A Conserved Salt Bridge between Transmembrane Segments 1 and 10 Constitutes an Extracellular Gate in the Dopamine Transporter

Neurotransmitter transporters play an important role in termination of synaptic transmission by mediating reuptake of neurotransmitter, but the molecular processes behind translocation are still unclear. The crystal structures of the bacterial homologue, LeuT, provided valuable insight into the structural and dynamic requirements for substrate transport. These structures support the existence of gating domains controlling access to a central binding site. On the extracellular side, access is controlled by the “thin gate” formed by an interaction between Arg-30 and Asp-404. In the human dopamine transporter (DAT), the corresponding residues are Arg-85 and Asp-476. Here, we present results supporting the existence of a similar interaction in DAT. The DAT R85D mutant has a complete loss of function, but the additional insertion of an arginine in opposite position (R85D/D476R), causing a charge reversal, results in a rescue of binding sites for the cocaine analogue [³H]CFT. Also, the coordination of Zn²⁺ between introduced histidines (R85H/D476H) caused a ∼2.5-fold increase in [³H]CFT binding (B_max). Importantly, Zn²⁺ also inhibited [³H]dopamine transport in R85H/D476H, suggesting that a dynamic interaction is required for the transport process. Furthermore, cysteine-reactive chemistry shows that mutation of the gating residues causes a higher proportion of transporters to reside in the outward facing conformation. Finally, we show that charge reversal of the corresponding residues (R104E/E493R) in the serotonin transporter also rescues [³H](S)-citalopram binding, suggesting a conserved feature. Taken together, these data suggest that the extracellular thin gate is present in monoamine transporters and that a dynamic interaction is required for substrate transport.     

Numerical simulations of the pulsating flow of cerebrospinal fluid flow in the cervical spinal canal of a Chiari patient

The flow of cerebrospinal fluid (CSF) in a patient-specific model of the subarachnoid space in a Chiari I patient was investigated using numerical simulations. The pulsating CSF flow was modeled using a time-varying velocity pulse based on peak velocity measurements (diastole and systole) derived from a selection of patients with Chiari I malformation. The present study introduces the general definition of the Reynolds number to provide a measure of CSF flow instability to give an estimate of the possibility of turbulence occurring in CSF flow. This was motivated by the fact that the combination of pulsating flow and the geometric complexity of the spinal canal may result in local Reynolds numbers that are significantly higher than the commonly used global measure such that flow instabilities may develop into turbulent flow in these regions. The local Reynolds number was used in combination with derived statistics to characterize the flow. The results revealed the existence of both local unstable regions and local regions with velocity fluctuations similar in magnitude to what is observed in fully turbulent flows. The results also indicated that the fluctuations were not self-sustained turbulence, but rather flow instabilities that may develop into turbulence. The case considered was therefore believed to represent a CSF flow close to transition.     

Highly reduced mass loss rates and increased litter layer in radioactively contaminated areas

The effects of radioactive contamination from Chernobyl on decomposition of plant material still remain unknown. We predicted that decomposition rate would be reduced in the most contaminated sites due to an absence or reduced densities of soil invertebrates. If microorganisms were the main agents responsible for decomposition, exclusion of large soil invertebrates should not affect decomposition. In September 2007 we deposited 572 bags with uncontaminated dry leaf litter from four species of trees in the leaf litter layer at 20 forest sites around Chernobyl that varied in background radiation by more than a factor 2,600. Approximately one quarter of these bags were made of a fine mesh that prevented access to litter by soil invertebrates. These bags were retrieved in June 2008, dried and weighed to estimate litter mass loss. Litter mass loss was 40 % lower in the most contaminated sites relative to sites with a normal background radiation level for Ukraine. Similar reductions in litter mass loss were estimated for individual litter bags, litter bags at different sites, and differences between litter bags at pairs of neighboring sites differing in level of radioactive contamination. Litter mass loss was slightly greater in the presence of large soil invertebrates than in their absence. The thickness of the forest floor increased with the level of radiation and decreased with proportional loss of mass from all litter bags. These findings suggest that radioactive contamination has reduced the rate of litter mass loss, increased accumulation of litter, and affected growth conditions for plants.     

Incidence of Otitis Media in a Contemporary Danish National Birth Cohort

Objectives

In recent years welfare in Denmark has increased which might be expected to reduce otitis media (OM) incidence. We examined the age-specific incidence of OM in a nation-wide cohort of children aged 0–7 years born in 1996–2003 (Danish National Birth Cohort, DNBC). Only selection was ability to understand and speak Danish.

Methods

Information of OM and ventilation tubes (VT) was collected through three maternal interviews at 6-month, 18-month and 7-years of age and based on this age-specific and cumulative incidence of OM was calculated. As different numbers of the total population answered the different interviews, the calculations are done with different denominators. The information in DNBC was validated against two population based registries containing information of VT insertions.

Results

Cumulative incidence of OM at 7 years was 60.6% (31,982/52,755). For children with OM, 16.2% (7143/44194) had their first OM episodes between 0–6 months of age, 44.3% (19579/44194) between 7–18 months, and 39.5% (17472/44194) between 19 months and 7 years. Four or more OM episodes before 7 years were reported by 39.5% (12620/31982) and by 64.0% (2482/3881) of those who had their OM debut between 0–6 months; by 48.2% (4998/10378) with debut between 7–18 months; and by 28.7% (4996/17344) with debut between 19 months and 7 years. These figures are essentially unchanged from earlier figures from Denmark. VT insertion at least once was reported by 26,1% in the 7-year interview. Assuming recordings in the Danish National Patient Registry to be gold standard, maternal self-reportings in DNBC of insertion of VT showed high sensitivity (96.4%), specificity (98.2%), and positive (94.8%) and negative predictive values (98.8%).

Conclusion

OM affects nearly 2/3 of preschool children in Denmark despite reduction in known OM risk factors.     

Monitoring Changes in Membrane Polarity,Membrane Integrity,and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC₄(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca²⁺, K⁺, and H⁺, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.     

Prey vulnerability to a gape-size limited predator: behavioural and morphological impacts on northern pike piscivory

The northern pike ( Esox lucius ) is a selective and important predator in lake ecosystems. Prey size in pike is limited by pike gape size, which is a linear function of pike body length. Here we show that the absolute gape-size limit in pike is greater than previously considered, and that maximum ingestible prey size is limited by prey body depth. Further, we experimentally show that pike prefer shallow-bodied roach before deeper-bodied common bream, and small prey sizes within each prey species. Handling time in pike increases with prey body depth, and since common bream are deeper-bodied than roach, handling time is longer for bream than for roach of the same length, but equal considering body depth. Prey handling time is suggested to be a major cost to the pike, since it increases the risk of losing the prey, as well as exposure to predation, kleptoparasitism and cannibalism. Consequently, prey vulnerability is determined by risk of predation and intraspecific interactions, and behavioural preferences in the pike, and not by pike gape-size limits. The consequences for natural populations is evaluated by analysing size structures of predator and prey fish populations in a eutrophic lake.     

A folding variant of α-lactalbumin with bactericidal activity against Streptococcus pneumoniae

As the result of a typesetting error, Fig.2 in the article by Håkansson et al. on pages 589–600 of Molecular Microbiology , Volume 35, Issue 3, was unfortunately reproduced as a low-resolution image. The correct version is reproduced here.     

Parental nepotism enhances survival of retained offspring in the Siberian jay

The evolutionary payoff accruing to parents from breeding offspringcould be an incentive for prolonged investments in the offspring.Enhanced survival for offspring as a result of such a prolongedparental investment would increase the value of remaining inthe natal territory for the offspring. Here we show that first-yearsurvival in Siberian jays is higher in the company of theirparents. Two observations point to that the enhanced survivalof retained offspring is due to nepotistic parents rather thanto the quality of a shared habitat. First, winter survivalis higher only for those retained offspring whose parents havesurvived too ; this precludes the possibility that the linkbetween timing of dispersal and survival should reflect a higher phenotypic quality of retained offspring in general. Second,there is no support for the more parsimonious explanation thatthis link between the survival of parents and retained offspringreflects habitat quality of a shared territory. We could, withhigh statistical power, reject the possibility of a correlationbetween the survival of parental birds and unrelated immigrantsto the territory. Such a correlation would have been expectedif survival reflected habitat quality and not kinship. Our data instead suggest a direct fitness gain to retained offspringin enhanced survival through parental nepotism (parental facilitation).The behavior of parents in allowing retained offspring accessto food that is denied to immigrants is one proximate mechanismmediating a benefit of delayed dispersal.     

Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: an evaluation of compound specificity, inner filter effect and signal interpretation

Fixed wavelength fluorescence (FF) of bile has been evaluated as a monitoring tool for the screening of polyaromatic hydrocarbon (PAH) contamination in fish. The methodology was studied through laboratory and field experiments with Atlantic cod (Gadus morhua L.) and flounder (Platichthys flesus L.) exposed to various forms of PAH contamination. The present study demonstrates the ability of FF screening to discriminate between 2-, 4- and 5-ring PAH metabolites by using the wavelength pairs 290/335 nm, 341/383 nm and 380/430 nm, respectively. In general, the degree of fluorescence interference between these metabolite groups appears to be low. Dose- and time-response patterns of the FF signals were shown to give a good reflection of the PAH exposure. Further, the necessity of an appropriate dilution of bile samples prior to fluorescence measurements is demonstrated by a study of inner filter effect. Normally a dilution of 1000-2000-fold is necessary. Individual differences in the bile density, e.g. measured as the concentration of the bile pigment biliverdin, have to be allowed for when applying the FF method. However, it is shown that normalizing the FF signals to biliverdin concentrations on an individual basis added extra error to the data set. The simple, rapid and cost-effective FF method is found to be well suited for screening fish for PAH contamination.     

Butterfly Host Plant Choice in the Face of Possible Confusion

We tested predictions from the theory that ovipositing females of phytophagous insects are limited by their neural capacity for information processing. Previous studies have found that relatively specialized insects make faster and/or more accurate identifications of host plants compared to generalists. The study species was the polyphagous comma butterfly, Polygonia c-album (Nymphalidae). We compared females originating from two populations (Sweden and England) which differ in degree of specialization on the preferred host Urtica dioica (Urticaceae). Females were given a choice between this plant and a very similar nonhost, white dead nettle, Laminum album (Lamiacease), or a choice between a relatively poor host, Betuala pubescens, and the nonhost Betula pendula (Betulaceae). Oviposition rate was lower in cages with Betula compared to cages with Urtica, demonstrating that P. c-album females will withhold eggs when preferred hosts are not available. As predicted, females originating from the Swedish generalist population oviposited more often on the nonhost Lamium. However, females of both populations discriminated very strongly against oviposition on B. pendula. We found that newly hatched larvae have some ability to move from herbaceous nonhost to hosts. Although alternative interpretations are possible, the results give further support to the hypothesis that there are trade-offs between diet breadth and the ability to discriminate among plants.