A Novel Gβγ-Subunit Inhibitor Selectively Modulates μ-Opioid- Dependent Antinociception and Attenuates Acute Morphine-Induced Antinociceptive Tolerance and Dependence

The Gβγ subunit has been implicated in many downstream signaling events associated with opioids. We previously demonstrated that a small molecule inhibitor of Gβγ-subunit-dependent phospholipase (PLC) activation potentiated morphine-induced analgesia (Bonacci et al., 2006). Here, we demonstrate that this inhibitor, M119 (cyclohexanecarboxylic acid [2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-(9Cl)]), is selective for μ-opioid receptor-dependent analgesia and has additional efficacy in mouse models of acute tolerance and dependence. When administered by an intracerebroventricular injection in mice, M119 caused 10-fold and sevenfold increases in the potencies of morphine and the μ-selective peptide, DAMGO, respectively. M119 had little or no effect on analgesia induced by the κ agonist U50,488 or δ agonists DPDPE or Deltorphin II. Similar results were obtained in vitro, as only activation of the μ-opioid receptor stimulated PLC activation, whereas no effect was seen with the κ- and δ-opioid receptors. M119 inhibited μ-receptor-dependent PLC activation. In studies to further explore the in vivo efficacy of M119, systemic administration M119 also resulted in a fourfold shift increase in potency of systemically administered morphine. Of particular interest, M119 was also able to attenuate acute, antinociceptive tolerance and dependence in mice treated concomitantly with both M119 and morphine. These studies suggest that small organic molecules, such as M119, that specifically regulate Gβγ subunit signaling may have important therapeutic applications in enhancing opioid analgesia, while attenuating the development of tolerance and dependence

Effect of phosphorylation on the interaction of calcium with leucine‐rich amelogenin peptide

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90185/1/j.1600-0722.2011.00900.x.pd

Access, Participation, and Empowerment: Exploring Leadership Practices that Promote Social Inclusion

Students with intellectual disabilities have limited options for continuing their education after high school. Inclusive postsecondary education (IPSE) programs provide access for students with intellectual disabilities to the college campus. However, it takes more than an opportunity to have a meaningful college experience. Gidley et al. (2010) outline a framework for promoting Social Inclusion for Quality Higher Education that uses the underlying theories of Neoliberalism, Social Justice, and Human Potential that inform practices. This qualitative research study used a single case study design to explore research questions that ask what specific leadership practices promoting social inclusion for individuals with intellectual disabilities at the postsecondary level are used at an IPSE program located at Metro University, a pseudonym for a large public university located in the Southeast. Data were collected through six individual interviews with the IPSE program staff, four focus group interviews with IPSE program students, and one focus group with members of the mentorship team, using a total of twenty-one participants. A document review of the parent and student manual and additional information retrieved from the program webpage contributed to the results of this study. The major findings revealed that through relationship building, problem-solving, curriculum development, staff support, and Person-Centered Planning, the IPSE program at Metro University provides an inclusive environment for its students that follows the framework of Social Inclusion for Quality Higher Education

DNA sequencing and site-directed mutagenesis of the drug-metabolizing enzyme, cytochrome P450-2B4

This project focuses on the development of an undergraduate-level laboratory procedure involving EMU\u27s new automated DNA sequencer. The gene used for this procedure is CYP2B4 from the rabbit genome, which codes for Cytochrome P450-2B4 (CVP450). Cytochrome P450s are an important class of proteins found in many species throughout the animal kingdom, including humans. The wild-type CYP2B4 will be sequenced, and then mutated to replace threonine-302 with alanine. Threonine-302 is suspected to playa key role in P450 function. Thus far, procedures have been developed for site-directed mutagenesis to convert threonine-302 to an alanine residue, transformation of bacteria with the target gene, isolation of the plasmid from the transformed bacteria, harvesting the bacteria to express the gene, and preparation of the protein for spectrophotometric analysis. While development of a viable DNA sequencing protocol has met with limited success, the protocol used for the site-directed mutagenesis of CVP2B4 has brought promising results

Synchrotron imaging and Markov Chain Monte Carlo reveal tooth mineralization patterns

