{"id":25688,"date":"2007-09-01T00:00:00","date_gmt":"2007-09-01T00:00:00","guid":{"rendered":"http:\/\/revistapesquisaclone.fapesp.br\/2007\/09\/01\/the-cellular-postman\/"},"modified":"2015-09-10T13:52:23","modified_gmt":"2015-09-10T16:52:23","slug":"the-cellular-postman","status":"publish","type":"post","link":"https:\/\/revistapesquisa.fapesp.br\/en\/the-cellular-postman\/","title":{"rendered":"The cellular postman"},"content":{"rendered":"
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ALEXANDER AND IRINA KERKIS\/ BUTANTAN INSTITUTE<\/span>Direct to the nucleus: cell division in the lungs of mouse turns green on producing the fluorescent protein whose gene crotalin transported it ALEXANDER AND IRINA KERKIS\/ BUTANTAN INSTITUTE<\/span><\/p><\/div>\n

Skilled in finding talented people who he allows to work as they please, chemist Tetsuo Yamane formed a research team in Brazil, with ramifications in other countries that in just a couple of years has led to the identification of very rare properties in the protein of a snake typical of the Brazilian Cerrado <\/em>and Caatinga, <\/em>the rattlesnake. Crotalin <\/em>as this protein is called, traverses the cellular membrane and transports genes and other molecules to the interior and even into the nucleus of the cells \u2013 not just any cell, but\u00a0 those that are multiplying. For this reason, this protein may be used in the diagnostics of diseases, in conveying medication and, as the latest experiments seem to indicate, in destroying tumors.<\/p>\n

Yamane, currently 76, as\u00a0 head of the biotechnological laboratory at the Institute of Energy and Nuclear Research Institute (Ipen) at the Biotechnology Center in the Amazon Region (CBA), began to create new sources for the study of crotalin <\/em>\u00a0some time in 1993, when he was considering the idea of returning to Brazil, after 40 years\u00a0 in the United States. Isolated in the 1950s by the biochemist Jos\u00e9 Moura Gon\u00e7alves, crotalin <\/em>had already been thoroughly studied, due to its capacity of paralyzing the muscles of rodents. Forty years later, it did not seem to pose major enigmas \u2013 except for a chemist, of Japanese origin, whose boldness had been fed on day-to-day basis by his association with scientists at the level of Richard Feyman and Linus Pauling, during his graduation and post-graduation\u00a0 years at California\u2019s Institute of Technology (Caltech). His propensity to ask\u00a0 new questions perfected itself even more during his ten years working with\u00a0 physicists\u00a0 at the Bell Laboratories, where the transistor, the laser, the integrated circuit and communications by satellite were invented. On becoming acquainted with crotalin<\/em>, Yamane became intrigued about the mechanisms, at that time still undeciphered, by which the protein acts on the system and by the possible interactions of this molecule whose structure reminds one of a woollen dragon.<\/p>\n

Could crotalin <\/em>interfere with the division of cells? It was this question by Ymane, active since 1994 in the Butantan Institute and at that time working only with the biochemists Gandhi R\u00e1dis-Baptista and \u00c1lvaro Prieto da Silva, that drew the attention of the cellular biologist Alexander Kerkis and\u00a0 his wife, biologist. Both are Russian. They had been active in one of the most important Russian research centers in Siberia, before perestroika fragmented the scientific knowledge structure there. After spending some time at the State University of Northern Rio de Janeiro, the Kerkis came to S\u00e3o Paulo in 1999. They took up the study of mice stem cells at the Institute of Biomedical Sciences (ICB) at the University of S\u00e3o Paulo USP. It was then that they met Yamane and, jointly, discovered new properties in a toxin that seemed not to have anything further to yield. In very small doses, they verified, that crotalin<\/em> very quickly reached \u2013 in only five minutes \u2013 the nucleus not only the embryonic stem-cells of mice, but also\u00a0 other types of cells.<\/p>\n

