Crystal Structure of an Ammonia-Permeable Aquaporin

Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.     

Ovulation and Embryonic Developmental Rate Following hCG-stimulation in Sows

The hCG induced ovulation in sows was studied by use of ultrasonography, and an investigation of the development and diversity of the zygotes/embryos was performed at 24 h after ovulation. Crossbred sows (N=48) were weaned (day 0) and checked for heat twice daily from day 3 onwards. From day 4, the ovaries were transrectally scanned twice daily On day 4, the sows were given an injection of 750 iu hCG im and inseminated 27 ± 2 h (X ± SD) and 38 ± 1 h later. From 38 to 48 h after the hCG injection, the ovaries were scanned at 60 to 90 min intervals. At 24 h after ovulation the oviducts were surgically flushed in 18 sows. Out of the 48 sows, 34 showed heat at 12–36 h after the hCG-treatment and 14 showed heat before the hCG treatment. In the former group of sows, 20 (59%) ovulated within the interval of 38 to 48 h after the hCG treatment, and the follicular size immediately before ovulation was 7.8 ± 0.6 mm. Among the sows which showed heat before hCG treatment only 7 (50%) ovulated within the above interval and the preovulatory follicle size was larger (8.3 ± 0.5, p<0.05) than in the former group of sows, which showed heat after the hCG treatment. The flushing of 18 sows yielded a total of 243 ova, 70 (29 %) 1-cell stages, 160 (66 %) 2-cell stages and 13 (5%) 4-cell stages. A pronounced difference in the degree of variation in embryonic development was seen between sows: 4 animals yielded 1- to 4-cell stages, one exclusively 2-cell stage. In conclusion, the control of ovulation in sows by hCG treatment will affect the follicular growth and the exact timing of ovulation can not always be relied on. It is strongly recommended to use ultrasonography to monitor the time of ovulation if this parameter is important. Ova recovered at 24±1 h after the median time of ovulation revealed a pronounced diversity (1- to 4- cell stage) within sows. No obvious relation with this embryonic diversity and the follicular size at ovulation was seen in these data.     

NMR and computational characterization of the N-(deoxyguanosin-8-yl)aminofluorene adduct [(AF)G] opposite adenosine in DNA: (AF)G[syn].A[anti] pair formation and its pH dependence

This paper reports on a combined two-dimensional NMR and energy minimization computational characterization of the conformation of the N-(deoxyguanosyl-8-yl)aminofluorene adduct [(AF)G] positioned across adenosine in a DNA oligomer duplex as a function of pH in aqueous solution. This study was undertaken on the d[C1-C2-A3-T4-C5-(AF)G6-C7-T8-A9-C10-C11].[G12-G13-T14 -A15-G16-A17-G18- A19-T20-G21-G22] complementary undecamer [(AF)G 11-mer duplex]. The modification of the single G6 on the pyrimidine-rich strand was accomplished by reaction of the oligonucleotide with N-acetoxy-2-(acetylamino)fluorene and subsequent deacetylation under alkaline conditions. The HPLC-purified modified strand was annealed with the unmodified purine-rich strand to generate the (AF)G 11-mer duplex. The exchangeable and nonexchangeable protons are well resolved and narrow in the NMR spectra of the (AF)G 11-mer duplex so that the base and the majority of sugar nucleic acid protons, as well as several aminofluorene ring protons, have been assigned following analysis of two-dimensional NOESY and COSY data sets at pH 6.9, 30 degrees C in H2O and D2O solution. The NOE distance constraints establish that the glycosidic torsion angle is syn at (AF)G6 and anti at A17, which results in the aminofluorene ring being positioned in the minor groove. A very large downfield shift is detected at the H2' sugar proton of (AF)G6 associated with the (AF)G6[syn].A17[anti] alignment in the (AF)G 11-mer duplex. The NMR parameters demonstrate formation of Watson-Crick C5.G18 and C7.G16 base pairs on either side of the (AF)G6[syn].A17[anti] modification site with the imino proton of G18 more stable to exchange than the imino proton of G16. Several nonexchangeable aminofluorene protons undergo large downfield shifts as do the imino and H8 protons of G16 on lowering of the pH from neutrality to acidic values for the (AF)G 11-mer duplex. Both the neutral and acidic pH conformations have been defined by assigning the NOE constraints in the [C5-(AF)G6-C7].[G16-A17-G18] segment centered about the modification site and incorporating them in distance constrained minimized potential energy calculations in torsion angle space with the DUPLEX program. A series of NOEs between the aminofluorene protons and the DNA sugar protons in the neutral pH conformation establish that the aminofluorene ring spans the minor groove and is directed toward the G16-A17-G18 sugar-phosphate backbone on the partner strand.(ABSTRACT TRUNCATED AT 400 WORDS)     

La cytométrie dans l’exploration de l’allergie

Les tests cellulaires pour l’explorationin vitro de l’hypersensibilité immédiate, utilisent le polynucléaire basophile du sang périphérique. Ce leucocyte qui possède la plupart des propriétés du mastocyte, un récepteur membranaire de forte affinité pour les IgE (FceR1) et des granulations contenant des médiateurs préformés de l’anaphylaxie, est un bon témoin de la sensibilisation mastocytaire contre un allergène. Mais comme ce dernier est d’un accès difficile, pour les étudesin vitro chez l’homme on préfère travailler sur le basophile.