Quantification of endocannabinoids, arachidonic acid and prostaglandins in tissues from mice treated with the substrate specific COX-2 inhibitor 4'-O-methylhonokiol

Il sistema endocannabinoide è rimasto sconosciuto fino agli anni ’90 quando il primo componente di questo sistema, il recettore per i cannabinoidi di tipo 1 è stato identificato. Il sistema endocannabinoide è implicato in molti processi fisiologici a carico del sistema immunitario, sistema nervoso centrale e sistema circolatorio. E’ ormai noto che disfunzioni del sistema endocannabinoide siano implicate in varie patologie e questo ha accresciuto l’interesse nell’investigare nuove strategie farmacologiche atte a ripristinare la normale attività di questo sistema. Il sistema endocannabinoide è composto principalmente da due recettori accoppiati alle proteine G, recettore di tipo 1 (CB1) prevalentemente presenti nel sistema nervoso centrale, e di tipo 2 (CB2) la cui più alta densità si riscontra in cellule ed organi del sistema immunitario. Successivamente, tutta una serie di ligandi endogeni per i recettori dei cannabinoidi è stata identificata e tali molecole sono state definite endocannabinoidi. Tra queste, il 2-arachidonoil glicerolo (2-AG) e l’N-arachidonoiletanolammina (andandamide, AEA) sono i rappresentati più abbondanti a livello tissutale e più studiati. Nel tempo, ligandi di natura sintetica per recettori CB1 e CB2 sono stati sintetizzati e studiati a scopo terapeutico. Le molecole attive sui recettori CB1, sono caratterizzate da effetti psicotropici e per questo impiegate solamente per studi in vitro e in vivo come controlli. Recentemente gran parte della ricerca si è concentrata sullo sviluppo di ligandi selettivi per i recettori CB2 che non presentano gli effetti collaterali a livello del sistema nervoso centrali tipici dell’attivazione del recettore CB1. Gli endocannabinoidi vengono rilasciati “on demand” a partire da precursori fosfolipidici. Una volta attivati i recettori, queste molecole vengono ricaptate all’interno della cellula dove vengono successivamente degradati. Il 2-AG agisce da agonista totale sia sui recettori CB1 che CB2 e il principale enzima responsabile della sua degradazione è la monoacil-glicerol lipasi (MAGL). L’ AEA agisce invece da agonista parziale per entrambe i recettori e la sua degradazione avviene principalmente ad opera della idrolasi delle ammidi degli acidi grassi (FAAH). 2AG e AEA posso andare incontro ad un altro processo enzimatico ad opera della ciclo-ossigenasi 2 (COX-2) che catalizza la loro ossigenazione in prodotti prostaglandine-like. La COX-2 è un enzima chiave nella regolazione del processo infiammatorio ed è bersaglio dei comuni farmaci anti-infiammatori non steroidei utilizzati nel trattamento di infiammazione, dolore e febbre. La COX-2 è responsabile della formazione delle prostaglandine, importanti nella mediazione del processo infiammatorio, a partire da acido arachidonico. Recentemente è stato identificato che tale enzima è anche responsabile della ossigenazione dei due principali endocannabinoidi 2-AG e AEA, per formare rispettivamente, prostaglandin-glicerol-esteri (PG-GE) e prostaglandin-etanolammidi (PG-EA o prostamidi). Queste molecole, sono risultate avere degli effetti come mediatori dell’infiammazione agendo su recettori diversi da quelli delle prostaglandine ma non ancora identificati. Di recente la ricerca si sta focalizzando sulla inibizione selettiva dell’ossigenazione degli endocannabinoidi senza inibire l’ossigenazione dell’acido arachidonico. Questo effetto può risultare di interesse terapeutico per problemi a carico di tessuti che esprimono in modo costitutivo la COX-2 e dove le prostaglandine svolgono un importante ruolo fisiologico, come il rene. Molecole con queste proprietà vengono chiamate Substrate-Specific Inhibitors (SSIs) della ossigenazione degli endocannabinoidi mediata da COX-2. Il 4’-O-methylhonokiol (MH) è un composto bi-fenilico estratto dall’olio che si ricava dai semi della Magnolia grandiflora che agisce sia in vitro che in vivo come COX-2 SSI. A questa molecola sono state riconosciute proprietà anti-infiammatorie, anti-ossidanti, ansiolitiche e ha dimostrato effetti di neuroprotezione. Il MH agisce da agonista selettivo per i recettori CB2 e come detto sopra, inibisce in modo selettivo l’ossigenazione degli endocannabinoidi mediata dall’enzima COX-2. Ho svolto il mio lavoro di tesi nel laboratorio di ricerca all’Istituto di Biochimica e Medicina Molecolare (IBMM) dell’università di Berna, Svizzera e lo scopo della mia tesi è stato quello di quantificare i livelli di endocannabinoidi e prostaglandine a livello renale per approfondire gli effetti del MH sulla COX-2 in questo organo. Per studiare gli effetti di questa molecola in vivo, topi SWISS albino sono stati trattati con differenti dosi di MH o controllo somministrate per via intraperitoneale. L’esperimento ha coinvolto 4 gruppi di animali, ciascuno costituito da 6 individui che sono stati iniettati con 3mg/Kg, 10mg/kg, 20mg/kg di MH oppure DMSO come controllo. Dopo 2 ore gli animali sono stati sacrificati. La quantificazione degli endocannabinoidi e delle prostaglandine nel rene, cervello, fegato, polmone e milza, è stata fatta applicando un metodo LC-MS/MS correntemente utilizzato nel gruppo del Prof. J. Gertsch dove ho svolto il lavoro di tesi. Questa tecnica prevede l’accoppiamento di un sistema HPLC ad uno spettrometro di massa che permette di quantificare ed identificare i diversi metaboliti a seguito di un processo di ionizzazione. Il metodo utilizzato è stato validato determinando precisione, accuratezza ed effetto della matrice. Dai controlli di qualità effettuati il metodo è risultato preciso, accurato e tutti i valori sono rientrati nei limiti. I risultati hanno mostrato che nel rene i livelli di acido arachidonico e prostaglandine non subiscono nessun cambiamento significativo rispetto al controllo, mentre i livelli di 2-AG e AEA aumentano significativamente. Questo effetto è probabilmente dovuto ad una inibizione selettiva da parte del MH sulla ossigenazione degli endocannabinoidi ad opera della COX-2. Durante questo lavoro ho eseguito la quantificazione di questi metaboliti anche in altri organi e misurato la concentrazione tissutale del MH. Questi dati hanno confermato, ad esempio, la penetrazione del MH a livello cerebrale anche in condizioni di non infiammazione, raggiungendo concentrazioni in linea con le sue attività biologiche. Sempre a livello cerebrale, il trattamento con MH induce una diminuzione dose-dipendente dei livelli di AEA, un effetto che è in linea con uno studio precedente riportato in letteratura. A livello epatico, l’effetto indotto dal MH è simile al rene, infatti si nota un incremento dose-dipendente dei livelli di AEA e 2-AG mentre i livelli di prostaglandine sono per tutte le condizioni, incluso i controlli, al limite o sotto il limite di rilevamento. A differenza del rene, il MH induce un significativo incremento dei livelli di acido arachidonico a livello epatico. Le concentrazioni più alte di MH sono state trovate nella milza (circa 5-10 volte superiori agli altri organi). In questo organo si riscontra un’alta densità di cellule del sistema immunitario e, quindi, di recettori di tipo CB2 ai quali si lega il MH e che potrebbe spiegare tale effetto. Il 2-AG mostra una tendenza a crescere che però non è statisticamente significativa rispetto al controllo. L’acido arachidonico non cambia, mentre i livelli di prostaglandine, che sono relativamente bassi (ma misurabili) nei controlli, aumentano significativamente in seguito al trattamento con MH

