Early warning signals detect critical impacts of experimental warming

Earth's surface temperatures are projected to increase by ~1–4°C over the next century, threatening the future of global biodiversity and ecosystem stability. While this has fueled major progress in the field of physiological trait responses to warming, it is currently unclear whether routine population monitoring data can be used to predict temperature‐induced population collapse. Here, we integrate trait performance theory with that of critical tipping points to test whether early warning signals can be reliably used to anticipate thermally induced extinction events. We find that a model parameterized by experimental growth rates exhibits critical slowing down in the vicinity of an experimentally tested critical threshold, suggesting that dynamical early warning signals may be useful in detecting the potentially precipitous onset of population collapse due to global climate change.     

Early warning signals also precede non‐catastrophic transitions

Synthesis The quickly expanding literature on early warning signals for critical transitions in ecosystems suggests that critical slowing down is a key phenomenon to measure the distance to a tipping point in ecosystems. Such work is broadly misinterpreted as showing that slowing down is specific to tipping points. In this contribution, we show why this is not the case. Early warning signals based on critical slowing down indicate a broader class of situations where a system becomes increasingly sensitive to perturbations. Ecosystem responses to external changes can surprise us by their abruptness and irreversibility. Models have helped identifying indicators of impending catastrophic shifts, referred to as ‘generic early warning signals’. These indicators are linked to a phenomenon known as ‘critical slowing down’ which describes the fact that the recovery rate of a system after a perturbation decreases when the system approaches a bifurcation – such as the classical fold bifurcation associated to catastrophic shifts. However, contrary to what has sometimes been suggested in the literature, a decrease in recovery rate cannot be considered as specific to approaching catastrophic shifts. Here, we analyze the behavior of early warning signals based on critical slowing down in systems approaching a range of catastrophic and non‐catastrophic situations. Our results show that slowing down generally happens in situations where a system is becoming increasingly sensitive to external perturbations, independently of whether the impeding change is catastrophic or not. These results highlight that indicators specific to catastrophic shifts are still lacking. More importantly, they also imply that in systems where we have no reason to expect catastrophic transitions, slowing down may still be used in a more general sense as a warning signal for a potential decrease in stability.     

p-Chloroamphetamine induces serotonin release through serotonin transporters.

p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.     

Early warning signals for critical transitions: a generalized modeling approach

Critical transitions are sudden, often irreversible, changes that can occur in a large variety of complex systems; signals that warn of critical transitions are therefore highly desirable. We propose a new method for early warning signals that integrates multiple sources of information and data about the system through the framework of a generalized model. We demonstrate our proposed approach through several examples, including a previously published fisheries model. We regard our method as complementary to existing early warning signals, taking an approach of intermediate complexity between model-free approaches and fully parameterized simulations. One potential advantage of our approach is that, under appropriate conditions, it may reduce the amount of time series data required for a robust early warning signal.     

Broad lipid phase transitions in mammalian cell membranes measured by Laurdan fluorescence spectroscopy

Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show that linear changes of cell membrane order in the physiological temperature regime are part of broad order-disorder-phase transitions which extend over a much broader temperature range. Even though these extreme temperatures are usually not object of live science research due to failure of cellular functions, our findings help to understand and predict cell membrane properties under physiological conditions as they explain the underlying physics of a broad order-disorder phase transition. Therefore, we analyzed the membranes of various cell lines, red blood cell ghosts and lipid vesicles by spectral decomposition in a custom-made setup in a temperature range from ?40 °C to +90 °C. While the generalized polarization as a measure for membrane order of artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed changes of the generalized polarization are equivalent to temperature changes dT in the range of +12 K < dT < -6 K.     

Des-keto lobeline analogs with increased potency and selectivity at dopamine and serotonin transporters

A series of des-keto lobeline analogs has been synthesized and evaluated for their ability to inhibit the dopamine transporter (DAT) and serotonin transporter (SERT) function and for their affinity for the synaptic vesicle monoamine transporter (VMAT2), as well as for alpha4beta2( *) and alpha7( *) neuronal nicotinic acetylcholine receptors (nAChRs). The enantiomers 8R-hydroxylobel-9-ene (3a) and 10S-hydroxylobel-7-ene (3c) exhibited high potency and selectivity at SERT and DAT, respectively.     

Structure-activity studies of 3'-4'-dichloro-meperidine analogues at dopamine and serotonin transporters

The structure-activity relationships of 3',4'-dichloro-meperidine were investigated at dopamine (DAT) and serotonin transporters (SERT). Large ester substituents and lipophilic groups at the 4-position favored molecular recognition at the SERT. The benzyl ester of 3',4'-dichloro-meperidine exhibited high potency and high selectivity for the SERT (DAT/SERT=760). Chemical modification of the ester group and N-substitution generally led to compounds with decreased DAT affinity. Only small esters and alkyl groups were tolerated at the 4-position of the meperidine ring system by the DAT. Overall, the meperidine analogues were generally more selective for the SERT than for the DAT.     

