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TWO TOPICS OF PHYSIOPATHOLOGICAL INTEREST IN MITOCHONDRIAL RESEARCH: INNER MEMBRANE CHANNELS AND MITOCHONDRIOTROPIC REDOX-ACTIVE COMPOUNDS
Full text linkThe group I have been part of during my doctorate thesis research is interested in various aspects of mitochondrial function. The projects I have been involved in are concerned with two very different topics; both studies have however as long-term objective the discovery and validation of compounds potentially useful for an anti-cancer action at the mitochondrial level.
The first of these projects concerns the potassium-selective channels of the inner mitochondrial membrane. A few have been discovered over the past several years. Besides participating in the regulation of mitochondrial processes and parameters these channels have attracted attention because of their involvement in other aspects of cellular physiology. Two of them seem to be able to afford protection from ischemic damage (preconditioning), while a recent study by our group in collaboration with German researchers has provided evidence that a third, mtKV1.3, plays an important role in Bax-mediated apoptosis in lymphocytes. Hence our interest in this channel.
I have verified whether mtKV1.3 might be present in the mitochondria not only of T lymphocytes, where it has been discovered, but also in those of a few other cancerous cell lines. So far I have succeeded in establishing its presence in two human lines (PC3 and MCF7) (Gulbins et al., 2010 – Chapt. 1). I have then investigated whether inhibition of the channel might cause cell death in these lines, a finding which would have been of considerable interest for the development of new chemotherapeutic drugs. Unfortunately I have not been able to obtain evidence of a death-inducing effect by a specific inhibitor of mtKV1.3 (Chapt. 2).
Within this project we have also discovered another mitochondrial channel: Ca2+-activated mtKCa3.1, which we have observed in the mitochondria of a human colon tumor line (HCT116) both by patch-clamping the inner membrane and by Wester blotting (De Marchi et al., 2009 – Chapt. 3). I have then investigated whether its inhibition might be cytotoxic or at least cytostatic, but in this case also the answer has so far been negative. I have checked for the presence of mtKCa3.1 in two other cell lines of colonic origin (C26 and Caco2) to try and understand whether this channel might be expressed in a cancer-specific manner by subsequently performing a comparison with non-tumoral cells or tissue. However, in both cases the channel has turned out to be present only in the plasma membrane (Sassi et al., 2010 – Chapt. 4).
The other project I have taken part in concerns the pharmacological exploitation of plant polyphenols, contained in many foods and beverages. A vast literature shows that these compounds have interesting biological properties which could be useful in health care endeavours such as protection from cardiovascular damage or neurodegeneration, or to prevent the onset and inhibit the growth of many types of cancer. Practical applications for these potentially useful compounds are made difficult by their low bioavailability. A priori, one way to circumvent this problem may be to cause their accumulation in an opportune site of action. The mitochondrion is an obvious choice, since polyphenols are redox-active molecules (whose activity may be anti- or pro-oxidant depending on conditions such as pH, presence of Fe2+/3+ o Cu+/2+ and concentration of the polyphenol itself) and mitochondria are the main cellular site of production of radicals as well as being mechanistically involved in cell death. Mitochondriotropic compounds might turn out to have biomedical relevance regardless of whether their activity may be anti-oxidant/cytoprotective or pro-oxidant/cytotoxic.
Our group has therefore synthesized a few derivatives of quercetin and resveratrol – two much-studied model polyphenols – capable of accumulating into mitochondria. This property is conferred by the triphenylphosphonium group (TPP), a lipophylic cation which can diffuse through biomembranes and accumulate in regions held at negative electrical potential, such as the mitochondrial matrix and the cytoplasm. We have verified that these derivatives indeed accumulate in mitochondria as expected (Mattarei et al., 2008 – Chapt. 5; Biasutto et al., 2008 – Chapt. 6). A first exploration of the biological properties of these new compounds has been carried out with two quercetin derivatives (Q3BTPI, QTA3BTPI). We have observed that these compounds supplied to isolated mitochondria are potential co-inducers of the mitochondrial permeability transition (MPT), as well as inhibitors of respiration and of the F0F1 ATPase. At the cellular level, both compounds induce an MPT-independent depolarization of mitochondria and a modest increase in the cellular production of ROS at least in the case of QTA3BTPI (Biasutto et al., 2010 – Chapt. 7).
