High- and Low-Affinity α-[3H]Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid ([3H]AMPA) Binding Sites Represent Immature and Mature Forms of AMPA Receptors and Are Composed of Differentially Glycosylated Subunits

Abstract: Quantitative α-[³H]amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid ([³H]AMPA) binding autoradiography was performed on frozen-thawed sections from rat brain after preincubation at 0 or 35°C for 1 h. Preincubation at 35°C instead of 0°C resulted in a selective decrease of [³H]AMPA binding assayed at a low concentration of [³H]-AMPA (50 nM) and an enhancement of binding at a high concentration (500 nM). The decrease in [³H]AMPA binding after preincubation at 35°C was accompanied with the loss of the lighter organelles of P3 (microsomal) fractions. These organelles were found to contain a small subpopulation of AMPA/GluR receptors exhibiting a high affinity for [³H]AMPA(K_D～14 nM), whereas heavier organelles exhibited lower affinity for AMPA (K_D～190 nM). This small subpopulation of AMPA/GluR receptors contained almost exclusively a structurally distinct species of GluR2/3 subunits with an apparent molecular mass of 103.5 kDa (assessed with anti-GluR2/3, C-terminal antibodies). Experiments using two deglycosylating enzymes, N-glycopeptidase F and endoglycosidase H, clearly indicated that the 103.5-kDa species represented a partially unglycosylated form of GluR2/3 subunits containing the high-mannose type of oligosaccharide moiety, whereas receptors present in synaptosomal fractions were composed of subunits with complex oligosaccharides. A similar result was obtained by using an antibody recognizing the N-terminal domain of GluR2(4). The same enzymatic treatment indicated that GluR1 subunits also exhibited a partially glycosylated form. These data indicate that high-affinity [³H]AMPA binding sites represent nonsynaptic, intracellular membrane-bound AMPA receptors that differ from synaptic receptors by at least the glycosylation state of GluR2 (and GluR1) subunits. In addition, our results provide a relatively simple way of assessing changes in two spatially and structurally distinct [³H]AMPA binding/GluR sites.     

Gating kinetics of the quisqualate-sensitive glutamate receptor of locust muscle studied using agonist concentration jumps and computer simulations.

Outside-out patches excised from extrajunctional membrane of locust muscle were subjected to "concentration jumps" of L-glutamate, using the liquid filament switch technique, to study channel opening and closing rates, desensitization onset, and recovery from desensitization of a quisqualate-sensitive glutamate receptor (qGluR). Based on data obtained from these experimental studies, computer modeling techniques have been used in an attempt to simulate the behavior of qGluR during a concentration jump of L-glutamate. A linear model with three closed states (one unliganded, one monoliganded, and one biliganded), one open state (binding two molecules of L-glutamate), and two desensitization states (the one monoliganded, the other biliganded) leading from the unliganded closed state simulated all of the experimentally observed behavior. The results are discussed in the context of previous equilibrium studies in which desensitization was inhibited with concanavalin A and for which a ten-state model was required to simulate the behavior of qGluR.     

Safely resuming neglected tropical disease control activities during COVID-19: Perspectives from Nigeria and Guinea

Since its early spread in early 2020, the disease caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Coronavirus Disease 2019 (COVID-19) has caused mass disruptions to health services. These have included interruptions to programs that aimed to prevent, control, and eliminate neglected tropical diseases (NTDs). In March 2020, the World Health Organization (WHO) released interim guidelines recommending the temporary cessation of mass drug administration (MDA), community-based surveys, and case detection, while encouraging continuation of morbidity management and vector control where possible. Over the course of the following months, national programs and implementing partners contributed to COVID-19 response efforts, while also beginning to plan for resumption of NTD control activities. To understand the challenges, opportunities, and recommendations for maximizing continuity of disease control during public health emergencies, we sought perspectives from Nigeria and Guinea on the process of restarting NTD control efforts during the COVID-19 pandemic. Through semistructured interviews with individuals involved with NTD control at the local and national levels, we identified key themes and common perspectives between the 2 countries, as well as observations that were specific to each. Overall, interviewees stressed the challenges posed by COVID-19 interruptions, particularly with respect to delays to activities and related knock-on impacts, such as drug expiry and prolonged elimination timelines, as well as concerns related to funding. However, respondents in both countries also highlighted the benefits of a formal risk assessment approach, particularly in terms of encouraging information sharing and increasing coordination and advocacy. Recommendations included ensuring greater availability of historical data to allow better monitoring of how future emergencies affect NTD control progress; continuing to use risk assessment approaches in the future; and identifying mechanisms for sharing lessons learned and innovations between countries as a means of advancing postpandemic health systems and disease control capacity strengthening.     

Quality determination and the repair of poor quality spots in array experiments

Background  

A common feature of microarray experiments is the occurence of missing gene expression data. These missing values occur for a variety of reasons, in particular, because of the filtering of poor quality spots and the removal of undefined values when a logarithmic transformation is applied to negative background-corrected intensities. The efficiency and power of an analysis performed can be substantially reduced by having an incomplete matrix of gene intensities. Additionally, most statistical methods require a complete intensity matrix. Furthermore, biases may be introduced into analyses through missing information on some genes. Thus methods for appropriately replacing (imputing) missing data and/or weighting poor quality spots are required.     

A Taxonomic Search Engine: Federating taxonomic databases using web services

Background  

The taxonomic name of an organism is a key link between different databases that store information on that organism. However, in the absence of a single, comprehensive database of organism names, individual databases lack an easy means of checking the correctness of a name. Furthermore, the same organism may have more than one name, and the same name may apply to more than one organism.     

Radiographic joint damage in rheumatoid arthritis is associated with differences in cartilage turnover and can be predicted by serum biomarkers: an evaluation from 1 to 4 years after diagnosis

Introduction  

The objective of this study was to determine whether serum biomarkers for degradation and synthesis of the extracellular matrix of cartilage are associated with, and can predict, radiographic damage in patients with rheumatoid arthritis (RA).     

Insulin stimulates membrane fusion and GLUT4 accumulation in clathrin coats on adipocyte plasma membranes

Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by approximately 4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.