Human Alzheimer’s disease synaptic <Emphasis Type=Italic>O</Emphasis>-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry

Neuronal synaptic functional deficits are linked to impaired learning and memory in Alzheimer’s disease (AD). We recently demonstrated that O-GlcNAc, a novel cytosolic and nuclear carbohydrate post-translational modification, is enriched at neuronal synapses and positively regulates synaptic plasticity linked to learning and memory in mice. Reduced levels of O-GlcNAc have been observed in AD, suggesting a possible link to deficits in synaptic plasticity. Using lectin enrichment and mass spectrometry, we mapped several human cortical synaptic O-GlcNAc modification sites. Overlap in patterns of O-GlcNAcation between mouse and human appears to be high, as previously mapped mouse synaptic O-GlcNAc sites in Bassoon, Piccolo, and tubulin polymerization promoting protein p25 were identified in human. Novel O-GlcNAc modification sites were identified on Mek2 and RPN13/ADRM1. Mek2 is a signaling component of the Erk 1/2 pathway involved in synaptic plasticity. RPN13 is a component of the proteasomal degradation pathway. The potential interplay of phosphorylation with mapped O-GlcNAc sites, and possible implication of those sites in synaptic plasticity in normal versus AD states is discussed. iTRAQ is a powerful differential isotopic quantitative approach in proteomics. Pulsed Q dissociation (PQD) is a recently introduced fragmentation strategy that enables detection of low mass iTRAQ reporter ions in ion trap mass spectrometry. We optimized LTQ ion trap settings for PQD-based iTRAQ quantitation and demonstrated its utility in O-GlcNAc site mapping. Using iTRAQ, abnormal synaptic expression levels of several proteins previously implicated in AD pathology were observed in addition to novel changes in synaptic specific protein expression including Synapsin II.     

Phosphorus dynamics within agricultural drainage ditches in the lower Mississippi Alluvial Valley

Excessive phosphorus loading from fertilizers in agriculture results in enriched runoff and downstream aquatic system eutrophication. This study evaluated phosphorus dynamics in agricultural drainage ditches across eight sites within the Lower Mississippi Alluvial Valley (LMAV). The objective of the study was to examine the capacity of drainage ditches across the LMAV to sorb P. Spatially and temporally, all drainage ditch sediments had very low immediately bioavailable phosphorus (P_w), and a very low degree of phosphorus saturation (DPS < 20%) throughout the LMAV. Phosphorus binding energy (K) (0.34-0.60 L/mg) and P sorption maxima (17.8-26.6 L/mg) were low, with very little variation in space and time. Using these metrics, drainage ditches sampled within the LMAV could be described as P sinks, capable of sorbing varying degrees of P seasonally as a result to changes in the Fe-P pool. Sorption, however, will likely be low due to low P sorption maxima and low binding energies. These results will help in P management within primary aquatic systems (such as drainage ditches) within the agricultural landscape and enhance P mitigation strategies at the source, prior to runoff reaching downstream aquatic systems.     

A TEST OF TWO POSSIBLE MECHANISMS FOR PHOTOTACTIC STEERING IN VOLVOX CARTERI (CHLOROPHYCEAE)

We tested two competing models that could explain how differential flagellar activity leads to phototactic turning in spheroids of Volvox carteri f. weismannia (Powers) Iyengar. In one model, turning results from the flagella of anterior cells in the lighted and shadowed hemispheres beating at different frequencies. In a competing model, turning results from a change in beat direction in these flagella. Both models successfully explain phototactic steering under constant illumination, but they make different predictions when colonies are exposed to abrupt changes in light intensity. If turning is due to control of flagellar beat frequency, both progression and rotation rates will change in the same direction and with similar magnitudes. If spheroid turning is due to a change in flagellar beat direction, a decreased rate of progression will accompany an increased rate of rotation and vice versa. We used video-microscopy to observe the behavior of positively phototactic V. carteri spheroids exposed to 10× step-up and step-down stimuli. After a step-up stimulus, spheroids slow their progression and rotation by equal amounts. No significant changes are reported in these parameters after the reciprocal step-down response. These observations are consistent with the variable flagellar frequency model and inconsistent with the variable flagellar direction model for phototactic turning. Switching the direction of light stimulus by 180° results in reorientation of positively phototactic spheroids. The kinetics of this reorientation did not precisely match the predictions of either model.     

Aluminium,boron and molybdenum availability and uptake by rice in acid sulfate soils

Although Al toxicity is believed to be a problem in acid sulfate soils cropped to rice (Oryza, sativa L.), little is known about the behavior of other trace metals such as B and Mo in these soils. The objectives of this study were to measure the availability of Al, B, and Mo in these soils, to determine what governs the availability of these metals and to investigate the relationships between metal availability and uptake by rice. Metal availability and uptake by rice were evaluated in 134 flooded acid sulfate soils in the Central Plains region of Thailand and in a growth chamber study using 50 of the same soils. Soil and plant metal analyses were conducted at the panicle differentiation stage of growth in both studies and in the soil prior to transplanting in the growth chamber study. Metal activities were determined with GEOCHEM. The mineral phases believed to be governing Al³⁺ activities were jurbanite under low pH conditions and amorphous Al(OH)₃ at high pH. The Al chemistry is believed to be intimately linked to the redox-pH cycle, which is driven by the monsoonal climate. Mortality of rice associated with Al toxicity was observed under field and growth chamber conditions. Interference in P uptake and/or assimilation was believed to be the mechanism of Al toxicity. Activities of B(OH) ₄ ^– and B(OH) ₃ ⁰ were found to be highly correlated to pH and ionic strength, respectively, with the latter being the dominant B ion found in these soils. Activities of MoO ₄ ^2– were positively correlated to pH and appeared to be controlled by wulfenite. Leaf Mo contents were found to be positively correlated with MoO ₄ ^2– activity.     

Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species

Acute cessation of flow (ischemia) leads to depolarization of the endothelial cell (EC) membrane mediated by K_ATP channels and followed by production of reactive oxygen species (ROS) from NADPH oxidase. We postulated that ROS are a signal for initiating EC proliferation associated with the loss of shear stress. Flow cytometry was used to identify proliferating CD31-positive pulmonary microvascular endothelial cells (mPMVECs) from wild-type, Kir6.2^–/–, and gp91^phox–/– mice. mPMVECs were labeled with PKH26 and cultured in artificial capillaries for 72 h at 5 dyn/cm² (flow adaptation), followed by 24 h of stop flow or continued flow. ROS production during the first hour of ischemia was markedly diminished compared with wild-type mice in both types of gene-targeted mPMVECs. Cell proliferation was defined as the proliferation index (PI). After 72 h of flow, >98% of PKH26-labeled wild-type mPMVECs were at a single peak (PI 1.0) and the proportion of cells in the S+G₂/M phases were at 5.8% on the basis of cell cycle analysis. With ischemia (24 h), PI increased to 2.5 and the ratio of cells in S+G₂/M phases were at 35%. Catalase, diphenyleneiodonium, and cromakalim markedly inhibited ROS production and cell proliferation in flow-adapted wild-type mPMVECs. Significant effects of ischemia were not observed in Kir6.2^–/– and gp91^phox–/– cells. ANG II activation of NADPH oxidase was unaffected by K_ATP gene deletion. Thus loss of shear stress in flow-adapted mPMVECs results in cell division associated with ROS generated by NADPH oxidase. This effect requires a functioning cell membrane K_ATP channel. cell signaling; ischemia; mechanotransduction; K_ATP channels; NADPH oxidase