The C-terminal domain of MinC inhibits assembly of the Z ring in Escherichia coli.

In Escherichia coli, the Min system, consisting of three proteins, MinC, MinD, and MinE, negatively regulates FtsZ assembly at the cell poles, helping to ensure that the Z ring will assemble only at midcell. Of the three Min proteins, MinC is sufficient to inhibit Z-ring assembly. By binding to MinD, which is mostly localized at the membrane near the cell poles, MinC is sequestered away from the cell midpoint, increasing the probability of Z-ring assembly there. Previously, it has been shown that the two halves of MinC have two distinct functions. The N-terminal half is sufficient for inhibition of FtsZ assembly, whereas the C-terminal half of the protein is required for binding to MinD as well as to a component of the division septum. In this study, we discovered that overproduction of the C-terminal half of MinC (MinC(122-231)) could also inhibit cell division and that this inhibition was at the level of Z-ring disassembly and dependent on MinD. We also found that fusing green fluorescent protein to either the N-terminal end of MinC(122-231), the C terminus of full-length MinC, or the C terminus of MinC(122-231) perturbed MinC function, which may explain why cell division inhibition by MinC(122-231) was not detected previously. These results suggest that the C-terminal half of MinC has an additional function in the regulation of Z-ring assembly.

Crystal structures of progressive Ca2+ binding states of the Ca2+ sensor Ca2+ binding domain 1 (CBD1) from the CALX Na+/Ca2+ exchanger reveal incremental conformational transitions.

Na(+)/Ca(2+) exchangers (NCX) constitute a major Ca(2+) export system that facilitates the re-establishment of cytosolic Ca(2+) levels in many tissues. Ca(2+) interactions at its Ca(2+) binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na(+)/Ca(2+) exchange activity. The structure of the Ca(2+)-bound form of CBD1, the primary Ca(2+) sensor from canine NCX1, but not the Ca(2+)-free form, has been reported, although the molecular mechanism of Ca(2+) regulation remains unclear. Here, we report crystal structures for three distinct Ca(2+) binding states of CBD1 from CALX, a Na(+)/Ca(2+) exchanger found in Drosophila sensory neurons. The fully Ca(2+)-bound CALX-CBD1 structure shows that four Ca(2+) atoms bind at identical Ca(2+) binding sites as those found in NCX1 and that the partial Ca(2+) occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca(2+) binding at CBD1. The structures also predict that the primary Ca(2+) pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu(455), which coordinates the primary Ca(2+) pair, produces dramatic reductions of the regulatory Ca(2+) affinity for exchange current, whereas mutagenesis of Glu(520), which coordinates the secondary Ca(2+) pair, has much smaller effects. Furthermore, our structures indicate that Ca(2+) binding only enhances the stability of the Ca(2+) binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca(2+) regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins.

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (a-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

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. ^

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.

The Frontal Eye Field Is Involved in Visual Vector Inversion in Humans – A Theta Burst Stimulation Study

In the antisaccade task, subjects are requested to suppress a reflexive saccade towards a visual target and to perform a saccade towards the opposite side. In addition, in order to reproduce an accurate saccadic amplitude, the visual saccade vector (i.e., the distance between a central fixation point and the peripheral target) must be exactly inverted from one visual hemifield to the other. Results from recent studies using a correlational approach (i.e., fMRI, MEG) suggest that not only the posterior parietal cortex (PPC) but also the frontal eye field (FEF) might play an important role in such a visual vector inversion process. In order to assess whether the FEF contributes to visual vector inversion, we applied an interference approach with continuous theta burst stimulation (cTBS) during a memory-guided antisaccade task. In 10 healthy subjects, one train of cTBS was applied over the right FEF prior to a memory-guided antisaccade task. In comparison to the performance without stimulation or with sham stimulation, cTBS over the right FEF induced a hypometric gain for rightward but not leftward antisaccades. These results obtained with an interference approach confirm that the FEF is also involved in the process of visual vector inversion.

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.

Use of contrast-enhanced fluid-attenuated inversion recovery sequence to detect brain lesions in dogs and cats.

BACKGROUND The diagnostic value of a contrast-enhanced T2-weighted FLAIR sequence (ceFLAIR) in brain imaging is unclear. HYPOTHESIS/OBJECTIVES That the number of brain lesions detected with ceFLAIR would be no greater than the sum of lesions detected with nFLAIR and ceT1W sequence. ANIMALS One hundred and twenty-nine animals (108 dogs and 21 cats) undergoing magnetic resonance imaging (MRI) of the head between July 2010 and October 2011 were included in the study. METHODS A transverse ceFLAIR was added to a standard brain MRI protocol. Presence and number of lesions were determined based on all available MRI sequences by 3 examiners in consensus and lesion visibility was evaluated for nFLAIR, ceFLAIR, and ceT1W sequences. RESULTS Eighty-three lesions (58 intra-axial and 25 extra-axial) were identified in 51 patients. Five lesions were detected with nFLAIR alone, 2 with ceT1W alone, and 1 with ceFLAIR alone. Significantly higher numbers of lesions were detected using ceFLAIR than nFLAIR (76 versus 67 lesions; P = 0.04), in particular for lesions also detected with ceT1W images (53 versus 40; P =.01). There was no significant difference between the number of lesions detected with combined nFLAIR and ceT1W sequences compared to those detected with ceFLAIR (82 versus 76; P =.25). CONCLUSION AND CLINICAL IMPORTANCE Use of ceFLAIR as a complementary sequence to nFLAIR and ceT1W sequences did not improve the detection of brain lesions and cannot be recommended as part of a routine brain MRI protocol in dogs and cats with suspected brain lesions.

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.