Multilobular tumor of the zygomatic bone in a dog

Multilobular tumor of bone (MTB) (also known as Multilobular Osteochondrosarcoma) is an uncommon bone tumor frequently located on the skull of dogs, rarely on the ribs or pelvis. These neoplasms are slow growing, locally invasive, and have the potential to compress and invade the brain. A 10-year-old mixed breed dog was presented with a history of approximately 4 months of progressive growth of a left zygomatic mass. Radiographic investigation revealed a finely granular or stippled non homogeneous radiopaque mass involving the zygomatic arch. After surgery, grossly the neoplasm consisted of multiple, variably sized, grayish-white to yellow nodules separated by collagenous septa of different thickness. Histologically, the tumor was characterized by the presence of multiple lobules containing osteoid and cartilage, separated by a net of fibrous septae. This neoplastic pattern was consistent with a typical multilobular tumor of bone and based on clinical, radiographical, gross and light microscopic findings the definitive diagnosis was made. While reviewing veterinary literature only few cases of MTB were found in dogs

C. elegans expressing human β2-microglobulin: a novel model for studying the relationship between the molecular assembly and the toxic phenotype.

Availability of living organisms to mimic key step of amyloidogenesis of human protein has become an indispensable tool for our translation approach aiming at filling the deep gap existing between the biophysical and biochemical data obtained in vitro and the pathological features observed in patients. Human β(2)-microglobulin (β(2)-m) causes systemic amyloidosis in haemodialysed patients. The structure, misfolding propensity, kinetics of fibrillogenesis and cytotoxicity of this protein, in vitro, have been studied more extensively than for any other globular protein. However, no suitable animal model for β(2)-m amyloidosis has been so far reported. We have now established and characterized three new transgenic C. elegans strains expressing wild type human β(2)-m and two highly amyloidogenic isoforms: P32G variant and the truncated form ΔN6 lacking of the 6 N-terminal residues. The expression of human β(2)-m affects the larval growth of C. elegans and the severity of the damage correlates with the intrinsic propensity to self-aggregate that has been reported in previous in vitro studies. We have no evidence of the formation of amyloid deposits in the body-wall muscles of worms. However, we discovered a strict correlation between the pathological phenotype and the presence of oligomeric species recognized by the A11 antibody. The strains expressing human β(2)-m exhibit a locomotory defect quantified with the body bends assay. Here we show that tetracyclines can correct this abnormality confirming that these compounds are able to protect a living organism from the proteotoxicity of human β(2)-m

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses

Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals

The molecular defect of albumin Tagliacozzo: 313 Lys → Asn

AbstractAlbumin Tagliacozzo is a fast-moving genetic variant of human serum albumin found in 19 unrelated families. The protein was isolated from the serum of a heterozygous healthy subject. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to CNBr fragment IV (residues 299–329). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by RP-HPLC, revealed the variant was caused by 313 Lys → Asn substitution, probably due to a point mutation in the structural gene. The lack of a lysine residue accounts for the electrophoretic behavior of albumin Tagliacozzo

Plasminogen activation triggers transthyretin amyloidogenesis in vitro

Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro. In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo. Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro. Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo. Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation

Disability, anxiety and depression associated with medication-overuse headache can be considerably reduced by detoxification and prophylactic treatment. Results from a multicentre, multinational study (COMOESTAS project)

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Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics.

Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6fu displaying fluorescence rise and decay times (t1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In HEK293T cells, GCaMP6fu revealed a 4-fold faster decay of ATP-evoked intracellular calcium transients than GCaMP6f. Stimulation of hippocampal CA1 pyramidal neurons with five action potentials fired at 100 Hz resulted in a single dendritic calcium transient with a 2-fold faster rise and 7-fold faster decay time (t1/2 of 40 ms) than GCaMP6f, indicating that tracking high frequency action potentials may be limited by calcium dynamics. We propose that the design strategy used for generating GCaMP6fu is applicable for the acceleration of the response kinetics of GCaMP-type calcium indicators

Neuroprotective Potential of Biphalin, Multireceptor Opioid Peptide, Against Excitotoxic Injury in Hippocampal Organotypic Culture

Biphalin is a dimeric opioid peptide that exhibits affinity for three types of opioid receptors (MOP, DOP and KOP). Biphalin is undergoing intensive preclinical study. It was recognized that activation of δ-opioid receptor elicits neuroprotection against brain hypoxia and ischemia. We compare the effect of biphalin and morphine and the inhibition of opioid receptors by naltrexone on survival of neurons in rat organotypic hippocampal cultures challenged with NMDA. Findings: (1) 0.025–0.1 μM biphalin reduces NMDA-induced neuronal damage; (2) biphalin neuroprotection is abolished by naltrexone; (3) reduced number of dead cells is shown even if biphalin is applied with delay after NMDA challenge

High-Affinity Naloxone Binding to Filamin A Prevents Mu Opioid Receptor–Gs Coupling Underlying Opioid Tolerance and Dependence

Ultra-low-dose opioid antagonists enhance opioid analgesia and reduce analgesic tolerance and dependence by preventing a G protein coupling switch (Gi/o to Gs) by the mu opioid receptor (MOR), although the binding site of such ultra-low-dose opioid antagonists was previously unknown. Here we show that with approximately 200-fold higher affinity than for the mu opioid receptor, naloxone binds a pentapeptide segment of the scaffolding protein filamin A, known to interact with the mu opioid receptor, to disrupt its chronic opioid-induced Gs coupling. Naloxone binding to filamin A is demonstrated by the absence of [3H]-and FITC-naloxone binding in the melanoma M2 cell line that does not contain filamin or MOR, contrasting with strong [3H]naloxone binding to its filamin A-transfected subclone A7 or to immunopurified filamin A. Naloxone binding to A7 cells was displaced by naltrexone but not by morphine, indicating a target distinct from opioid receptors and perhaps unique to naloxone and its analogs. The intracellular location of this binding site was confirmed by FITC-NLX binding in intact A7 cells. Overlapping peptide fragments from c-terminal filamin A revealed filamin A2561-2565 as the binding site, and an alanine scan of this pentapeptide revealed an essential mid-point lysine. Finally, in organotypic striatal slice cultures, peptide fragments containing filamin A2561-2565 abolished the prevention by 10 pM naloxone of both the chronic morphine-induced mu opioid receptor–Gs coupling and the downstream cAMP excitatory signal. These results establish filamin A as the target for ultra-low-dose opioid antagonists previously shown to enhance opioid analgesia and to prevent opioid tolerance and dependence