For the first time it was demonstrated that a protein until then considered only as a toxin also acted as a cellular postman: it traversed the membrane of cells in the splitting up process and reached the nucleus, where the chromosomes are situated. Once there, this little protein adheres to the centromeres, the agent whereby the chromosomes are duplicated and remain united during the cellular division. Thereupon, the chromosomes separate themselves into independent cells; the crotalin<\/em> leaves the cells remaining in the intercellular space, as if waiting for another moment to become active once more. The results, published by the FASEB journal in July 2004, opened new prospects for research and for the use of\u00a0\u00a0 this protein. \u201cWe began to regard the toxin from a new perspective\u201d, states pharmacologist Mirian Hayashi, who worked with Yamane at the\u00a0 Butantan Institute for three years on this research project, subsequent to another three in the development of pharmaceutical products in Japan.<\/p>\n

Against pain and parasites
\n<\/strong>It was also at Butantan that another team found a substance in the rattlesnake with an analgesic power 600 times that of morphine and, apparently, without relevant side effects.. In an experiment carried out at USP, the poison of the Crotalus durissus terrificus<\/em>, which inhabits\u00a0 the south and west of Brazil, was the one to show itself the most effective against the parasite causing da leishmaniasis, in comparison with two other subspecies, one typical of the Caatinga<\/em> and the other of the south western and central western regions.. The fractions most active in tests within cells and mice were gyrotoxin <\/em>and crotalin<\/em>.<\/p>\n

Crotalin<\/em>, the major component of a rattlesnake\u2019s poison, is a small protein. It contains 42 amino acids, almost as many as insulin, the hormone of 51 amino acids that controls the\u00a0 sugar level in the blood. However, it is minuscule when compared, for example, with hemoglobin, the portentous molecule of four chains of 140 amino acids each, which conveys oxygen to all of the body\u2019s cells. Given that it is so small, it becomes understandable that it would easily traverse the membranes of the cells. But how?<\/p>\n

Here is the answer: by coupling itself to the molecules of the cellular surface known as proteoglycans of heparin-sulfates that circulate within the cell\u2019s interior. This coupling is not fortuitous. \u201cThe moment in which the cells most produce heparin-sulfates is during their reproductive cycle\u201d, states biochemist Ivarne Tersariol, professor of the Federal University of the State of S\u00e3o Paulo (Unifesp) and of the University of Mogi das Cruzes (UMC). As this group demonstrated in an article by the Journal of Biological Chemistry, the affinity between crotalin<\/em> and proteoglycan of heparin-sulfate molecules is due, essentially, to electrostatic forces: crotalin<\/em> is a molecule with a positive electrical charge, whereas the proteoglycan of heparin-sulfate molecules are negatively charged.<\/p>\n

It so happens that crotalin <\/em>is not entirely positive: one side is electrically neutral. This peculiarity serves the purpose of connecting other molecules both to the positive and neutral sides. However, this was still only theory, as was the hypothesis of crotalin<\/em>, given its positive electrical charge, connecting itself to the negative DNA. There were indications that this might be possible; however, crotalin <\/em>might couple itself, not directly to, but to some other protein associated with the DNA.<\/p>\n

To clear up the doubt, Mirian spoke with Vitor Oliveira. A chemist at the Federal University of S\u00e3o Carlos (UFSCar), he was working in projects relative to the analysis of protein structures in a private university. It was he who operated\u00a0 equipment at Unifesp that analyses how molecules absorb components of a special type of light. The answer became available within five minutes, by means of a graph: yes, crotalin <\/em>coupled itself directly to the DNA. Soon after, an experiment by the group showed that crotalin <\/em>might in fact convey a form of circular DNA known as plasmid to the nucleus of liver, lung and bone marrow cells, which find themselves under continuous multiplication. The alien DNA functioned normally, as if natural, within one of each four cells: a noteworthy result.<\/p>\n

There is also growing evidence that crotalin <\/em>might be connected to the system\u2019s defense \u2013 and not just because it is part of a poison. Its three-dimensional structure is similar to that of beta-defensine<\/em>, a protein found in the saliva and in the mucous (of the nose, for example) of human beings and of other animals. \u201cMolecules, such as these, are part of the first defense line of organisms\u201d, states Irina. In a manner similar to the beta-defensines<\/em>, crotalin <\/em>might integrate the innate immune system, which functions intensely during pregnancy and the first months after birth when the organism does not yet produce antibodies against microorganisms. Therefore, snakes also use something of themselves, albeit in a larger volume, to defend themselves: the poison gland is a modified salivary gland, states Mirian. However, not all Crotalus durissus terrificus <\/em>produce crotalin. <\/em>Gandhi R\u00e1dis-Baptista verified that all carry the gene responsible for the production of crotalin<\/em>; some of the representatives of this species \u2013 measuring up to 1.5 meters in length and easily identified by their tail rattles \u2013 produce a protein with a similar structure, called crotasin<\/em>,\u00a0 the effect of which is not yet known.<\/p>\n

R\u00e1dis-Baptista is now with the Federal University of Pernambuco, complying with what seems to be\u00a0 the fate of the participants of this story; not to put down roots or, at least, to\u00a0 go after even more challenging projects. Mirian left Butantan a year ago, soon after Yamane, and is now at Unifesp as a professor of pharmacology; Vitor Oliveira left the private university and returned to Unifesp as a professor of biophysics. Irina Kerkis left USP and is now at Butantan, whereas Alexander Kerkis now works in the lab of a medical clinic.\u00a0 F\u00e1bio Nascimento, the biochemist who carried out the heparin-sulfate experiments, is currently working in a biotechnological company in Switzerland<\/p>\n

Uncertainties ahead
\n<\/strong>Even if at a distance, apparently they did not lose enthusiasm to work together on problems they might not solve alone. Extracted and purified with dexterity by Eduardo Oliveira at USP in Ribeir\u00e3o Preto, crotalin <\/em>forms aggregates of two or three units that reduce its capacity to convey molecules. To avoid the formation of these aggregates is, perhaps, more difficult than to produce a synthetic version of crotalin<\/em>, which would avoid dependence on the purification of snake poison.<\/p>\n

Although they have a lot of work ahead of them, the researchers are already negotiating with companies interested in employing crotalin <\/em>sections as gene conveyers. In 2004, they requested the patent for the potential uses of this molecule, given that they do not wish to repeat the story of other molecules discovered by Brazilians, which, because they had not been patented nor relied on corporate support, ended up being taken over by other research groups. They are aware that being the owner of a patent is only one of the requisites in the long road\u00a0 to the development of a pharmaceutical product.<\/p>\n

Further ahead, should the next laboratory tests confirm the crotalin\u2019s <\/em>potential, the group shall be faced with another challenge: to produce the molecule in greater quantities \u2013 on a trial scale and, thereafter, on an industrial scale \u2013 so as to facilitate negotiations with companies or institutions interested in carrying out the final tests, prior to the molecule becoming a medication or a marker in cancer diagnoses. \u201cThose with good ideas are welcome\u201d, states Yamane. \u201cWe are an open group.\u201d Little by little, Yamane has already attracted other groups \u2013 from Germany. Poland, the United States and Japan \u2013 to work with crotalin<\/em>. \u201cEach one may contribute; only in this manner does science advance\u201d, he adds, without giving up the daring that has marked his scientific career: \u201cLinus Pauling always reminded one that, on beginning a project, one should think of how to contribute in an original manner\u201d.<\/p>\n

The project<\/strong>
\nStudy of the transportation system mediated by cationic peptides (n\u00ba\u00a003\/13225-9<\/a>);\u00a0Modality:\u00a0<\/strong>Regular Line of Support to Research; Coordinator:\u00a0<\/strong>Tetsuo Yamane \u2013 Ipen;\u00a0Investment:\u00a0<\/strong>R$ 65,649.56 (FAPESP)<\/p>\n","protected":false},"excerpt":{"rendered":"Protein from the rattlesnake’s poison shows potential to transport medication","protected":false},"author":17,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_exactmetrics_skip_tracking":false,"_exactmetrics_sitenote_active":false,"_exactmetrics_sitenote_note":"","_exactmetrics_sitenote_category":0,"footnotes":""},"categories":[159],"tags":[],"coauthors":[5968],"class_list":["post-25688","post","type-post","status-publish","format-standard","hentry","category-science"],"acf":[],"_links":{"self":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/25688","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/users\/17"}],"replies":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/comments?post=25688"}],"version-history":[{"count":0,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/25688\/revisions"}],"wp:attachment":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/media?parent=25688"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/categories?post=25688"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/tags?post=25688"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/coauthors?post=25688"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}