Clinical, Radiographic and Histologic Evaluation of 40 Cystic Oral Lesions in 37 Cats.

Feline cystic oral lesions are uncommon and include odontogenic cysts and cystic odontogenic tumors. Accurate diagnosis requires close collaboration between the clinician's clinical and radiographic findings and the pathologist's histologic interpretations. The odontogenic cysts identified in this series include a periapical cyst, dentigerous cysts and a type of unclassified collateral cyst that appears to be a previously undefined, distinct entity in cats (UCC). Many of the cysts (52%) were unable to be classified due to insufficient diagnostic information, which often related to the associated tooth being unavailable for evaluation. Cystic odontogenic tumors included ameloblastomas, amyloid producing ameloblastomas (APA), and feline inductive odontogenic tumors (FIOT). The purpose of this case series was to assess correlations between clinical and radiographic findings, histopathologic interpretation and signalment to identify common characteristics and provide recommendations for clinicians and pathologists to optimize diagnostic efficiency and accuracy for cystic oral lesions in cats.

Single ß³-amino acid substitutions to MOG peptides suppress the development of experimental autoimmune encephalomyelitis.

CD4(+) T-cells play a key role in the pathogenesis of multiple sclerosis (MS). Altered peptide ligands capable of modulating T-cell autoreactivity are considered a promising strategy for development of antigen-specific therapies for MS. Since peptides are inherently unstable, the current study explored single ß-amino acid substitution as a means of stabilizing an epitope of myelin oligodendrocyte glycoprotein. ß-Amino acid substitution at position 44, the major T-cell receptor contact residue, increased the half-life of active metabolites. Vaccination with one altered peptide, MOG44ßF, conferred protection from EAE, decreased T-cell autoreactivity and pro-inflammatory cytokine production. Additional studies using MOG44ßF in an oral treatment regimen, administered after EAE induction, also attenuated disease severity. Thus, altered peptides such as those reported here may lead to the development of novel and more specific treatments for MS.

Structural basis of receptor sulfotyrosine recognition by a CC chemokine: the N-terminal region of CCR3 bound to CCL11/eotaxin-1.

Trafficking of leukocytes in immune surveillance and inflammatory responses is activated by chemokines engaging their receptors. Sulfation of tyrosine residues in peptides derived from the eosinophil chemokine receptor CCR3 dramatically enhances binding to cognate chemokines. We report the structural basis of this recognition and affinity enhancement. We describe the structure of a CC chemokine (CCL11/eotaxin-1) bound to a fragment of a chemokine receptor: residues 8­23 of CCR3, including two sulfotyrosine residues. We also show that intact CCR3 is sulfated and sulfation enhances receptor activity. The CCR3 sulfotyrosine residues form hydrophobic, salt bridge and cation-p interactions with residues that are highly conserved in CC chemokines. However, the orientation of the chemokine relative to the receptor N terminus differs substantially from those observed for two CXC chemokines, suggesting that initial binding of the receptor sulfotyrosine residues guides subsequent steps in receptor activation, thereby influencing the receptor conformational changes and signaling.

Design and synthesis of truncated EGF-A peptides that restore LDL-R recycling in the presence of PCSK9 in vitro.

Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 µM) compared with wild-type EGF-A (KD = 1.2 µM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.

Dual transmission grating based imaging radiometer for tokamak edge and divertor plasmas.

The designs of single transmission grating based extreme ultraviolet (XUV) and vacuum ultraviolet (VUV) imaging spectrometers can be adapted to build an imaging radiometer for simultaneous measurement of both spectral ranges. This paper describes the design of such an imaging radiometer with dual transmission gratings. The radiometer will have an XUV coverage of 20-200 Å with a ∼10 Å resolution and a VUV coverage of 200-2000 Å with a ~50 Å resolution. The radiometer is designed to have a spatial view of 16°, with a 0.33° resolution and a time resolution of ~10 ms. The applications for such a radiometer include spatially resolved impurity monitoring and electron temperature measurements in the tokamak edge and the divertor. As a proof of principle, the single grating instruments were used to diagnose a low temperature reflex discharge and the relevant data is also included in this paper.