The progressive character of tooth formation records aspects of mammalian life history, diet, seasonal behavior and climate. Tooth mineralization occurs in two stages: secretion and maturation, which overlap to some degree. Despite decades of study, the spatial and temporal pattern of elemental incorporation during enamel mineralization remains poorly characterized. Here we use synchrotron X-ray microtomography and Markov Chain Monte Carlo sampling to estimate mineralization patterns from an ontogenetic series of sheep molars (n = 45 M1s, 18 M2s). We adopt a Bayesian approach that posits a general pattern of maturation estimated from individual- and population-level mineral density variation over time. This approach converts static images of mineral density into a dynamic model of mineralization, and demonstrates that enamel secretion and maturation waves advance at nonlinear rates with distinct geometries. While enamel secretion is ordered, maturation geometry varies within a population and appears to be driven by diffusive processes. Our model yields concrete expectations for the integration of physiological and environmental signals, which is of particular significance for paleoseasonality research. This study also provides an avenue for characterizing mineralization patterns in other taxa. Our synchrotron imaging data and model are available for application to multiple disciplines, including health, material science, and paleontological research.Full Tex

A robot vision system for object identification, localization, and manipulation using 3D geometric models

The successful implementation of sensor based robots in dynamic environments will depend largely upon the immunity of the system to incomplete and erroneous sensory information. This paper introduces a six module, 3D model based robot vision system that utilizes 3D geometric models of the objects expected to appear in a scene and can tolerate incomplete and noisy image features. Unique attributes of the work presented include; 1) a matching strategy that is robust to handling incomplete and noisy image features, 2) a procedure for calculating object pose from arbitrary viewing perspectives, 3) the development of a powerful Geometric Modeling and Sensor Simulation System, and 4) the design and development of an integrated 3D geometric model-based robot vision system. Object identification and localization is independent of the particular robot pose and object pose, as long as the object is within view of the sensor. The system effectively utilizes topology during the matching phase to significantly reduce the number of mappings between the domain of image features to that of the object features (object models). These mappings are represented by the mazimal cliques of an association graph. In the context of our system, the maximal cliques represent the topologically best object feature-image feature mappings. Geometric information is then employed during pose determination to calculate the object\u27s unconstrained pose. Our pose determination procedure borrows theories and techniques from camera calibration and automated cartography to generate pose vectors defining the objects pose relative to the robot. Object pose vectors are derived by solving the perspective-three-point problem for all object feature-image feature triplets. Using these pose vectors, an iterative K-means clustering procedure finds the correct object pose. We verify the performance of the system through experimentation involving several objects being viewed from arbitrary position and location, including images involving multiple objects with occlusion. The initial utility of the Geometric Modeling and Sensor Simulation system was two fold. First, the geometric models contain the object features employed during matching and pose determination. Second, the simulation of edge based intensity imagery is necessary for proper (and efficient) object pose verification. However, other important uses for geometric modeling and sensor simulation naturally developed (for example, the testing of multisensor robotic vision systems), hence the sensor simulation capabilities were extended. The sensor simulation routines generate synthetic data from each of the available simulated sensors given a geometrically modeled environment and given the sensor specifications and sensor locations. The five simulated sensory modalities are laser range imagery, point laser range, ultrasonic range, proximity, and edge based intensity imagery. It is clear that the design and integration of a complete robot vision system is not trivial. One must engineer a system consisting of cooperating subsystems that when merged together work efficiently and in a robust manner. Although in this system any one component by itself may be of little utility, the cooperation of all the components as a system is what gives it value. Indeed, we are making strides in this direction; building an integrated multi-component 3D model-based robot vision system

Phylogeography and population genetics of northern flying squirrels (Glaucomys sabrinus) in Southeast Alaska

Thesis (M.S.) University of Alaska Fairbanks, 2000The Prince of Wales flying squirrel (Glaucomys sabrinus griseifrons), a forest associated species, is endemic to several islands in the Alexander Archipelago of Southeast Alaska. Mitochondrial and nuclear markers were examined to assess the genetic uniqueness of this subspecies and its geographic extent and to investigate gene flow among island and mainland populations of flying squirrels. Data from both sets of markers are congruent, and agree with the subspecific designation. The data also indicate that the Prince of Wales subspecies is isolated from other populations in Southeast Alaska, but that there may be gene flow among islands on which it occurs. This island lineage is likely the result of a founder event after the retreat of the Pleistocene ice sheets. The fact that this subspecies is isolated and divergent from mainland populations has potential implications for the design and planning of timber harvests on these islands

Preparative SDS PAGE as an Alternative to His–Tag Purification of Recombinant Amelogenin

Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralisation researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond