Yif1B Is Involved in the Anterograde Traffic Pathway and the Golgi Architecture

Yif1B is an intracellular membrane‐bound protein belonging to the Yip family, shown previously to control serotonin 5‐HT_1A receptor targeting to dendrites. Because some Yip proteins are involved in the intracellular traffic between the ER and the Golgi, here we investigated the precise localization of Yif1B in HeLa cells. We found that Yif1B is not resident into the Golgi, but rather belongs to the IC compartment. After analyzing the role of Yif1B in protein transport, we showed that the traffic of the VSVG protein marker was accelerated in Yif1B depleted HeLa cells, as well as in hippocampal neurons from Yif1B KO mice. Conversely, Yif1B depletion in HeLa cells did not change the retrograde traffic of ShTx. Interestingly, in long term depletion of Yif1B as in Yif1B KO mice, we observed a disorganized Golgi architecture in CA1 pyramidal hippocampal neurons, which was confirmed by electron microscopy. However, because short term depletion of Yif1B did not change Golgi structure, it is likely that the implication of Yif1B in anterograde traffic does not rely on its role in structural organization of the Golgi apparatus, but rather on its shuttling between the ER, the IC and the Golgi compartments.      

Subunit-specific coupling between gamma-aminobutyric acid type A and P2X2 receptor channels

ATP and gamma-aminobutyric acid (GABA) are two fast neurotransmitters co-released at central synapses, where they co-activate excitatory P2X and inhibitory GABAA (GABA type A) receptors. We report here that co-activation of P2X2 and various GABAA receptors, co-expressed in Xenopus oocytes, leads to a functional cross-inhibition dependent on GABAA subunit composition. Sequential applications of GABA and ATP revealed that alphabeta- or alphabetagamma-containing GABAA receptors inhibited P2X2 channels, whereas P2X2 channels failed to inhibit gamma-containing GABAA receptors. This functional cross-talk is independent of membrane potential, changes in current direction, and calcium. Non-additive responses observed between cation-selective GABAA and P2X2 receptors further indicate the chloride independence of this process. Overexpression of minigenes encoding either the C-terminal fragment of P2X2 or the intracellular loop of the beta3 subunit disrupted the functional cross-inhibition. We previously demonstrated functional and physical cross-talk between rho1 and P2X2 receptors, which induced a retargeting of rho1 channels to surface clusters when co-expressed in hippocampal neurons (Boue-Grabot, E., Emerit, M. B., Toulme, E., Seguela, P., and Garret, M. (2004) J. Biol. Chem. 279, 6967-6975). Co-expression of P2X2 and chimeric rho1 receptors with the C-terminal sequences of alpha2, beta3, or gamma2 subunits indicated that only rho1-beta3 and P2X2 channels exhibit both functional cross-inhibition in Xenopus oocytes and co-clustering/retargeting in hippocampal neurons. Therefore, the C-terminal domain of P2X2 and the intracellular loop of beta GABAA subunits are required for the functional interaction between ATP- and GABA-gated channels. This gamma subunit-dependent cross-talk may contribute to the regulation of synaptic activity.     

Ovarian follicular activity and hormonal profile during estrous cycle in cows: the development of 2 versus 3 waves

Most estrous cycles in cows consist of 2 or 3 waves of follicular activity. Waves of ovarian follicular development comprise the growth of dominant follicles some of which become ovulatory and the others are anovulatory. Ovarian follicular activity in cows during estrous cycle was studied with a special reference to follicular waves and the circulating concentrations of estradiol and progesterone. Transrectal ultrasound examination was carried out during 14 interovulatory intervals in 7 cows. Ovarian follicular activity was recorded together with assessment of serum estradiol and progesterone concentrations. Three-wave versus two-wave interovulatory intervals was observed in 71.4% of cows. The 3-wave interovulatory intervals differed from 2-wave intervals in: 1) earlier emergence of the dominant follicles, 2) longer in length, and 3) shorter interval from emergence to ovulation. There was a progressive increase in follicular size and estradiol production during growth phase of each wave. A drop in estradiol concentration was observed during the static phase of dominant anovulatory follicles. The size of the ovulatory follicle was always greater and produced higher estradiol compared with the anovulatory follicle. In conclusion, there was a predominance of 3-wave follicular activity that was associated with an increase in length of interovulatory intervals. A dominant anovulatory follicle during its static phase may initiate the emergence of a subsequent wave. Follicular size and estradiol concentration may have an important role in controlling follicular development and in determining whether an estrous cycle will have 2 or 3-waves.     