Synthesis and Structure Elucidation of Glutamyl-Queuosine

Queuosine is one of the most complex hypermodified RNA nucleosides found in the Wobble position of tRNAs. In addition to Queuosine itself, several further modified derivatives are known, where the cyclopentene ring structure is additionally modified by a galactosyl-, a mannosyl-, or a glutamyl-residue. While sugar-modified Queuosine derivatives are found in the tRNAs of vertebrates, glutamylated Queuosine (gluQ) is only known in bacteria. The exact structure of gluQ, particularly with respect to how and where the glutamyl side chain is connected to the Queuosine cyclopentene side chain, is unknown. Here we report the first synthesis of gluQ and, using UHPLC-MS-coinjection and NMR studies, we show that the isolated natural gluQ is the α-allyl-connected gluQ compound

Noxious gases in rabbit housing systems: effects of cross and longitudinal ventilation

Animal welfare is a matter of increasing interest due to ethical and economical worries regarding animal rights and the sustainability of meat production. Ammonia, carbon dioxide, and methane can be produced in the livestock buildings and, if not adequately controlled by ventilation, can be dangerous for animals and farmers. The aim of the present paper is to study the effects of different ventilation systems in rabbit buildings based on the temporal patterns and the spatial distribution of these noxious gases. The experimental measurements were conducted in two rabbit farms with genetically homogeneous animals subjected to the same diet. Two buildings with different forced ventilation layouts (cross ventilation - building A and longitudinal ventilation - building B) were subjected to the monitoring of indoor environmental conditions (temperature, relative humidity, ammonia, carbon dioxide, methane) over a whole year. In both the buildings, ventilation was adjusted automatically by means of electronic control units, which were controlled by temperature sensors, located at the centre of the buildings. Gas concentrations inside the buildings followed clearly defined sinusoidal patterns on a daily basis with the highest values reached in winter during the morning hours for ammonia and during the night hours for carbon dioxide and methane. In particular, ammonia revealed a maximum concentration of 30.7 mg m–3 in building A (cross ventilation) and 12.9 mg m–3 in building B (longitudinal ventilation), whereas the minimum values were 6.0 and 4.2 mg m–3, in building A and B, respectively. As a consequence, daily mean concentrations of noxious gases, solely could not be considered representative of the actual conditions of air quality in the buildings. The airflow direction clearly influenced the spatial concentration of ammonia, which showed different patterns in the two buildings. In building A, the highest ammonia concentration was in a diffuse central area, whereas in building B, it was determined to be less extended and located in the proximity of the wall equipped with extraction fans. The results of this study provide important indications for the planning and management of housing systems for rabbits including: the correct positioning of gas sensors for regulating ventilation systems must be central in case of cross ventilation, but close to the suction fans in case of longitudinal ventilation; the cross ventilation can lead to ammonia concentration higher than longitudinal ventilation, which is caused by the close and prolonged contact of airflow with manure surface in the channels; fans for longitudinal ventilation must be positioned in the same side of the building where scrapers discharge manure; furthermore, manure scraping has to be performed daily in winter during the hours of the day when ventilation rate is at its maximum

Reduction of odorous compounds emissions from swine slurry by electrolytic treatments and copper addition

Swine livestock farms represent potential sources of emissions of gaseous compounds and odors in relation to slurry management, manure treatment, and particularly, storage. Electrolytic treatments of slurries were proposed to solve this pressing problem. Electrolytic treatment consists of the passage of a low electric current through the liquid phase of swine manure in storage basins determined by two or more electrodes with alternated polarisation. We investigated the mechanisms of odour reduction and evaluated the effects of current intensity and of anodically dissolved copper (Cu2+) ions. Fresh swine slurry was subject to electrolytic treatment in a bench top experiment, at 25 mA and 75 mA for 32 days with either copper or graphite electrodes. Emissions of methane, hydrogen sulphide, and volatile fatty acids were measured. Effects of copper addition to fresh swine slurry were also monitored in a bench top scale trial. Reduction of emissions was found to be due to both current passage and copper dissolution. Reduction of foul emissions in graphite electrodes assays resulted lower than in copper electrodes assays, although the direct additions of Cu2+ ions had a much larger effect than could be predicted from the results of electrolytic treatments with copper electrodes, probably due to the greater uniformity in distribution in swine manure. A simple empiric odour threshold value normalised index, odour offensiveness index of slurries, was found to be effective in differentiating treatments