Functional coupling of serotonin and noradrenaline transporters

Re-uptake of the neurotransmitters serotonin and noradrenaline out of the synaptic cleft is mediated by selective transporter proteins, the serotonin transporter and the noradrenaline transporter respectively. Both are integral membrane proteins that are have a high degree of homology and represent members of a larger neurotransmitter transporter superfamily. Several studies have indicated that the serotonin transporter has an an oligomeric structure. To determine whether monoamine transporters can also function in oligomeric structures in situ, we constructed a concatenate consisting of one molecule of serotonin transporter covalently linked to one molecule of noradrenaline transporter. Heterologous expression of this hybrid construct allowed us to analyse the function, i.e. transport activity, and the structure, i.e. the molecular weight of the total construct and of its single components, at the same time. We showed that serotonin-noradrenaline transporter fusion proteins are fully active and exhibit the pharmacological profile of both their individual components. These findings support the hypothesis that monoamine transporters are expressed and may function as oligomeric proteins composed of non-interacting monomers.     

The ABCA subclass of mammalian transporters

We describe here a subclass of mammalian ABC transporters, the ABCA subfamily. This is a unique group that, in contrast to any other human ABC transporters, lacks a structural counterpart in yeast. The structural hallmark of the ABCA subfamily is the presence of a stretch of hydrophobic amino acids thought to span the membrane within the putative regulatory (R) domain. As for today, four ABCA transporters have been fully characterised but 11 ABCA-encoding genes have been identified. ABCA-specific motifs in the nucleotide binding folds can be detected when analysing the conserved sequences among the different members. These motifs may reveal functional constraints exclusive to this group of ABC transporters.     

State transitions in cyanobacteria studied with picosecond fluorescence at room temperature

Cyanobacteria can rapidly regulate the relative activity of their photosynthetic complexes photosystem I and II (PSI and PSII) in response to changes in the illumination conditions. This process is known as state transitions. If PSI is preferentially excited, they go to state I whereas state II is induced either after preferential excitation of PSII or after dark adaptation. Different underlying mechanisms have been proposed in literature, in particular i) reversible shuttling of the external antenna complexes, the phycobilisomes, between PSI and PSII, ii) reversible spillover of excitation energy from PSII to PSI, iii) a combination of both and, iv) increased excited-state quenching of the PSII core in state II. Here we investigated wild-type and mutant strains of Synechococcus sp. PCC 7942 and Synechocystis sp. PCC 6803 using time-resolved fluorescence spectroscopy at room temperature. Our observations support model iv, meaning that increased excited-state quenching of the PSII core occurs in state II thereby balancing the photochemistry of photosystems I and II.     

Physiological characteristics of serotonin transporters on rat platelets

Physiological characteristics of serotonin (5-hydroxytryptamine, 5HT) transport through the platelet membrane was investigated in Wistar rats with our recently developed method permitting repetitive measurements of transporter kinetics in individual animals. Full kinetic analysis in the population of 91 animals revealed Michaelis constant (K_m) of 0.158±0.025 μM and maximal velocity (V_max) of 5HT uptake of 225±32 pmol per 10⁸platelets min⁻¹ (mean±S.D.). Both kinetic parameters demonstrated normal distribution curves, which for V_max were slightly skewed toward higher than average values. No gender effect was shown in frequency distributions, mean values and variability of kinetic parameters. A significant intraindividual correlation between kinetic parameters was found suggesting compensation at the level of the plasma membrane. Kinetic parameters were not influenced by age (until the middle age) or annual cycle (under laboratory conditions) and were shown to be fairly stable in time, supporting the view that platelet 5HT transport kinetics could be a useful biological trait marker.     

Identification of a primary metabolite of ibogaine that targets serotonin transporters and elevates serotonin

Ibogaine is a hallucinogenic indole with putative efficacy for the treatment of cocaine, stimulant and opiate abuse. The purported efficacy of ibogaine following single dose administrations has led to the suggestion that a long-acting metabolite of ibogaine may explain in part how the drug reduces craving for psychostimulants and opiates. We report here that 12-hydroxyibogamine, a primary metabolite of ibogaine, displays high affinity for the 5-HT transporter and elevates extracellular 5-HT. In radioligand binding assays, 12-hydroxyibogamine was 50-fold more potent at displacing radioligand binding at the 5-HT transporter than at the DA transporter. Ibogaine and 12-hydroxyibogamine were equipotent at the dopamine transporter. In vivo microdialysis was used to evaluate the acute actions of ibogaine and 12-hydroxyibogamine on the levels of DA and 5-HT. Administration of 12-hydroxyibogamine produced a marked dose-related elevation of extracellular 5-HT. Ibogaine and 12-hydroxyibogamine failed to elevate DA levels in the nucleus accumbens over the dose range tested. The elevation in synaptic levels of 5-HT by 12-hydroxyibogamine may heighten mood and attenuate drug craving. The effects of the active metabolite on 5-HT transmission may account in part for the potential of ibogaine to interrupt drug-seeking behavior in humans.     