Identification of purple acid phosphatase inhibitors by fragment-based screening: promising new leads for osteoporosis therapeutics.

Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K(i) values in the 30-60 µm range. Ligand efficiency values are 0.39-0.44 kcal/mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.

Structural basis of binding by cyclic nonphosphorylated peptide antagonists of Grb7 implicated in breast cancer progression.

Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7.

Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3.

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.

Uptake of a cell permeable G7-18NATE contruct into cells and binding with the Grb-7-SH2 domain.

Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display-derived cyclic nonphosphorylated inhibitor peptide, G7-18NATE, when linked to Penetratin, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7-18NATE covalently attached to just the last seven residues of Penetratin (G7-18NATE-P-Biotin). We demonstrate that this construct is taken up efficiently into MDA-MB-468 breast cancer cells and colocalizes with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7-18NATE-P-Biotin to the Grb7-SH2 domain, and showed that it binds with micromolar affinity (K(d) = 14.4 microM), similar to the affinity of G7-18NATE (K(d) = 35.4 microM). Together this shows that this shorter G7-18NATE-P-Biotin construct is suitable for further studies of the antiproliferative and antimigratory potential of this inhibitor.

Preparation and crystallization of the Grb7 SH2 domain in complex with the G7-18NATE nonphosphorylated cyclic inhibitor peptide.

Grb7 is an adapter protein that is involved in signalling pathways that mediate eukaryotic cell proliferation and migration. Its overexpression in several cancer types has implicated it in cancer progression and led to the development of the G7-18NATE cyclic peptide inhibitor. Here, the preparation of crystals of G7-18NATE in complex with its Grb7 SH2 domain target is reported. Crystals of the complex were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant at room temperature. X-ray diffraction data were collected from crystals to 2.4 Šresolution using synchrotron X-ray radiation at 100 K. The diffraction was consistent with space group P2(1), with unit-cell parameters a=52.7, b=79.1, c=54.7 Å, α=γ=90.0, ß=104.4°. The structure of the G7-18NATE peptide in complex with its target will facilitate the rational development of Grb7-targeted cancer therapeutics.

Synthesis of stapled beta3-peptides through ring-closing metathesis.

The first synthesis of carbon-stapled beta(3)-peptides is reported. The precursor beta(3)-peptides, with O-allyl beta-serines located in an i/i+3 relationship, were prepared on solid phase. We show that efficient ring-closing metathesis (RCM) of these new beta(3)-peptides proceeds smoothly either in solution or on an appropriate solid support. All products were generated with high selectivity for the E-isomer.

Response to Malassezia pachydermatis by peripheral blood mononuclear cells from clinically normal and atopic dogs.

OBJECTIVE: To investigate the potential cell-mediated immune response of atopic dogs to the yeast Malassezia pachydermatis and to correlate it with the type-1 hypersensitivity (humoral) response of the same population of dogs. ANIMALS: 16 clinically normal dogs, 15 atopic dogs with Malassezia dermatitis, 5 atopic dogs with Malassezia otitis, and 7 atopic control (ie, without Malassezia dermatitis or otitis) dogs. PROCEDURE: A crude extract of M pachydermatis was extracted for use as an intradermal allergy testing reagent and for stimulation of isolated peripheral blood mononuclear cells in vitro. Flow cytometry was also used to assess cell surface antigenic determinants (CD3, CD4, CD8, CD14, CD21, CD45RA, surface immunoglobulin) on peripheral blood mononuclear cells. RESULTS: Atopic dogs with cytologic evidence of Malassezia dermatitis had an increased lymphocyte blastogenic response to crude M pachydermatis extract, compared with clinically normal dogs and dogs with Malassezia otitis. Atopic control dogs did not differ significantly in their responses from atopic dogs with Malassezia dermatitis or otitis. A significant correlation was not found between the lymphocyte blastogenic response and the type-1 hypersensitivity response to M pachydermatis within any of the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Cell-mediated and humoral reactivities to M pachydermatis contribute to the pathogenesis of atopic dermatitis in dogs but are not directly correlated. Modification of the dysregulated immune response toward M pachydermatis may assist in the reduction of pathologic changes associated with an atopic dermatitis phenotype in dogs.