and 3.2 M(-1) min(-1) at 37 and 10 degrees C, respectively.

Paraoxonase and arylesterase activities in the serum of two hyperlipoproteinaemic patients after repeated extracorporal lipid precipitation.

The effect of heparin-induced extracorporal lipid precipitation (HELP) on the activities of paraoxonase (EC 3.1.8.1) and arylesterase (EC 3.1.1.2) was studied in serum of a patient with hyperlipoproteinaemia (A) and of a patient with non-insulin dependent diabetes mellitus and hyperlipoproteinaemia (B). The enzyme activities were measured spectrophotometrically (Tris-HCl buffer, pH 7.4, 37 degrees C) with paraoxon and phenylacetate as substrates of paraoxonase and arylesterase, respectively. Both patients underwent HELP applications once a week over a period of 7 weeks. Over that period no overall change was observed either in enzyme activities or in the lipid and protein content of the sera. However, each HELP session caused an immediate decrease of EDTA-insensitive arylesterase activity (on average 56% in A and 42% in B), while EDTA-sensitive arylesterase remained almost unaltered. Paraoxonase remained unchanged in A, but decreased in B (approximately 60%). Of the atherogenic lipoprotein parameters, the most pronounced decrease was found in VLDL-cholesterol and in triglycerides (on average 45% in A and 32% in B), while the anti-atherogenic HDL-cholesterol decreased < 10%. Possible implications of the effect of HELP on the enzyme activities studied remain to be explained.