Partial agonism and antagonism of the ionotropic glutamate receptor iGLuR5: structures of the ligand-binding core in complex with domoic acid and 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid

More than 50 structures have been reported on the ligand-binding core of the ionotropic glutamate receptor iGluR2 that belongs to the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid-type of receptors. In contrast, the ligand-binding core of the kainic acid-type receptor iGluR5 has only been crystallized with three different ligands. Hence, additional structures of iGluR5 are needed to broaden the understanding of the ligand-binding properties of iGluR5, and the conformational changes leading to channel opening and closing. Here, we present two structures of the ligand-binding core of iGluR5; one as a complex with the partial agonist (2S,3S,4S)-3-carboxymethyl-4-[(1Z,3E,5R)-5-carboxy-1-methyl-hexa-1,3-dienyl]-pyrrolidine-2-carboxylic acid (domoic acid) and one as a complex with the antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid ((S)-ATPO). In agreement with the partial agonist activity of domoic acid, the ligand-binding core of the iGluR5 complex is stabilized by domoic acid in a conformation that is 11 degrees more open than the conformation observed in the full agonist (S)-glutamic acid complex. This is primarily caused by the 5-carboxy-1-methyl-hexa-1,3-dienyl moiety of domoic acid and residues Val685-Thr690 of iGluR5. An even larger domain opening of 28 degrees is introduced upon binding of the antagonist (S)-ATPO. It appears that the span of domain opening is much larger in the ligand-binding core of iGluR5 (30 degrees) compared with what has been observed in iGluR2 (19 degrees ). Similarly, much larger variation in the distances between transmembrane linker residues in the two protomers comprising the dimer is observed in iGluR5 as compared with iGluR2.     

Characterisation of the external features ofSchistocephalus solidus (Müller, 1776) (Cestoda) from different geographical regions and an assessment of the status of the Baltic ringed sealPhoca hispida botnica (Gmelin) as a definitive host

A comparative study of some morphological (segment number, scolex morphology and biometry, length and weight) and biological (maturation in different hosts) features ofSchistocephalus solidus plerocercoids and adults from different geographical regions (Baltic Sea and the British Isles) was carried out. The length of the plerocercoids fromGasterosteus aculeatus was shown to be the variable that best correlated with segment number. A very clear bimodal distribution of segment numbers separated the majority of British and Baltic plerocercoids (British n=21, mean length 25.48, SD 5.63, range 14–34 mm; mean segment number 66.33, SD 8.68, range 51–86. Baltic n=30, mean length 33.23, SD 4.64, range 23–48 mm; mean segment number 117.27, SD 10.30, range 99–138). AdultS. solidus from the intestines of Baltic ringed sealsPhoca hispida botnica and from a Welsh cormorantPhalacrocorax carbo carbo were also compared, and a similar bimodal distribution of segment numbers was found (Baltic n=70, mean segment number 106.16, SD 10.60, range 77–136; Welsh n=98, mean segment number 73.13, SD 8.78, range 54–97). Neither the morphology nor measurements of the scolex from apical-view scanning electron microphotographs provided distinguishing features for taxonomic purposes. Of 580 adult worms from Baltic ringed seals only 2.9% were gravid, 2.1% from spring and 10.5% from autumn samples. By contrast, of 98 adults from the Welsh cormorant 46.7% were gravid. The proportion of gravid worms did not increase with increasing worm numbers in seals. Reasons for poor maturation are discussed. Plerocercoids of BritishS. solidus were fromleiurus (gymnurus) forms ofG. aculeatus, which were relatively small, whereas in the northern Baltic plerocercoids were fromsemiarmatus ortrachurus forms, which were larger. As segment number was definitively established during the growth of the plerocercoid in the stickleback, the hypothesis is proposed that segment number is a phenotypic variable related to stickleback length (size).     