Radio-guided occult lesion localization of cervical recurrences from differentiated thyroid cancer

Background: The aim of this work was to evaluate the application of he concept of ROLL (radio-guided occult lesion localisation) in order to identify non-palpable recurrences in the cervical region by differentiated hyroid cancer (DTC). This procedure makes it possible to perform minimally- invasive radio-guided surgery in a number of selected cases. Material and Methods: The new ROLL procedure was used in 32 DTC patients with loco-regional recurrent lesions. Three of the patients were subjected twice to the procedure. The patients had undergone total thyroidectomy and post-thyroidectomy 131I-ablation, and in somecases had been subjected to one or more cervical lymphadenectomies. Pre-operative labelling consists of ultrasound-guided intra-lesional administration of 99mTc-labeled human albumin macroaggregates (99mTc-MAA, 5e10MBq in 100e200 mL of saline), and was followed by scintigraphy of the cervical region, 2 to 18 hours before programmed surgery. A hand-held gamma-probe used intra-operatively made it possible to identify the labelled lesions. ROLL was employed by means of two different approaches: the former consisted of a selective minimally invasive excision of radiolabelled lesions in 12 patients (s-ROLL); the latter consisted of a modified radical neck dissection (MRND) after excision of radiolabelled lesions in 20 patients (d-ROLL). Results: A total of 59 lesions detected by ultrasound examination were radio-labelled by intra-lesional injection of 99mTc-MAA: of the 59 lesions, 41 were metastatic lymph nodes located in the latero-cervical regions, while 17 recurrent metastatic lesions were spotted in the suprajugular region (at VI level) and 1 lesion was in the high mediastinum. The mean echographic size of the lesions was 11.1_4.1 mm. Using the ROLL procedure, it was possible to localise and remove 59 out of 59 lesions (identification rate 100%), including those of 3 patients previously subjected to non-radioguided surgery, which had failed. Leakage of 99mTc-MAA in to the surrounding tissues during pre-operative labelling relatively hindered precise gamma-probe-guided identification of 3 lesions (in 2 patients). However, these lesions were identified and surgically removed on the basis of both radioactivity and palpation. Histopathological examination confirmed metastatic involvement in all the lesions and further metastases in the other nodes that had been removed. Furthermore, ROLL allows the extent of the cervical dissection to be maintained at a minimum. In our case, the procedure was particularly useful in the paratracheal region as there was no injury of the nerve or parathyroidectomy. After a median follow-up of 29 months, 19 patients showed no evidence of disease, whereas 12 patients developed loco-regional recurrences (in sites near to and/or different from those operated with ROLL), and 2 patients presented distant metastases. Statistical analyses showed no differences between the two surgical applications with regard to in-field relapse, diseasefree survival and overall survival. Conclusions: Radio-guided surgery carried out withROLLis possible in selected patients presenting with recurrent loco-regional DTC (including iodine- negative lesions). The ROLL procedure permits lesions that can be removed with limited invasiveness to be identified, in particular in patients who have already undergone cervical dissections. ROLL has proved to be particularly suitable in those cases in which the lesions were small, nonpalpableand/ or situated in sites that were surgically difficult to reach. Therefore, it is possible to use minimally invasive approaches, followed by shorter operating times and with reduced morbidity complications. Similar to other ROLLprocedures, the low radioactivity dose used does not produce significant radiation exposure either to the patients or to the staff members. In order to obtain optimal performance of the procedure, it is necessary to dispense with experts in ultrasound-guided tissue sampling in the cervical region, and to ensure close collaboration of a team that includes nuclear medicine specialists, surgeons and pathologists. The clinical importance of the ROLLprocedure in managing recurrentDTC should be debated, taking into account the information concerning the prognostic factors of the disease

