oncovirokiller

Cosa non si impara dai virus…

Partiamo dai meccanismi di invasione e replicazione virale. I virus, per potersi replicare, hanno bisogno dell’apparato cellulare. Le nostre cellule, d’altro canto, si sacrificano per salvare l’organismo quando siano in stato di stress o pericolo. Un gene fondamentale nei processi di tumorigenesi, il gene di inattivazione tumorale TP53, si mette in movimento ed attiva la proteina p53 ed i programmi di apoptosi (suicidio programmato) quando la cellula diventi pericolosa, o perché tumorale o perché infetta.

Gli adenovirus (responsabili per il comune raffreddore) sono in grado di inattivare la p53 comprimendo e rendendo illeggibile i suoi geni, e la stessa p53 è inattivata nella maggior parte delle cellule tumorali. Come piegare a nostro vantaggio questi fenomeni?

Un team di ricerca presso il Salk Institute for Biological Studies (qui un video, e qui la press release) ha pensato che selezionando un adenovirus a cui manca una proteina fondamentale per l’inattivazione preventiva di p53 (E1B-55K), esso non sarebbe capace di replicarsi nelle cellule normodotate di p53, ma solo in quelle nelle quali p53 è inattiva, ovvero le cellule tumorali. Se questo approccio avesse successo, si tratterebbe di una terapia oncolitica molto promettente e mirata.

Quello che è successo poi è molto istruttivo. Nonostante l’esperimento non sia stato un successo, questo insuccesso ha permesso di comprendere meglio i meccanismi di inattivazione di p53. E’ quindi un caso di scuola nel quale l’insuccesso di ottenere l’effetto B agendo sul supposto agente causale semplice A permette di rendere visibile la complessità di A, di esplicitarne le articolazioni.

Gli adenovirus mancanti di E1B-55K non hanno impedito a p53 di attivarsi, ma la cellula non ha comunque attivato il processo di apoptosi. Le indagini per capire le ragioni dell’insuccesso hanno rivelato che gli adenovirus si difendono dall’apoptpsi  attraverso meccanismi multipli (almeno una seconda proteina, E4-ORF3, è implicata). Eliminare E1B-55K aveva eliminato solo la prima componente dell’agente causale. Quello che si spera è che una volta meglio compresa la complessità dei meccanismi causali sia possibile utilizzare queste conoscenze per una terapia oncolitica promettente.

A novel mechanism used by adenovirus to sidestep the cell’s suicide program, could go a long way to explain how tumor suppressor genes are silenced in tumor cells and pave the way for a new type of targeted cancer therapy, report researchers at the Salk Institute for Biological Studies in the Aug. 26, 2010 issue of Nature.

When a cell is under stress, the tumor suppressor p53 springs into action activating an army of foot soldiers that initiate a built-in “auto-destruct” mechanism that eliminates virus-infected or otherwise abnormal cells from the body. Just like tumor cells, adenoviruses, which cause upper-respiratory infections, need to get p53 out of the way to multiply successfully.

“Instead of inactivating p53 directly, adenovirus renders the ‘guardian of the genome’ powerless by targeting the genome itself,” explains Clodagh O’Shea, Ph.D., an assistant professor in the Molecular and Cell Biology Laboratory, who led the study. “It literally creates zip files of p53 target genes by compressing them till they can no longer be read.”

The p53 tumor suppressor pathway is inactivated in almost every human cancer, allowing cells to escape normal growth controls. Yet there is still no rationally designed targeted cancer therapy to treat patients based on the loss of p53.

“All of the targeted therapies we have are based on small molecules that inactivate oncogenes, but cancer is not solely caused by the gain of growth-promoting genes,” says O’Shea. “The loss of tumor suppressors is just as important. The big question is how do you target something that’s no longer there?”

Adenovirus seemed to provide the answer. It brings along a viral protein, E1B-55K, which binds and degrades p53 in infected cells. Without E1B-55K to inactivate p53, adenovirus should only be able to replicate in p53-deficient tumor cells. Then, each time it bursts open the host cell to release thousands of viral progenies, the next generation of viruses is ready to seek out remaining cancer cells while leaving normal cells unharmed.

“This makes adenovirus a perfect candidate for oncolytic cancer therapy,” says O’Shea. “Although these viruses did their job, to everybody’s surprise, the patients’ responses did not correlate with the p53 status of their tumors,” says O’Shea. Intrigued, she and her team followed up on this unexpected finding.

Conrado Soria, Ph.D., a research assistant and co-first author of the study, quickly realized that E1B-55K was only half of the story. “The inability of the E1B-55K-mutant virus to replicate in normal cells was not because the virus failed to degrade p53,” he explains.

In unstressed normal cells, p53 is only found at low levels due to rapid degradation. In response to DNA damage, the activation of oncogenes or infection by DNA viruses, p53 degradation is halted and as a result p53 protein levels accumulate. This increase activates p53 target genes, which arrest the cell cycle or induce apoptosis.

Just as predicted, p53 started to build up in normal cells that had been infected with adenovirus lacking E1B-55K but it was still unable to turn on its target genes and start the cell on the path to apoptosis. He eventually discovered why: Adenovirus brings along another protein, E4-ORF3, which neutralizes the p53 checkpoint through a completely different mechanism.

Instead of inactivating p53 directly, the tiny protein prevents the tumor suppressor from binding to its target genes in the genome by modifying chromatin, the dense histone/DNA complex that keeps everything neatly organized within the cells’ nucleus. “These modifications cause parts of chromosomes to condense into so-called heterochromatin, burying the regulatory regions of p53 target genes deep within,” says graduate student and co-first author Fanny E. Estermann. “With access denied, p53 is powerless to pull the trigger on apoptosis.”

O’Shea hopes to exploit these new insights to understand how high levels of wild type p53 might be inactivated in cancer as well as the mechanisms that induce aberrant silencing of tumor suppressor gene loci in cancer cells. “Our study really changes the longstanding definition of how p53 is inactivated in adenovirus-infected cells and will finally allow us to develop true p53 tumor selective oncolytic therapies.”

Tidbits: un gusto repellente

Un altro tassello del mosaico complesso che descrive il funzionamento ed il ruolo dei sensi chimici. Naturalmente si parla di recettori TRP (Transient Receptor Potential) descritti altre volte (qui uno recente) come importanti per la traduzione di segnali chimici alimentari in effetti fisiologici.

In due studi (uno pubblicato su Neuron ed il secondo su Current Biology) il team di Craig Montell, ha testato due repellenti per insetti (DEET e citronellale, una aldeide presente in mote spp. di Cymbopogon). In entrambi i casi la repellenza è fortemente correlata con la presenza e funzionalità dei canali TRP (ed altri), responsabili per la percezione gustativa (DEET) e olfattiva (DEET e citronellale) delle sostanze. Fondamentalmente la repellenza sarebbe una vera e propria reazione di disgusto verso il sapore e l’odore delle sostanze testate.

Three taste receptors on the insects’ tongue and elsewhere are needed to detect DEET. Citronellal detection is enabled by pore-like proteins known as TRP (pronounced “trip”) channels. When these molecular receptors are activated by exposure to DEET or citronellal, they send chemical messages to the insect brain, resulting in “an aversion response,” the researchers report.

“DEET has low potency and is not as long-lasting as desired, so finding the molecules in insects that detect repellents opens the door to identifying more effective repellents for combating insect-borne disease,” says Craig Montell, Ph.D., a professor of biological chemistry and member of Johns Hopkins’ Center for Sensory Biology.

Scientists have long known that insects could smell DEET, Montell notes, but the new study showing taste molecules also are involved suggests that the repellant deters biting and feeding because it activates taste cells that are present on the insect’s tongue, legs and wing margins.

“When a mosquito lands, it tastes your skin with its gustatory receptors, before it bites,” Montell explains. “We think that one of the reasons DEET is relatively effective is that it causes avoidance responses not only through the sense of smell but also through the sense of taste. That’s pretty important because even if a mosquito lands on you, there’s a chance it won’t bite.”

The Johns Hopkins study of the repellants, conducted on fruit flies because they are genetically easier to manipulate than mosquitoes, began with a “food choice assay.”

The team filled feeding plates with high and low concentrations of color-coded sugar water (red and blue dyes added to the sugar), allowing the flies to feed at will and taking note of what they ate by the color of their stomachs: red, blue or purple (a combination of red and blue). Wild-type (normal) flies preferred the more sugary water to the less sugary water in the absence of DEET. When various concentrations of DEET were mixed in with the more sugary water, the flies preferred the less sugary water, almost always avoiding the DEET-laced sugar water.

Flies that were genetically engineered to have abnormalities in three different taste receptors showed no aversion to the DEET-infused sugar water, indicating the receptors were necessary to detect DEET.

“We found that the insects were exquisitely sensitive to even tiny concentrations of DEET through the sense of taste,” Montell reports. “Levels of DEET as low as five hundredths of a percent reduced feeding behavior.”