I have extended the study to evaluate the possible cytostatic/cytotoxic action of the various mitochondriotropic compounds on tumoral (C26) and non-tumoral (MEF) cell lines. I have furthermore investigated what impact these compounds have on the mitochondria of another tumoral cell line (Jurkat lymphocytes). The results of cell vitality determinations showed that the various mitochondriotropic derivatives, administered at concentrations in the μM range, can have a cytotoxic/cytostatic activity vs. rapidly growing cells, while their effect on slowly dividing non-tumoral ones is much more modest. The initial findings of a mechanistic study have revealed that relatively high (10-5 M range) concentrations can elicit structural alterations, mitochondrial depolarization and generation of radical oxygen species (Chapt. 8).Il gruppo presso cui ho svolto il mio corso di dottorato si interessa di vari aspetti della funzionalità mitocondriale. I progetti che ho seguito riguardano due ambiti molto diversi tra loro; tuttavia in entrambi i casi si tratta di studi il cui obiettivo a lungo termine è l’individuazione di composti potenzialmente utili per un’azione antitumorale a livello mitocondriale.
Uno di questi progetti riguarda i canali del potassio presenti nella membrana mitocondriale interna. Negli ultimi anni ne sono stati individuati diversi. Oltre ad aver un ruolo nella regolazione di processi e parametri mitocondriali questi canali son risultati interessanti per il loro coinvolgimento in altri aspetti della fisiologia cellulare. Due di questi sembrano aver un ruolo nella protezione dal danno ischemico (precondizionamento), mentre un recente studio svolto dal nostro gruppo in collaborazione con un gruppo tedesco ha messo in evidenza come un terzo, il mtKV1.3, svolga un ruolo importante nell’apoptosi mediata da Bax in linfociti. Da qui l’interesse da parte nostra verso quest’ultimo canale.
Ho verificato se il mtKV1.3 fosse presente nei mitocondri non solo dei linfociti T, dove è stato individuato inizialmente, ma anche in alcune altre linee tumorali. Sono riuscito finora ad individuarlo in due linee umane (PC3 e MCF7) (Gulbins et al., 2010 - Cap. 1). Ho successivamente indagato se l’inibizione del canale potesse indurre morte cellulare in tali linee. Ciò sarebbe di notevole interesse nello sviluppo di nuovi chemioterapici. Tuttavia dai risultati ottenuti l’utilizzo di inibitori specifici del canale nelle differenti linee non ha mostrato effetti rilevanti di induzione di morte cellulare (Cap. 2).
Sempre nell’ambito di questo progetto abbiamo individuato un altro canale mitocondriale: il mtKCa3.1 che abbiamo osservato nei mitocondri di una linea tumorale del colon umana (HCT116) sia mediante patch-clamp che Western blot. (De Marchi et al., 2009 - Cap. 3). Ho poi verificato se la sua inibizione potesse essere citotossica o almeno citostatica, tuttavia anche in questo caso non abbiamo ottenuto una risposta positiva. Ho verificato la presenza o meno del canale anche in due altre linee tumorali del colon (C26 e Caco2) per comprendere se fosse un canale espresso in modo tumore-specifico procedendo successivamente a confronti con cellule non tumorali, tuttavia in ambedue i casi il canale è risultato presente solo nella plasma membrana (Sassi et al., 2009 - Cap 4).
L’altro progetto da me intrapreso riguarda lo sfruttamento farmacologico dei polifenoli vegetali presenti in molti cibi e bevande. Una notevole letteratura mostra come tali composti possiedano interessanti proprietà biologiche che potrebbero esser utili in diversi ambiti, come la protezione cardiovascolare o dalla neurodegenerazione, oppure nel prevenire l’insorgere e nell’inibire la crescita di molti tipi di cancro. Questi composti dalle interessanti potenzialità trovano una notevole difficoltà d’utilizzo a causa della loro scarsa biodisponibilità. A priori, un modo per ovviare a questo problema è accumulare tali composti in un’opportuna sede d’azione. Una scelta ovvia è il mitocondrio, dato che i polifenoli sono molecole redox-attive (la cui attività può essere anti- o pro-ossidante in base a differenti condizioni come pH, presenza di Fe2+/3+ o Cu+/2+ e concentrazione del composto) e che i mitocondri son il principale sito di produzione dei radicali, oltre ad esser coinvolti nei processi di morte cellulare. Questi composti mitocondriotropici potrebbero aver una rilevanza biomedica sia che dimostrino un’attività anti-ossidante/citoprotettiva sia invece che si comportino da pro-ossidanti citotossici.