Antagonistic roles of ESCRT and Vps class C/HOPS complexes in the recycling of yeast membrane proteins

In Saccharomyces cerevisiae, deficiencies in the ESCRT machinery trigger the mistargeting of endocytic and biosynthetic ubiquitinated cargoes to the limiting membrane of the vacuole. Surprisingly, impairment of this machinery also leads to the accumulation of various receptors and transporters at the plasma membrane in both yeast and higher eukaryotes. Using the well-characterized yeast endocytic cargo uracil permease (Fur4p), we show here that the apparent stabilization of the permease at the plasma membrane in ESCRT mutants results from an efficient recycling of the protein. Whereas several proteins as well as internalized dyes are known to be recycled in yeast, little is known about the machinery and molecular mechanisms involved. The SNARE protein Snc1p is the only cargo for which the recycling pathway is well characterized. Unlike Snc1p, endocytosed Fur4p did not pass through the Golgi apparatus en route to the plasma membrane. Although ubiquitination of Fur4p is required for its internalization, deubiquitination is not required for its recycling. In an attempt to identify actors in this new recycling pathway, we found an unexpected phenotype associated with loss of function of the Vps class C complex: cells defective for this complex are impaired for recycling of Fur4p, Snc1p, and the lipophilic dye FM4-64. Genetic analyses indicated that these phenotypes were due to the functioning of the Vps class C complex in trafficking both to and from the late endosomal compartment.     

Glutamate Dehydrogenase Isoforms with N-Terminal (His)6- or FLAG-Tag Retain Their Kinetic Properties and Cellular Localization

Glutamate dehydrogenase (GDH) is a crucial enzyme on the crossroads of amino acid and energy metabolism and it is operating in all domains of life. According to current knowledge GDH is present only in one functional isoform in most animals, including mice. In addition to this housekeeping enzyme (hGDH1 in humans), humans and apes have acquired a second isoform (hGDH2) with a distinct tissue expression profile. In the current study we have cloned both mouse and human GDH constructs containing FLAG and (His)₆ small genetically-encoded tags, respectively. The hGDH1 and hGDH2 constructs containing N-terminal (His)₆ tags were successfully expressed in Sf9 cells and the recombinant proteins were isolated to ≥95 % purity in a two-step procedure involving ammonium sulfate precipitation and Ni²⁺-based immobilized metal ion affinity chromatography. To explore whether the presence of the FLAG and (His)₆ tags affects the cellular localization and functionality of the GDH isoforms, we studied the subcellular distribution of the expressed enzymes as well as their regulation by adenosine diphosphate monopotassium salt (ADP) and guanosine-5′-triphosphate sodium salt (GTP). Through immunoblot analysis of the mitochondrial and cytosolic fraction of the HEK cells expressing the recombinant proteins we found that neither FLAG nor (His)₆ tag disturbs the mitochondrial localization of GDH. The addition of the small tags to the N-terminus of the mature mitochondrial mouse GDH1 or human hGDH1 and hGDH2 did not change the ADP activation or GTP inhibition pattern of the proteins as compared to their untagged counterparts. However, the addition of FLAG tag to the C-terminus of the mouse GDH left the recombinant protein fivefold less sensitive to ADP activation. This finding highlights the necessity of the functional characterization of recombinant proteins containing even the smallest available tags.     

Pediatric Invasive Pneumococcal Disease Caused by Vaccine Serotypes following the Introduction of Conjugate Vaccination in Denmark

A seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in the Danish childhood immunization program (2+1 schedule) in October 2007, followed by PCV13 starting from April 2010. The nationwide incidence of IPD among children younger than 5 years nearly halved after the introduction of PCV7 in the program, mainly due to a decline in IPD caused by PCV7-serotypes. We report the results from a nationwide population-based cohort study of laboratory confirmed IPD cases in children younger than 5 years during October 1, 2007 to December 31, 2010 and describe the characteristics of children suspected to present with a vaccine failure. The period between April 19 and December 31, 2010 was considered a PCV7/PCV13 transitional period, where both vaccines were offered. We identified 45 episodes of IPD caused by a PCV7 serotype (23% of the total number) and 105 (55%) caused by one of the 6 additional serotypes in PCV13. Ten children had received at least one PCV7 dose before the onset of IPD caused by a PCV7 serotype. Seven children were considered to be incompletely vaccinated before IPD, but only three cases fulfilled the criteria of vaccine failure (caused by serotypes 14, 19F and 23F). One case of vaccine failure was observed in a severely immunosuppressed child following three PCV7 doses, and two cases were observed in immunocompetent children following two infant doses before they were eligible for their booster. None of the IPD cases caused by the additional PCV13 serotypes had been vaccinated by PCV13 and there were therefore no PCV13-vaccine failures in the first 8-months after PCV13 introduction in Denmark.     

