Eric S. Eberhardt

Microtubule-associated protein 2 (MAP2) associates with the NMDA receptor and is spatially redistributed within rat hippocampal neurons after oxygen-glucose deprivation

MAP2 (microtubule-associated protein 2) is a cytoskeletal phosphoprotein that regulates the dynamic assembly characteristics of microtubules and appears to provide scaffolding for organelle distribution into the dendrites and for the localization of signal transduction apparatus in dendrites, particularly near spines. MAP2 is degraded after ischemia and other metabolic insults, but the time course and initial triggers of that breakdown are not fully understood. This study determined that MAP2 resides in a complex with the NMDA receptor, suggesting that spatially localized changes may be important in the mechanism of MAP2 redistribution and breakdown after oxygen-glucose deprivation (OGD). Using OGD in the adult rat hippocampal slice as a model system, this study demonstrated that MAP2 breakdown occurs very early after OGD, with the first statistical decrease in MAP2 levels within the first 30 min after the insult. There is a dramatic redistribution of MAP2 to the somata of pyramidal neurons, particularly neurons at the CA1-subiculum border. Free radicals and nitric oxide are not involved in the damage to MAP2. NMDA-receptor activation plays a prominent role in the MAP2 breakdown. In direct response to NMDA receptor activation, calcium influx, likely through the receptor ion channel complex, as well as release of calcium from the mitochondria through activation of the 2Na(+)-Ca(2+) exchanger of mitochondria, triggers MAP2 degradation. The proteolysis of MAP2 is limited by endogenous calpain activity, likely via the spatial access of calpain to MAP2.

Copine A Is Required for Cytokinesis, Contractile Vacuole Function, and Development in Dictyostelium

ABSTRACT Copines make up a family of soluble, calcium-dependent, membrane binding proteins found in a variety of eukaryotic organisms. In an earlier study, we identified six copine genes in the Dictyostelium discoideum genome and focused our studies on cpnA. Our previous localization studies of green fluorescent protein-tagged CpnA in Dictyostelium suggested that CpnA may have roles in contractile vacuole function, endolysosomal trafficking, and development. To test these hypotheses, we created a cpnA− knockout strain, and here we report the initial characterization of the mutant phenotype. The cpnA− cells exhibited normal growth rates and a slight cytokinesis defect. When placed in starvation conditions, cpnA− cells appeared to aggregate into mounds and form fingers with normal timing; however, they were delayed or arrested in the finger stage. When placed in water, cpnA− cells formed unusually large contractile vacuoles, indicating a defect in contractile vacuole function, while endocytosis and phagocytosis rates for the cpnA− cells were similar to those seen for wild-type cells. These studies indicate that CpnA plays a role in cytokinesis and contractile vacuole function and is required for normal development, specifically in the later stages prior to culmination. We also used real-time reverse transcription-PCR to determine the expression patterns of all six copine genes during development. The six copine genes were expressed in vegetative cells, with each gene exhibiting a distinct pattern of expression throughout development. All of the copine genes except cpnF showed an upregulation of mRNA expression at one or two developmental transitions, suggesting that copines may be important regulators of Dictyostelium development.

Thermodynamic origin of prolyl peptide bond isomers

The thermodynamic preference for the trans isomer of prolyl peptide bonds arises almost entirely from enthalpy in aqueous buffer and in toluene.

Comparison Between Air-Assisted and Single-Fluid Pressure Atomizers for Direct-Injection SI Engines Via Spatial and Temporal Mass Flux Measurements

Abstract : Two distinct atomization strategies are contrasted through the measurement of time and spatially dependent mass flux. The two systems investigated include a pressure atomizer (6.9 MPa opening pressure) and an air assist atomizer. Both systems have potential for use in direct injection spark ignition engines. The mass flux data presented were obtained using a spray patternator that was developed to allow phased sampling of the spray. The temporal mass related history of the spray was reconstructed as volume versus time plots and interpolated mass flux contour plots. Results indicate substantial differences in the distribution of both mass and mass flux in space and time for the two injection systems. For example, the pressure atomizer at high mass delivery rates produced a spray that collapsed into a dispersed cylindrical shape while at low rates, generated a hollow cone structure. In addition, the air-assist device discharges 87% of its injected volume within the first of three poppet oscillations while producing a wide hollow cone structure.

Preparing undergraduates to participate in the post‐genome era: A capstone laboratory experience in proteomics*

Proteomics is one of the important new disciplines to emerge from the genome sequencing projects of the last decade. In order to introduce our students to the techniques and promise of this emerging field, a capstone laboratory experience has been developed. The exercise involves multiple aspects of proteomics research including microbial culturing methods, two‐dimensional gel electrophoresis techniques, matrix‐assisted laser desorption‐ionization time‐of‐flight mass spectrometry, and database mining. Over a 12‐week semester, students design their own experiments and apply a proteomic approach to investigate the heat shock response in Escherichia coli. In the trial presented in this article, students successfully identified several major heat shock proteins. The laboratory outlined here can be readily adapted to explore a wide variety of responses in metabolic pathways or responses resulting from other environmental insults or stresses. Additionally, the laboratory can be modified to explore the proteomes of organelles, tissues, and other model organisms.