CONFORMATIONAL CHANGES IN THE EXTRACELLULAR DOMAIN OF GLUTAMATE RECEPTORS

The family of membrane protein called glutamate receptors play an important role in the central nervous system in mediating signaling between neurons. Glutamate receptors are involved in the elaborate game that nerve cells play with each other in order to control movement, memory, and learning. Neurons achieve this communication by rapidly converting electrical signals into chemical signals and then converting them back into electrical signals. To propagate an electrical impulse, neurons in the brain launch bursts of neurotransmitter molecules like glutamate at the junction between neurons, called the synapse. Glutamate receptors are found lodged in the membranes of the post-synaptic neuron. They receive the burst of neurotransmitters and respond by fielding the neurotransmitters and opening ion channels. Glutamate receptors have been implicated in a number of neuropathologies like ischemia, stroke and amyotrophic lateral sclerosis. Specifically, the NMDA subtype of glutamate receptors has been linked to the onset of Alzheimer’s disease and the subsequent degeneration of neuronal cells. While crystal structures of AMPA and kainate subtypes of glutamate receptors have provided valuable information regarding the assembly and mechanism of activation; little is known about the NMDA receptors. Even the basic question of receptor assembly still remains unanswered. Therefore, to gain a clear understanding of how the receptors are assembled and how agonist binding gets translated to channel opening, I have used a technique called Luminescence Resonance Energy Transfer (LRET). LRET offers the unique advantage of tracking large scale conformational changes associated with receptor activation and desensitization. In this dissertation, LRET, in combination with biochemical and electrophysiological studies, were performed on the NMDA receptors to draw a correlation between structure and function. NMDA receptor subtypes GluN1 and GluN2A were modified such that fluorophores could be introduced at specific sites to determine their pattern of assembly. The results indicated that the GluN1 subunits assembled across each other in a diagonal manner to form a functional receptor. Once the subunit arrangement was established, this was used as a model to further examine the mechanism of activation in this subtype of glutamate receptor. Using LRET, the correlation between cleft closure and activation was tested for both the GluN1 and GluN2A subunit of the NMDA receptor in response to agonists of varying efficacies. These investigations revealed that cleft closure plays a major role in the mechanism of activation in the NMDA receptor, similar to the AMPA and kainate subtypes. Therefore, suggesting that the mechanism of activation is conserved across the different subtypes of glutamate receptors.

Cytochrome P-450 reductase FAD binding domain: Cloning, enzymatic activity and flavin binding characterization

The Mixed Function Oxidase System metabolizes a wide range of biochemicals including drugs, pesticides and steroids. Cytochrome P450 reductase is a key enzymatic component of this system, supplying reducing equivalents from NADPH to cytochrome P450. The electrons are shuttled through reductase via two flavin moieties: FAD and FMN. Although the exact mechanism of flavins action is not known, the enzymatic features of reductase greatly depleted of either FMN of FAD have been characterized. Additionally, flavin location within reductase has been proposed by homology and chemical modification studies. This study seeks to extend the flavin depletion analysis in a more controlled system by eliminating the proposed FMN binding domain with recombinant DNA techniques and biochemical analysis. Two P450 reductase cDNA clones containing only the FMN and NADPH binding domain were isolated, expressed and the protein products purified and analysed. This study confirms the proposed FAD binding site, role of FAD in electron shuttling pathway and provides new methods to study the FAD binding domain. ^

Fibrillins: Sequence, phylogeny and carboxy terminal domain proteolytic processing

Fibrillin-1 and -2 are large secreted glycoproteins that are known to be components of extracellular matrix microfibrils located in the vasculature, basement membrane and various connective tissues. These microfibrils are often associated with a superstructure known as the elastic fiber. During the development of elastic tissues, fibrillin microfibrils precede the appearance of elastin and may provide a scaffolding for the deposition and crosslinking of elastin. Using RT/PCR, we cloned and sequenced 3.85Kbp of the FBN2 gene. Five differences were found between our contig sequence and that published by Zhang et al. (1995). Like many extracellular matrix proteins, the fibrillins are modular proteins. We compared analogous domains of the two fibrillins and also members of the latent TGF-$\beta$ binding protein (LTBP) family to determine their phylogenetic relationship. We found that the two families are homologous. LTBP-2 is the most similar to the fibrillin family while FBN-1 is the most similar to the LTBP family. The fibrillin-1 carboxy terminal domain is proteolytically processed. Two eukaryotic protein expression systems, baculoviral and CHO-K1, were developed to examine the proteolytic processing of the carboxy terminal domain of the fibrillin-1 protein. Both expression systems successfully processed the domain and both processed a mutant less efficiently. In the CHO-K1 cells, processing occurred intracellularly. ^

Functions and intramolecular interactions of ribosomal RNA in decoding of termination codons

Two regions in the 3$\prime$ domain of 16S rRNA (the RNA of the small ribosomal subunit) have been implicated in decoding of termination codons. Using segment-directed PCR random mutagenesis, I isolated 33 translational suppressor mutations in the 3$\prime$ domain of 16S rRNA. Characterization of the mutations by both genetic and biochemical methods indicated that some of the mutations are defective in UGA-specific peptide chain termination and that others may be defective in peptide chain termination at all termination codons. The studies of the mutations at an internal loop in the non-conserved region of helix 44 also indicated that this structure, in a non-conserved region of 16S rRNA, is involved in both peptide chain termination and assembly of 16S rRNA.^ With a suppressible trpA UAG nonsense mutation, a spontaneously arising translational suppressor mutation was isolated in the rrnB operon cloned into a pBR322-derived plasmid. The mutation caused suppression of UAG at two codon positions in trpA but did not suppress UAA or UGA mutations at the same trpA positions. The specificity of the rRNA suppressor mutation suggests that it may cause a defect in UAG-specific peptide chain termination. The mutation is a single nucleotide deletion (G2484$\Delta$) in helix 89 of 23S rRNA (the large RNA of the large ribosomal subunit). The result indicates a functional interaction between two regions of 23S rRNA. Furthermore, it provides suggestive in vivo evidence for the involvement of the peptidyl-transferase center of 23S rRNA in peptide chain termination. The $\Delta$2484 and A1093/$\Delta$2484 (double) mutations were also observed to alter the decoding specificity of the suppressor tRNA lysT(U70), which has a mutation in its acceptor stem. That result suggests that there is an interaction between the stem-loop region of helix 89 of 23S rRNA and the acceptor stem of tRNA during decoding and that the interaction is important for the decoding specificity of tRNA.^ Using gene manipulation procedures, I have constructed a new expression vector to express and purify the cellular protein factors required for a recently developed, realistic in vitro termination assay. The gene for each protein was cloned into the newly constructed vector in such a way that expression yielded a protein with an N-terminal affinity tag, for specific, rapid purification. The amino terminus was engineered so that, after purification, the unwanted N-terminal tag can be completely removed from the protein by thrombin cleavage, yielding a natural amino acid sequence for each protein. I have cloned the genes for EF-G and all three release factors into this new expression vector and the genes for all the other protein factors into a pCAL-n expression vector. These constructs will allow our laboratory group to quickly and inexpensively purify all the protein factors needed for the new in vitro termination assay. (Abstract shortened by UMI.) ^

Affinity retardation chromatography: characterization of the method and its application. The description of low affinity laminin self-interactions.

Affinity retardation chromatography (ARC), a method for the examination of low-affinity interactions, is mathematically described in order to characterize the method itself and to estimate binding coefficients of self-assembly domains of basement membrane protein laminin. Affinity retardation was determined by comparing the elutions on a "binding" and on a "nonreacting" column. It depends on the binding coefficient, the concentrations of both ligands, and the nonbinding elution position. Half maximal binding of the NH2-terminal domain of laminin B1-short arm to the A- and/or B2-short arms was estimated to occur at 10-17 microM for noncooperative and at < or = 3 microM for cooperative binding. A model of the laminin polymerization, postulating two levels of cooperative binding behavior, is described.

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

Coilin is the signature protein of the Cajal body (CB), a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Newly imported Sm-class snRNPs are thought to traffic through CBs before proceeding to their final nuclear destinations. Loss of coilin function in mice leads to significant viability and fertility problems. Coilin interacts directly with the spinal muscular atrophy (SMA) protein via dimethylarginine residues in its C-terminal domain. Although coilin hypomethylation results in delocalization of survival of motor neurons (SMN) from CBs, high concentrations of snRNPs remain within these structures. Thus, CBs appear to be involved in snRNP maturation, but factors that tether snRNPs to CBs have not been described. In this report, we demonstrate that the coilin C-terminal domain binds directly to various Sm and Lsm proteins via their Sm motifs. We show that the region of coilin responsible for this binding activity is separable from that which binds to SMN. Interestingly, U2, U4, U5, and U6 snRNPs interact with the coilin C-terminal domain in a glutathione S-transferase (GST)-pulldown assay, whereas U1 and U7 snRNPs do not. Thus, the ability to interact with free Sm (and Lsm) proteins as well as with intact snRNPs, indicates that coilin and CBs may facilitate the modification of newly formed snRNPs, the regeneration of 'mature' snRNPs, or the reclamation of unassembled snRNP components.

Softening the Lower Crust: Modes of Syn-Transport Transposition Around and Adjacent to a Deep Crustal Granulite Nappe, Parry Sound Domain, Grenville Province, Ontario, Canada

The Parry Sound domain is a granulite nappe-stack transported cratonward during reactivation of the ductile lower and middle crust in the late convergence of the Mesoproterozoic Grenville orogeny. Field observations suggest the following with respect to the ductile sheath: (1) Formation of a carapace of transposed amphibolite facies gneiss derived from and enveloping the western extremity of the Parry Sound domain and separating it from high-strain gneiss of adjacent allochthons. This ductile sheath formed dynamically around the moving granulite nappe through the development of systems of progressively linked shear zones. (2) Transposition initiated by hydration (amphibolization) of granulite facies gneiss by introduction of fluid along cracks accompanying pegmatite emplacement. Shear zones nucleated along pegmatite margins and subsequently linked and rotated. The source of the pegmatites was most likely subjacent migmatitic and pegmatite-rich units or units over which Parry Sound domain was transported. Comparison of gneisses of the ductile sheath with high-strain layered gneiss of adjacent allochthons show the mode of transposition of penetratively layered gneiss depended on whether or not the gneiss protoliths were amphibolite or granulite facies tectonites before initiation of transposition, resulting in, e.g., folding before shearing, no folding before shearing, respectively. Meter-scale truncation along high-strain gradients at the margins of both types of transposition-related shear zones observed within and marginal to Parry Sound domain mimic features at kilometer scales, implying that apparent truncation by transposition originating in a manner similar to the ductile sheath may be a common feature of deep crustal ductile reworking. Citation: Culshaw, N., C. Gerbi, and J. Marsh (2010), Softening the lower crust: Modes of syn-transport transposition around and adjacent to a deep crustal granulite nappe, Parry Sound domain, Grenville Province, Ontario, Canada, Tectonics, 29, TC5013, doi:10.1029/2009TC002537.

Precipitation isoscape of high reliefs: interpolation scheme designed and tested for monthly resolved precipitation oxygen isotope records of an Alpine domain

Stable oxygen isotope composition of atmospheric precipitation (δ18Op) was scrutinized from 39 stations distributed over Switzerland and its border zone. Monthly amount-weighted δ18Op values averaged over the 1995–2000 period showed the expected strong linear altitude dependence (−0.15 to −0.22‰ per 100 m) only during the summer season (May–September). Steeper gradients (~ −0.56 to −0.60‰ per 100 m) were observed for winter months over a low elevation belt, while hardly any altitudinal difference was seen for high elevation stations. This dichotomous pattern could be explained by the characteristically shallower vertical atmospheric mixing height during winter season and provides empirical evidence for recently simulated effects of stratified atmospheric flow on orographic precipitation isotopic ratios. This helps explain "anomalous" deflected altitudinal water isotope profiles reported from many other high relief regions. Grids and isotope distribution maps of the monthly δ18Op have been calculated over the study region for 1995–1996. The adopted interpolation method took into account both the variable mixing heights and the seasonal difference in the isotopic lapse rate and combined them with residual kriging. The presented data set allows a point estimation of δ18Op with monthly resolution. According to the test calculations executed on subsets, this biannual data set can be extended back to 1992 with maintained fidelity and, with a reduced station subset, even back to 1983 at the expense of faded reliability of the derived δ18Op estimates, mainly in the eastern part of Switzerland. Before 1983, reliable results can only be expected for the Swiss Plateau since important stations representing eastern and south-western Switzerland were not yet in operation.

A re-examination of petrogenesis and 40Ar/39Ar systematics in the the Chain of Ponds K-feldspar: "diffusion domain" archetype versus polyphase hygrochronology

K-feldspar (Kfs) from the Chain of Ponds Pluton (CPP) is the archetypal reference material, on which thermochronological modeling of Ar diffusion in discrete “domains” was founded. We re-examine the CPP Kfs using cathodoluminescence and back-scattered electron imaging, transmission electron microscopy, and electron probe microanalysis. 40Ar/39Ar stepwise heating experiments on different sieve fractions, and on handpicked and unpicked aliquots, are compared. Our results reproduce the staircase-shaped age spectrum and the Arrhenius trajectory of the literature sample, confirming that samples collected from the same locality have an identical Ar isotope record. Even the most pristine-looking Kfs from the CPP contains successive generations of secondary, metasomatic/retrograde mineral replacements that post-date magmatic crystallization. These chemically and chronologically distinct phases are responsible for its staircase-shaped age spectra, which are modified by handpicking. While genuine within-grain diffusion gradients are not ruled out by these data, this study demonstrates that the most important control on staircase-shaped age spectra is the simultaneous presence of heterochemical, diachronous post-magmatic mineral growth. At least five distinct mineral species were identified in the Kfs separate, three of which can be traced to external fluids interacting with the CPP in a chemically open system. Sieve fractions have size-shifted Arrhenius trajectories, negating the existence of the smallest “diffusion domains”. Heterochemical phases also play an important role in producing non-linear trajectories. In vacuo degassing rates recovered from Arrhenius plots are neither related to true Fick’s Law diffusion nor to the staircase shape of the age spectra. The CPP Kfs used to define the "diffusion domain" model demonstrates the predominance of metasomatic alteration by hydrothermal fluids and recrystallization in establishing the natural Ar distribution amongst different coexisting phases that gives rise to the staircase-shaped age spectrum. Microbeam imaging of textures is as essential for 40Ar-39Ar hygrochronology as it is for U-Pb geochronology.

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.

Quality Assessment and Enhancement Method for high-resolution Fourier-Domain OCT Imaging of Retina Laser Lesions

We present an image quality assessment and enhancement method for high-resolution Fourier-Domain OCT imaging like in sub-threshold retina therapy. A Maximum-Likelihood deconvolution algorithm as well as a histogram-based quality assessment method are evaluated.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

SMG6 mediated degradation of nonsense mRNA requires phosphorylation-independent interaction with the helicase domain of UPF1

Eukaryotic mRNAs with premature translation-termination codons (PTCs) are recognized and eliminated by nonsense-mediated mRNA decay (NMD). NMD targeted mRNAs can be degraded by different routes that all involve phosphorylated UPF1 (P-UPF1) as a starting point. The endonuclease SMG6, which cleaves mRNA near the PTC, is one of three known NMD factors thought to be recruited to nonsense mRNAs by interaction with P-UPF1, leading to eventual mRNA degradation. By MS2-mediated tethering of SMG6 and mutants thereof to a reporter RNA combined with knockdowns of various NMD factors, we demonstrate that besides its endonucleolytic activity, SMG6 also requires UPF1 and SMG1 for inducing RNA decay. Our experiments revealed a phosphorylation-independent interaction between SMG6 and UPF1 that is important for SMG6-mediated mRNA decay and using yeast two hybrid assays, we mapped this interaction to the unique stalk region of the UPF1 helicase domain. This region of UPF1 is essential for SMG6-mediated reporter RNA decay and also for NMD. Our results postulate that besides recruiting SMG6 to its RNA substrates, UPF1 is also required to activate its endonuclease activity.

Head movement synchrony in social interactions of patients with schizophrenia indicates symptoms, cognition and social functioning

Background: Disturbed interpersonal communication is a core problem in schizophrenia. Patients with schizophrenia often appear disconnected and "out of sync" when interacting with others. This may involve perception, cognition, motor behavior, and nonverbal expressiveness. Although well-known from clinical observation, mainstream research has neglected this area. Corresponding theoretical concepts, statistical methods, and assessment were missing. In recent research, however, it has been shown that objective, video-based measures of nonverbal behavior can be used to reliably quantify nonverbal behavior in schizophrenia. Newly developed algorithms allow for a calculation of movement synchrony. We found that the objective amount of movement of patients with schizophrenia during social interactions was closely related to the symptom profiles of these patients (Kupper et al., 2010). In addition and above the mere amount of movement, the degree of synchrony between patients and healthy interactants may be indicative of various problems in the domain of interpersonal communication and social cognition. Methods: Based on our earlier study, head movement synchrony was assessed objectively (using Motion Energy Analysis, MEA) in 378 brief, videotaped role-play scenes involving 27 stabilized outpatients diagnosed with paranoid-type schizophrenia. Results: Lower head movement synchrony was indicative of symptoms (negative symptoms, but also of conceptual disorganization and lack of insight), verbal memory, patients’ self-evaluation of competence, and social functioning. Many of these relationships remained significant even when corrected for the amount of movement of the patients. Conclusion: The results suggest that nonverbal synchrony may be an objective and sensitive indicator of the severity of symptoms, cognition and social functioning.