The physiology of bicarbonate transporters in mammalian reproduction

HCO(3)(-) plays critically important roles during virtually the entire process of reproduction in mammals, including spermatogenesis, sperm capacitation, fertilization, and development of early stage embryos. Therefore, the acid-base balance in the male and female reproductive tracts must be finely modulated. The fluid milieu in the epididymis is acidic, containing very low concentration of HCO(3)(-). In this acidic low HCO(3)(-) environment, mature sperm are rendered quiescent in the epididymis. In contrast, the luminal fluid in the female uterus and oviduct is alkaline, with very high concentration of HCO(3)(-) that is essential for sperm to fulfill fertilization. HCO(3)(-) transporter of solute carrier 4 (SLC4) and SLC26 families represent the major carriers for HCO(3)(-) transport across the plasma membrane. These transporters play critical roles in intracellular pH regulation and transepithelial HCO(3)(-) transport. The physiological roles of these transporters in mammalian reproduction are of fundamental interest to investigators. Here we review recent progress in understanding the expression of HCO(3)(-) transporters in reproductive tract tissues as well as the physiological roles of these transporters in mammalian reproduction.     

Cocaine inhibition of ligand binding at dopamine, norepinephrine and serotonin transporters: a structure-activity study

Structure-activity relationships for cocaine and analog binding at the dopamine, norepinephrine and serotonin transporters were determined. Cocaine inhibition of ligand binding to each of these sites has a stereospecific requirement for the levorotatory isomer. Binding potencies of cocaine derivatives involving N-substitution, C2 and C3 substituent modifications, however, revealed differences in structure-activity relationships for cocaine binding at the transporters. Removal of the N-methyl groups produced little change in binding potency at the dopamine transporter site but produced increases in binding potency at norepinephrine and serotonin transporter sites. Changes in structure at the C2 substituent produced changes in binding potency at the dopamine transporter which were generally similar in direction, but not necessarily in magnitude at the norepinephrine and serotonin transporters. Modifications to the C3 substituent, especially substitution of a hydroxyl moiety, produce changes in affinity at norepinephrine and serotonin transporters which are much larger than those observed at dopamine transporters. In general, our results indicate that unique structural requirements exist for each transporter site, but that cocaine binding at norepinephrine and dopamine transporters can be described by more similar structure-activity relationships than those found for the serotonin transporter. Requirements for cocaine binding to the dopamine transporter, which we have previously shown to be associated with the reinforcing effects of cocaine, include levorotatory stereospecificity, the benzene ring at C3, at least some portions of the tropane ring, and the presence of the C2 methyl ester group in the beta conformation.     

ATP hydrolysis at one of the two sites in ABC transporters initiates transport related conformational transitions

ABC transporters play important roles in all types of organisms by participating in physiological and pathological processes. In order to modulate the function of ABC transporters, detailed knowledge regarding their structure and dynamics is necessary. Available structures of ABC proteins indicate three major conformations, a nucleotide-bound "bottom-closed" state with the two nucleotide binding domains (NBDs) tightly closed, and two nucleotide-free conformations, the "bottom-closed" and the "bottom-open", which differ in the extent of separation of the NBDs. However, it remains a question how the widely open conformation should be interpreted, and whether hydrolysis at one of the sites can drive conformational transitions while the NBDs remain in contact. To extend our knowledge, we have investigated the dynamic properties of the Sav1866 transporter using molecular dynamics (MD) simulations. We demonstrate that the replacement of one ATP by ADP alters the correlated motion patterns of the NBDs and the transmembrane domains (TMD). The results suggest that the hydrolysis of a single nucleotide could lead to extracellular closure, driving the transport cycle. Essential dynamics analysis of simulations suggests that single nucleotide hydrolysis can drive the system toward a "bottom-closed" apo conformation similar to that observed in the structure of the MsbA transporter. We also found significant structural instability of the "bottom-open" form of the transporters in simulations. Our results suggest that ATP hydrolysis at one of the sites promotes transport related conformational changes leading to the "bottom-closed" apo conformation, which could thus be physiologically more relevant for describing the structure of the apo state.     

The bioassay of mammalian olfactory signals

The bioassay of chemical signals in mammals presents a number of problems. Many of these difficulties are the result of the complexity of the behaviour which has to be analysed and of the odiferous output itself. Additionally, it is usual for the response to the odour to be affected by information from other sensory modalities, by experience and by the spatial and social context. This degree of variability and of flexibility which is characteristic of mammals also has the result that concepts such as ‘pheromone’, which were originally developed in simpler organisms, can be seriously misleading. Although valuable information can be gained by less direct methods, a bioassay is only likely to be successful in the identification of active substances if: 1. there is a clearly defined behavioural or physiological response to the odour, 2. the method of testing actually measures this response, 3. the situation used for testing is as close as possible to that in which the response naturally occurs, 4. in the case of a behavioural response, the recording technique is as detailed as possible so that ambiguous results are avoided, 5. an adequate test population is available to allow repeated testing, 6. considerable resources are available for chemical analysis. Although it is not easy to fulfil all of these requirements at one time, olfaction is of such importance in mammalian communication that the attempt to do so will always be valuable.