Characterization and quantification of thiol-peptides in Arabidopsis thaliana using combined dilution and high sensitivity HPLC-ESI-MS-MS

Although thiol-peptide compounds, such as reduced glutathione (GSH), γ-glutamylcysteine (γ-EC), and phytochelatins, play fundamental roles in plants, their analytical determination and characterization is still somewhat problematic, mainly due to their high polarity and oxidation propensity. Thus, in this work a reliable and sensitive HPLC-ESI-MS-MS method was developed, in order to simultaneously assay, within 14-min instrumental runs, γ-EC, GSH, and phytochelatins up to phytochelatin 4. This analytical method was validated in shoot and root extracts of the model plant Arabidopsis thaliana (Brassicaceae) and guaranteed accurate quantification by using specific isotope labelled-internal standards for both GSH and phytochelatins, as well as standards for external calibration. Good linearities in the method performance were observed (R > 0.99), with a dynamic range over three orders of magnitude in thiol-peptide oncentrations. In MRM mode, the detection sensitivity of the thiol-peptides was equal to pproximately 16, 6, 7, 13, 10 fmol for γ-EC, GSH, phytochelatin 2, phytochelatin 3, and phytochelatin 4, respectively (20 μl injection each). The reproducibility of the method was confirmed by high intra- and inter-day accuracy and precision values. The recovery rates were estimated approximately in the range of 73.8–91.0% and the matrix effect evaluation revealed that all analytes exhibited ionization suppression. The use of stable isotope-labelled analogs of the thiol-peptides as internal standards was particularly worthy of note: it offered the considerable advantage of overcoming the consequences of matrix effect and thiol-peptide loss through sample preparation, by normalizing the analyte signal during the quantification process. Thus, by validating the method's sensitivity, accuracy, precision, reproducibility, stability, recovery, and matrix effect, data reliability and robustness were ensured

Delivery of Thyronamines (TAMs) to the Brain: A Preliminary Study

Recent reports highlighted the significant neuroprotective effects of thyronamines (TAMs), a class of endogenous thyroid hormone derivatives. In particular, 3-iodothyronamine (T1AM) has been shown to play a pleiotropic role in neurodegeneration by modulating energy metabolism and neurological functions in mice. However, the pharmacological response to T1AM might be influenced by tissue metabolism, which is known to convert T1AM into its catabolite 3-iodothyroacetic acid (TA1). Currently, several research groups are investigating the pharmacological effects of T1AM systemic administration in the search of novel therapeutic approaches for the treatment of interlinked pathologies, such as metabolic and neurodegenerative diseases (NDDs). A critical aspect in the development of new drugs for NDDs is to know their distribution in the brain, which is fundamentally related to their ability to cross the blood-brain barrier (BBB). To this end, in the present study we used the immortalized mouse brain endothelial cell line bEnd.3 to develop an in vitro model of BBB and evaluate T1AM and TA1 permeability. Both drugs, administered at 1 M dose, were assayed by high-performance liquid chromatography coupled to mass spectrometry. Our results indicate that T1AM is able to efficiently cross the BBB, whereas TA1 is almost completely devoid of this property