To add to the evidence that three taste receptors (Gr66a, Gr33a and Gr32a) are required for DEET detection, the team attached recording electrodes to tiny taste hairs (sensilla) on the fly tongue and measured the taste-induced spikes of electrical activity resulting from nerve cells responding to DEET. Consistent with the feeding studies, DEET-induced activity was profoundly reduced in flies with abnormal or mutated versions of Gr66a, Gr33a, and Gr32a.

In the second study, Montell and colleagues focused on the repellent citronellal. To measure repulsion to the vapors it emits, they applied the botanical compound to the inside bottom of one of the two connected test tubes, and introduced about 100 flies into the tubes. After a while, the team counted the flies in the two tubes. As expected, the flies avoided citronellal.

The researchers identified two distinct types of cell surface channels that are required in olfactory neurons for avoiding citronellal vapor. The channels let calcium and other small, charged molecules into cells in response to citronellal. One type of channel, called Or83b, was known to be required for avoiding DEET. The second type is a TRP channel.

The team tested flies with mutated versions of 11 different insect TRP channels. The responses of 10 were indistinguishable from wild-type flies. However, the repellent reaction to citronellal was reduced greatly in flies lacking TRPA1. Loss of either Or83b or TRPA1 resulted in avoidance of citronellal vapor.

The team then “mosquito-ized” the fruit flies by putting into them the gene that makes the mosquito TRP channel (TRPA1) and found that the mosquito TRPA1 substituted for the fly TRPA1.

“We found that the mosquito-version of TRPA1 was directly activated by citronellal,” says Montell who discovered TRP channels in 1989 in the eyes of fruit flies and later in humans.

Montell’s lab and others have tallied 28 TRP channels in mammals and 13 in flies, broadening understanding about how animals detect a broad range of sensory stimuli, including smells and tastes.

“This discovery now raises the possibility of using TRP channels to find better insect repellants.”

There is a clear need for improved repellants, Montell says. DEET is not very potent or long-lasting except at very high concentrations, and it cannot be used in conjunction with certain types of fabrics. Additionally, some types of mosquitoes that transmit disease are not repelled effectively by DEET. Citronellal, despite being pleasant-smelling (for humans, anyway), causes a rash when it comes into contact with skin.

Corso di Erboristeria Popolare

Ricevo e con piacere pubblico il programma del corso in Erboristeria Popolare organizato e tenuto dalla Dott.ssa Ilde Piccioli, autrice tra l’altro di un bel post sulle donne nella medicina antica.

Il corso è triennale, con frequenza mensile (una domenica al mese dalle 9 alle 18) da ottobre a giugno, e si terrà a Massa. Per maggiori informazioni rivolgetevi direttamente alla Dott. Piccioli, alla mail dafne1955@libero.it

L’erboristeria è l’antica arte della conoscenza delle piante, della loro coltivazione, raccolta e conservazione a scopi terapeutici e cosmetici. Era praticata maggiormente dalle donne, che coltivavano spezie ed erbe medicinali nei loro orti o raccoglievano piante spontanee. Le usavano fresche o le conservavano seccandole o facendo delle semplici estrazioni con vino o grappa.

Programma didattico

  • Storia e tradizioni.
  • Sistemi medici tradizionali.
  • Fisionomica e teoria delle segnature, guaritori, sciamani e streghe.
  • Orti botanici ed erbari.
  • Piante medicinali, piante officinali, piante arboree.
  • Principi attivi e fitocomplesso, fitochimica classi di composti, metaboliti primari e metaboliti secondari.
  • Preparati da piante officinali, tecniche estrattive, estratti e macerati, tinture, fitogemmoterapia.
  • Forme estrattive per uso esterno, formulazioni cosmetiche.
  • Estratti secchi, molli e fluidi.
  • Principi generali di erboristeria, tisane, infusi e decotti, apparati, organi e loro funzioni.
  • Menopausa.
  • Ansia e depressione.
  • Patologie da raffreddamento.
  • Piante esotiche e piante autoctone.
  • Olismo e visione d’insieme dell’uomo.
  • Alimentazione e salute.
  • Cibo come medicina, fitoalimurgia, le spezie alimenti e farmaci.
  • Piante tossiche.
  • Preparazione erbari secche.
  • Preparazione semplici rimedi.
  • Raccolta erbe mangerecce.
  • Schede monografiche delle piante maggiormente significative.