Pressure pain sensitivity and hardness along human normal and sensitized muscle

The spatial distribution of pressure sensitivity and muscle hardness was examined on normal muscle tissue and muscle tissue after induction of delayed onset muscle soreness (DOMS). The pressure sensitivity and muscle hardness were assessed at nine sites on the tibialis muscle from the proximal to distal tendon on two separate days. In total 37 healthy volunteers participated in three experiments. In the first experiment pressure pain threshold (PPT) and pressure pain tolerance (PPTO) were assessed. Decreased PPT and PPTO were found on day 2, 7 days after day 1. Proximal and distal stimulation sites were harder compared to muscle belly sites. In a second experiment two different probe sizes were used. Variation in PPT between the nine sites was found for the large probe with muscle belly being less sensitive to pressure stimulation compared to proximal and distal sites. The most proximal stimulation site was harder compared to muscle belly sites. In a third experiment PPT and muscle hardness were assessed before and 48?h after eccentric exercise. PPT at two muscle belly sites was significantly decreased during DOMS. No specific sites were harder during DOMS, the average muscle hardness across sites was however significantly increased. Decreased PPT and increased muscle hardness did not correlate. In conclusion, within subjects the pressure sensitivity varies along the musculoskeletal unit. In DOMS, specific muscle belly sites were more sensitive to pressure stimulation. Muscle–tendon sites were harder compared to muscle belly sites.     

Early Environmental Influences on Growth of Arcto-Norwegian Cod, Gadus Morhua, From The 0-group To Adults

A high growth rate for Arcto-Norwegian cod, Gadus morhua, in the Barents Sea and adjacent areas from the larva period to the 0-group enhances survival and ultimately recruitment to the fishery. However, it appeared that high growth rates for a cohort through the 0-group were not continued as the cohort ages. Based on survey data, there was a significant negative correlation between the average length at the 0-group and its average length at ages 2 through 8. We provided evidence suggesting that this phenomenon was caused by the inter-annual variability in inflow of warm, prey-rich Atlantic water into the Barents Sea from the Norwegian Sea. Enhanced inflow provided favorable conditions for cod growth during the larva and juvenile pelagic intervals. However, this same strong inflow carried a proportion of the cohort farther to the east in the Barents Sea, where the bottom water is colder than in the west. The colder conditions experienced by such cohorts, as compared to cohorts that have a more westerly settlement, led to slower growth prior to age 2. Slow growth during this interval appeared to be the reason for these cohorts' relatively smaller mean length at older ages.     

Growth efficiencies of freshwater bacterioplankton

The growth efficiency of freshwater bacteria was examined in continuous cultures. One series of experiments was carried out using generation times from 50 to 200 hours and aged, normal, and enriched media, all of natural origin. Another series of experiments examined the bacterial growth efficiency during the growth season in eutrophic Frederiksborg Slotssø, in relation to changes in the planktonic communities and to factors controlling the bacterial incorporation of ³H-thymidine. Attachment of bacteria to the inner surfaces of the experimental flasks was examined using various types of bottles, adding glass tubes to the bottles, and measuring ³H-thymidine incorporation and direct cell counts of attached and free-living bacteria. Attachment of bacteria varied, and in one example up to 36% of the thymidine incorporation was by attached bacteria after 4 days. It was calculated that 36% of attached bacteria caused an underestimation of the growth efficiency of 11%. The mean growth efficiency tended to decrease with generation time using enriched medium (47 to 19%) and aged medium (35 to 12%), and tended to decrease with medium quality (enriched > normal > aged media) from 37% to 27%. The only significant difference in growth efficiency occurred in relation to generation time, in samples with enriched medium (unpaired t-test, P < 0.05). The overall mean value for all generation times and media was 30% (SEM = 3%, n = 24). From April to October, the growth efficiency was determined 5 times in samples from Frederiksborg Slotssø. The overall mean value was 31% (SEM = 3%, n = 30), and there was no significant change in the growth efficiency during the period measured. In June, three bioassay experiments revealed that carbon limitation controlled bacterial incorporation of ³H-thymidine, whereas additions of phosphate and nitrate did not change the incorporation rates. The narrow range of growth efficiencies obtained in this study (mean 31%, SEM = 2%, n = 54) suggests that changes in substratequality in the media applied and in the eutrophic samples examined causes only subtle changes in the growth efficiency.