Rationally Evolving MCP-1/CCL2 into a Decoy Protein with Potent Anti-inflammatory Activity in Vivo

Leukocyte recruitment from the blood into injured tissues during inflammatory diseases is the result of sequential events involving chemokines binding to their GPC receptors as well as to their glycosaminoglycan (GAG) co-receptors. The induction and the crucial role of MCP-1/CCL2 in the course of diseases that feature monocyte-rich infiltrates have been validated in many animal models, and several MCP-1/CCL2 as well as CCR2 antagonists have since been generated. However, despite some of them being shown to be efficacious in a number of animal models, many failed in clinical trials, and therapeutically interfering with the activity of this chemokine is not yet possible. We have therefore generated novel MCP-1/CCL2 mutants with increased GAG binding affinity and knocked out CCR2 activity, which were designed to interrupt the MCP-1/CCL2-related signaling cascade. We provide evidence that our lead mutant MCP-1(Y13A/S21K/Q23R) exhibits a 4-fold higher affinity toward the natural MCP-1 GAG ligand heparan sulfate and that it shows a complete deficiency in activating CCR2 on THP-1 cells. Furthermore, a significantly longer residual time on GAG ligands was observed by surface plasmon resonance. Finally, we were able to show that MCP-1(Y13A/S21K/Q23R) had a mild ameliorating effect on experimental autoimmune uveitis and that a marginal effect on oral tolerance in the group co-fed with Met-MCP-1(Y13A/S21K/Q23R) plus immunogenic peptide PDSAg was observed. These results suggest that disrupting wild type chemokine-GAG interactions by a chemokine-based antagonist can result in anti-inflammatory activity that could have potential therapeutic implications.     

deBeitr?ge zur Naturgeschichte der V?gel Brasiliens

Ohne ZusammenfassungFazenda do Bom Valle, 15. Februar 1867.     

Beitr?ge zur Naturgeschichte der V?gel Brasiliens

Ohne ZusammenfassungS. Juli-Heft, S. 217–233.     

Type-3 copper proteins as biocompatible and reusable oxygen sensors

Fluorescently labeled type-3 copper proteins have been proposed previously as solution oxygen sensors by using a FRET mechanism. Herein, we describe how this principle can be adapted to sense O₂ by means of proteins immobilized in optically transparent silica matrices. Specifically, the protein, hemocyanin from Octopus vulgaris N-terminally labeled with Cy5, is immobilized in two different kinds of optically transparent silica matrices, which appear to be a promising platform for enzyme encapsulation. The presented results provide proof of principle that fluorescently labeled proteins immobilized in a silica matrix can be implemented in a reusable, biocompatible and stable oxygen measuring device that might lead to new developments in the field of optical biosensing.     

Identification of a Common Non-Apoptotic Cell Death Mechanism in Hereditary Retinal Degeneration

Cell death in neurodegenerative diseases is often thought to be governed by apoptosis; however, an increasing body of evidence suggests the involvement of alternative cell death mechanisms in neuronal degeneration. We studied retinal neurodegeneration using 10 different animal models, covering all major groups of hereditary human blindness (rd1, rd2, rd10, Cngb1 KO, Rho KO, S334ter, P23H, Cnga3 KO, cpfl1, Rpe65 KO), by investigating metabolic processes relevant for different forms of cell death. We show that apoptosis plays only a minor role in the inherited forms of retinal neurodegeneration studied, where instead, a non-apoptotic degenerative mechanism common to all mutants is of major importance. Hallmark features of this pathway are activation of histone deacetylase, poly-ADP-ribose-polymerase, and calpain, as well as accumulation of cyclic guanosine monophosphate and poly-ADP-ribose. Our work thus demonstrates the prevalence of alternative cell death mechanisms in inherited retinal degeneration and provides a rational basis for the design of mutation-independent treatments.     

Neurotensin Decreases the Affinity of Dopamine D2 Agonist Binding by a G Protein-Independent Mechanism

To examine whether GTP-binding proteins (G proteins) mediate the ability of neurotensin to lower the affinity of dopamine D2 agonist binding, the modulation by neurotensin in vitro of N-[3H]propylnorapomorphine [( 3H]-NPA) binding was investigated following pretreatment with pertussis toxin and N-ethylmaleimide in rat neostriatal membranes. Preincubation with N-ethylmaleimide (100 microM) markedly inhibited pertussis toxin-induced back-ADP ribosylation of three proteins with apparent molecular masses of 41, 40, and 39 kDa, respectively. This inhibition was prevented by adding dithiothreitol (250 microM) during the preincubation. N-Ethylmaleimide increased the KD (180 +/- 30%) and decreased the Bmax (-31 +/- 9%) of [3H]NPA binding sites but did not affect the binding properties of the selective D2 antagonist [3H]raclopride. N-Ethylmaleimide pretreatment did not affect the neurotensin (3 nM)-induced increase in the KD of [3H]NPA binding sites. Pertussin toxin treatment in vivo and in vitro was similarly ineffective. In conclusion, the present study indicates that neurotensin modulation of D2 agonist binding in neostriatal membranes is not mediated by G proteins.     

Atlanto-occipital fusion and spondylolisthesis in an Anasazi skeleton from Bright Angel Ruin, Grand Canyon National Park, Arizona

The skeleton of a middle-aged female showing an unusual pattern of congenital, traumatic, and degenerative pathology was recovered from a small Kayenta Anasazi site located near the confluence of Bright Angel Creek with the Colorado River in the Inner Gorge of Grand Canyon. The atlas is fused with the base of the skull and C2 is fused with C3. The cervical region was subjected to hyperextension, perhaps through use of a tumpline, with resultant reduction of the neural canal to 8 mm, a condition that quite likely led to neurological problems. The skeleton also includes a depression fracture of the lateral condyle of the left tibia. Complete, bilateral spondylolysis of L5 led to an olisthesis of approximately 15 mm. The disc between L5 and S1 then ossified, most likely from staphylococcus bacteremia, making the olisthesis permanent and thereby creating a unique arachaeological specimen. Although spondylolysis is usually viewed as a stress fracture, the general pattern of pathology in this individual makes it necessary to consider an etiology of acute trauma.