Finalità

La scuola si propone di fornire le conoscenze di base del mondo vegetale, con particolare

riferimento alle piante officinali ed alimentari spontanee usate nella tradizione anche locale.

  1. Si forniranno le conoscenze dal punto di vista botanico, storico, chimico ed erboristico, tenendo conto anche delle varie interpretazioni popolari (etnobotanica).
  2. Si acquisiranno competenze circa il riconoscimento di specie vegetali di interesse erboristico ed alimentare, supportate anche da uscite in località di interesse naturalistico.
  3. Verranno studiate anche le essenze arboree, con particolare attenzione a quelle di interesse fitogemmoterapico.
  4. Si forniranno conoscenze di base per la preparazione di tinture, macerati, unguenti ecc. dalle piante raccolte, in modo da poter essere utilizzate per uso personale.
  5. Si stimoleranno i partecipanti a creare piccoli “orti medicinali” e a coltivare le piante che andranno poi ad utilizzare: saranno fornite nozioni di progettazione e coltivazione di piccoli spazi verdi (giardini, terrazzi e balconi)

Docenti: dott. Ilde Piccioli, e altri

Attestato rilasciato: alla fine del triennio verrà rilasciato attestato di frequenza

Costi: 650€ per ogni annualità, pagabili in due soluzioni, 350€ all’iscrizione la restante quota a febbraio. Per pagamento all’iscrizione sconto del 10%

Tidbits: eugenolo

In un ancora più recente articolo su Applied and Environmental Microbiology, si esamina l’effetto antibatterico dell’eugenolo. E’ da molto tempo noto che questo composto (presente nell’olio essenziale di chiodi di garofano, di foglia di cannella, di alcune varietà di basilico, e in molte altre piante) mostra attività antibatterica diretta, come molti dei derivati volatili del percorso dell’acido shichimico, attraverso l’interazione con la membrana e la sua destabilizzazione o lisi. In questo articolo si osserva che anche concentrazioni al di sotto del livello inibitorio agiscono sui processi patologici associati alle infezioni batteriche. In particolare l’eugenolo sembra in grado di ridurre l’espressione di varie esoproteine (due enterotossine, SEA e SEB, e la toxic shock syndrome toxin 1) mediante azione a livello dell’espressiione genica.

Eugenol, an essential oil component in plants, has been demonstrated to possess activity against both Gram-positive and Gram-negative bacteria. This study examined the influence that subinhibitory concentrations of eugenol may have on the expression of the major exotoxins produced by Staphylococcus aureus. The results from a tumor necrosis factor (TNF) release assay and a hemolysin assay indicated that S. aureus cultured with graded subinhibitory concentrations of eugenol (16 to 128 µg/ml) dose dependently decreased the TNF-inducing and hemolytic activities of culture supernatants. Western blot analysis showed that eugenol significantly reduced the production of staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1 (the key exotoxins to induce TNF release), as well as the expression of {alpha}-hemolysin (the major hemolysin to cause hemolysis). In addition, this suppression was also evaluated at the transcriptional level via real-time reverse transcription (RT)-PCR analysis. The transcriptional analysis indicated that 128 µg/ml of eugenol remarkably repressed the transcription of the S. aureus sea, seb, tst, and hla genes. According to these results, eugenol has the potential to be rationally applied on food products as a novel food antimicrobial agent both to inhibit the growth of bacteria and to suppress the production of exotoxins by S. aureus.

Tidbits: Vinca

Qualche breve nota su alcuni articoli sbirciati in rete:

Un articolo appena pubblicato su PNAS illustra i meccanismi di sintesi, secrezione, compartimentazione ed escrezione degli alcaloidi della Vinca (Catharanthus roseus), e descrive gli effetti che questi meccanismi hanno sulla facilità (o meno) di ottenere farmaci per il trattamento dei tumori. Infatti, mentre i farmaci derivano dall’accoppiamento dei due alcaloidi catarantina e vindolina, i percorsi metabolici ed i meccanismi di secrezioni nella pianta risultano in una compartimentazione stretta che impedisce l’accoppiamento delle due molecole nella pianta. Una di esse, la catarantina, che mostra attività antifungina ed insettorepellente, viene secreta infatti nella cera cuticolare della foglia (dove queste attività hanno più senso), mentre la vindolina è presente esclusivamente all’interno delle cellule della foglia.

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cell-specific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity, provide an additional biological role for its secretion. We anticipate that this discovery will trigger a broad search for plants that secrete alkaloids, the biological mechanisms involved in their secretion to the plant surface, and the ecological roles played by them.