Il nostro gruppo ha quindi sintetizzato alcuni derivati di quercitina e resveratrolo - due polifenoli particolarmente studiati presi a modello - in grado di accumularsi nei mitocondri. Questo grazie alla funzionalizzazione con un gruppo trifenilfosfonio (TPP+), un catione lipofilico che può diffondere attraverso le membrane biologiche e accumularsi in regioni a potenziale negativo, quali la matrice mitocondriale e il citoplasma. Abbiamo verificato che questi derivati si accumulassero effettivamente nei mitocondri (Mattarei et al, 2008 - Cap. 5; Biasutto et al, 2008 – Cap. 6). Una prima indagine sugli effetti biologici di questi nuovi composti è stata condotta con due derivati della quercetina (Q3BTPI, QTA3BTPI). Abbiamo osservato come questi composti con mitocondri isolati siano potenziali co-induttori della transizione di permeabilità mitocondriale (MPT), nonché inibitori della respirazione e dell’ATP sintasi mitocondriale. Osservazioni a livello cellulare mostrano una depolarizzazione mitocondriale indipendente dalla MPT indotta da entrambi i composti e un modesto aumento della produzione cellulare di ROS da parte della QTA3BTPI (Biasutto et al., 2010 – Cap. 7).
Ho ampliato lo studio per valutare la possibile azione citotossica/citostatica su linee cellulari tumorali (C26) e non (MEF) dei vari composti mitocondriotropici. Ho anche indagato come tali composti influiscano sull’attività mitocondriale di un’altra linea cellulare tumorale (Jurkat). I risultati di esperimenti di vitalità cellulare mostrano come diversi composti mitocondriotropici, somministrati a concentrazioni in ambito micromolare, possano aver un’ attività citotossica/citostatica su cellule a crescita veloce, mentre il loro effetto su cellule non tumorali è molto modesto se esse hanno crescita lenta. I risultati iniziali di uno studio meccanicistico hanno evidenziato come concentrazioni relativamente elevate (nell’ambito di 10-5 M) di questi composti possano causare alterazioni morfologiche, depolarizzazione mitocondriale e generazione di specie radicaliche (Cap. 8)
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Improving the efficacy of plant polyphenols.
No full textPlant polyphenols exhibit potentially useful effects in a wide variety of pathophysiological settings. They interact with proteins such as signalling kinases, transcription factors and ion channels, and modulate redox processes, such as those taking place in mitochondria. Biomedical applications of these natural compounds are however severely hindered by their low bioavailability, rapid metabolism, and often by unfavourable physico-chemical properties, e.g. a generally low water solubility. Derivatives are under development with the aim of improving their bioavailability and/or bioefficacy. Various strategies can be adopted. An increase in circulating blood levels of non-metabolized natural compound may be attainable through prodrugs. In the ideal prodrug, phenolic hydroxyls are protected by capping groups which a) help or at least do not hinder permeation of epithelia; b) prevent conjugative modifications during absorption and first-pass through the liver; c) are eliminated with opportune kinetics to regenerate the parent compound. Moreover, prodrugs may be designed with the goals of modulating physical properties of the parent compound, and/or changing its distribution in the body. A more specific action may be achieved by concentrating the compounds at specific sites of action. An example of the second approach is represented by mitochondria-targeted redox-active polyphenol derivatives, designed to intervene on radical processes in these organelles and as a tool either to protect cells from oxidative insults or to precipitate their death. Mitochondrial targeting can be achieved through conjugation with a triphenylphosphonium lipophilic cation. Quercetin and resveratrol were chosen as model polyphenols for these proof-of-concept studies. Data available at the moment show that both quercetin and resveratrol mitochondria-targeted derivatives are pro-oxidant and cytotoxic in vitro, selectively killing fast-growing and tumoural cells when supplied in the low μM range; the mechanism of ROS generation appears to differ between the two classes of compounds. These approaches are emerging as promising strategies to obtain new efficient chemopreventive and/or chemotherapeutic drugs based on polyphenols derivatives
Development of mitochondria-targeted derivatives of resveratrol
No full textTo target natural polyphenols to the subcellular site where their redox properties might be exploited at best, that is, mitochondria, we have synthesised new proof-of-principle derivatives by linking resveratrol (3,4′,5-trihydroxy-trans-stilbene) to the membrane-permeable lipophilic triphenylphosphonium cation. The new compounds, (4-triphenylphosphoniumbutyl)-4′-O-resveratrol iodide and its bis-acetylated derivative, the latter intended to provide transient protection against metabolic conjugation, accumulate into energized mitochondria as expected and are cytotoxic for fast-growing but not for slower-growing cells. They provide a powerful potential tool to intervene on mitochondrial and cellular redox processes of pathophysiological relevance
Intermediate conductance Ca2+-activated potassium channel (K(Ca)3.1) in the inner mitochondrial membrane of human colon cancer cells
No full textPatch-clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca(2+)-activated K(+)-selective channel K(Ca)3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca(2+)] will cause mtK(Ca)3.1 opening, thus linking inner membrane K(+) permeability and transmembrane potential to Ca(2+) signalling. (C) 2009 Elsevier Ltd. All rights reserved
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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