Glucosylated N-acetyllactosamine O-antigen chain in the lipopolysaccharide from Helicobacter pylori strain UA861

The O-antigen chain from the lipopolysaccharide of Helicobacter pylori strain UA861 was determined to be composed of an elongated type 2 N - acetyllactosamine backbone, -[-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1- ]n-->, with approximately half of the GlcNAc units carrying a terminal alpha-d-Glc residue at the O -6 position. The O-chain of H.pylori UA861 was terminated by a N -acetyllactosamine [beta-D-Gal-(1-->4)-beta-D- GlcNAc] (LacNAc) epitope and did not express terminal Lewis X or Lewis Y blood-group determinants as previously found in other H.pylori strains. The absence of terminal Lewis X and Lewis Y blood-group epitopes and the replacement of Fuc by Glc as a side chain in the O- chain of H.pylori UA861 represents yet another type of lipopolysaccharide structure from H.pylori species. These structural differences in H.pylori lipopolysaccharide molecules carry implications with regard to possible different pathogenic events between strains and respective hosts.      

First record of lily thrips, Liothrips vaneeckei Priesner, in Australia (Thysanoptera: Phlaeothripidae)

The first Australian record of the lily thrips, Liothrips vaneeckei Priesner, is reported from a bulb farm in Warragul South, Victoria. It is an occasional pest of Lilium bulbs, both in the field and in storage, particularly in the USA and several European countries, and is also infrequently found in considerable numbers on the corms of orchids.     

Cellular penetration of fluorescently labeled superoxide dismutases of various origins.

BACKGROUND: Using fluorescently labeled superoxide dismutase (SOD) and flow cytometry, we have shown previously that the enzyme CuZn SOD (EC 1.15.1.1) from bovine erythrocytes binds rapidly to the cell surface with slow uptake into the cell during the following hours. The degree of labeling was most important for monocytes in comparison to other blood cells (erythrocytes, lymphocytes, and neutrophils) and fibroblasts. In agreement with the flow-cytometric findings, the inhibition of superoxide production was more important for SOD-pretreated monocytes than for neutrophils, as demonstrated with the cytochrome c reduction assay. It was thus of interest to confirm the observed differences between monocytes and neutrophils with confocal laser microscopy, study in greater detail the kinetics of binding, penetration, and intracellular localization of the enzyme, and compare the results obtained with bovine CuZn SOD with those from SODs of other origins and carrying different active sites. MATERIALS AND METHODS: Recombinant human (rh), bovine, and equine CuZn SODs, as well as rh and E. coli Mn SODs, were studied before use with respect to specific activity and purity (HPLC, SDS-PAGE electrophoresis). Fluorescein isothiocyanate was covalently conjugated to the various SODs for study with high-resolution confocal scanning laser microscopy. Superoxide production by monocytes and neutrophils was measured with the cytochrome c assay. RESULTS: As expected from our experiments with flow cytometry, only rare neutrophils were labeled with FITC-SOD, even with the longest incubation time of 3 hr and the highest dose of 1500 units/ml. In addition, they showed a localized fluorescence pattern that was quite different from the diffuse punctate fluorescence pattern of monocytes. Lymphocytes were not labeled at all. The rapid binding to the cellular surface of monocytes was confirmed, and even after 5 min of preincubation, FITC-SOD was found on a small percentage of monocytes. This was correlated with a reduction in superoxide release after phorbolmyristate acetate (PMA) stimulation by 40%. An interesting finding was the perinuclear accumulation of the penetrated SOD after the longest pretreatment of 3 hr, suggesting a barrier against further progression. Indeed, through confocal microscopy we were able to exclude any fluorescence at the nuclear level. While the fluorescence labeling patterns and the kinetics of penetration were quite similar for bovine, equine, and rh CuZn SOD, the Mn SODs showed poor labeling, correlated with a weak inhibitory effect on cytochrome c reduction, which was not statistically significant. CONCLUSIONS: The rapid binding of native CuZn SODs on the surface of monocytes, leading to reduced superoxide release by these cells, explains the observation that beneficial effects of injected SOD lasted for months despite rapid clearance of the enzyme from the bloodstream, according to pharmacodynamic studies. The preferential binding to monocytes, in contrast to neutrophils, may play a role in chronic inflammatory diseases in which the monocytes are in an activated state. The differences in binding capacity between CuZn SODs and Mn SODs, correlated with different inhibitory effects of superoxide production by monocytes, may also have therapeutic significance.