1,720,971 research outputs found
The role of p73 isoforms on DNA methylation in cancer cells
Get PDFUloga gena p73 u tumorigenezi nije u potpunosti razjašnjena. Korištenjem promotora P1 nastaju izoforme sa cjelovitim transaktivacijskim područjem (TAp73), odnosno korištenjem promotora P2 izoforme bez te regije (ΔNp73). Najčešća promjena u tumorima je povećana ekspresija izoforme ΔNp73, koja djeluje kao transdominantni inhibitor TAp73 i p53. Metiliranost DNA je oblik reverzibilne epigenetičke regulacije, kojim se nakon replikacije dodaju metilne skupine na citozinske ostatke DNA, mijenjajući aktivnost staničnih gena. Hipermetiliranost otoka CpG u promotorskim regijama pojedinih gena inhibira transkripciju utištavajući gene. Koristeći inducibilni sustav tet-on ispitali smo utjecaj pojačane ekspresije TAp73α i ΔNp73α na globalnu metiliranost DNA, no nismo pronašli statistički značajnu promjenu metiliranosti uzoraka s aktiviranim izoformama p73 u odnosu na njihove kontrole. Rezultate smo usporedili s rezultatima genske ekspresije nakon aktivacije ΔNp73α i TAp73α iz literature. Za gene koji pokazuju pojačanu ekspresiju našli smo, premda statistički ne značajno, smanjenje metiliranosti promotorskih CpG otoka, što potvrđuje činjenicu da metiliranje može utjecati na regulaciju aktivnosti gena.The role of p73 in tumorigenesis is not fully understood. Using P1 promoter transactivating (TAp73) isoforms are generated and P2 promoter the isoforms without this region (ΔNp73). The most common change in tumors is increased expression of ΔNp73, which acts as transdominant inhibitor of TAp73 and p53. DNA methylation is a form of epigenetic regulation, which adds methyl groups to cytosine residues, regulating gene activity. Hypermethylation of CpG islands in promoter regions inhibits transcription of genes silencing its expression. Using inducible tet-on system, we examined the impact of increased expression of TAp73α and ΔNp73α on global DNA methylation. However statistically significant change was not found upon induced expression of p73 isoforms. The results were compared with gene expression data obtained after induction of TAp73α or ΔNp73α from literature. For genes with increased expression we found, although not statistically significant, reduction of methylated CpG sites in promoter regions, which confirms the fact that methylation could affect the regulation of gene activity
Transcriptional activity of p53 isoforms
No full textProtein p53 kao tumor supresorima središnju ulogu u kontroli staničnog ciklusa i tumorigeneze. Nedavno je otkriveno da gen p53 kodira 9 izoformi (p53, p53 β , p53 γ , 133p53, 133p53 β , 133p53 γ , 40p53, 40p53 β i 40p53 γ ) nastalih alternativnim izrezivanjem ili prepisivanjem s alternativnog promotora u intronu 4. Ekspresija izoformi tkivno je specifična, a biološka funkcija im nije u potpunosti razjašnjena. Gen p53 ima dva srodnika velike strukturne i funkcionalne sličnosti, p63 i p73 . Spomenuti geni također imaju određen broj izoformi, koje djelovanje proteina p53 čine još složenijim. Testom luciferaze istražena je transkripcijska aktivnost izoformi p73 β , p53, p53 β , p53 γ , 133p53, 133p53 β , 133p53 γ i 40p53 transfeciranih u stanice H1299, koje nemaju eksprimiran protein p53 divljeg tipa. Dokazana je transkripcijska aktivnost svih istraživanih izoformi. Izoforme p73 β i p53 β pokazuju najveću transkripcijsku aktivnost. Izoforme p53 γ i 40p53 pokazuju značajnu razinu transkripcijske aktivnosti, dok izoforme 133p53, 133p53 β i 133p53 γ imaju izrazito nisku razinu transkripcijske aktivnosti. Metodom Western blot dokazana je ekspresija svih istraživanih izoformi na razini proteina.Protein p53 as a tumor suppressor plays a central role in the cell cycle control and tumorigenesis. It was recently discovered that the gene p53 encodes nine different protein isoforms (p53, p53β, p53γ, 133p53, 133p53β, 133p53γ, 40p53, 40p53β and 40p53γ) due to alternative splicing and usage of the alternative promoter in intron 4. The expression of isoforms is tissue-specific, and their biological function is not fully understood. p53 gene has two relatives of major structural and functional similarities, p63 and p73. They also have a number of isoforms, which make p53 activity more complex. Luciferase assay was used to detect transcriptional activity of isoforms, p73β, p53, p53β, p53γ, 133p53, 133p53β, 133p53γ and 40p53 that were trasfected into H1299 cells, which lack expression of wildtype p53 protein. Transcriptional activity of each isoform was detected. p73β and p53β isoforms show the greatest degree of ranscriptional activity, compared to full-length p53. p53γ and 40p53 show a significant level of transcriptional activity, whereas 133p53, 133p53β and 133p53γ have extremely low levels of transcription activity. Western blot method has shown the expression of all isoforms at the protein level analysis
Complex formation between mutant p53 and protein p63 in human tumor cell lines
No full textTumor supresorski gen TP53 mutiran je u preko 50% tumora čovjeka. Nakon mutacije može doći do gubitka funkcije p53 u stanici, ali i do promjene funkcije p53, koji može pokazivati onkogena svojstva. Neka onkogena svojstva mutiranih p53 mogu se objasniti interakcijom s drugim članovima obitelji p53, posebice s tumor supresorskim izoformama, TAp63 i TAp73. Na jačinu tih interakcija utječe polimorfizam na kodonu 72 gena TP53. Plazmidni vektori s ugrađenim genom za TAp63α, odnosno s genom za mutirani p53 koji imaju različit polimorfizam na kodonu 72 kotransfecirani su u stanice metastaza karcinoma pluća čovjeka (H1299) koje imaju djelomičnu deleciju gena TP53. Proteinski kompleksi su izolirani iz staničnih lizata metodom ko-imunoprecipitacije i analizirani metodom western blotting. Kompleksi su utvrđeni za mutirane oblike p53 L194F i R282W, te nisu utvrđeni za R175H, R280K i I332A. Ustanovljeno je da se mut p53 72R jače veže za TAp63α nego mut p53 72P.The tumor suppressor gene TP53 is mutated in over 50% of reported human tumor cases. The outcomes of the mutation are either loss of p53 function or gain of function resulting in oncogenic phenotype. Some of the mutant p53 oncogenic features are explained through interactions with other p53 family members, specifically tumor suppressor isoforms TAp63
and TAp73. A common polymorphism on codon 72 affects the strength of these interactions. Expression vectors coding for TAp63α or mutant p53 with different codon 72 polymorphism were cotransfected into H1299 cells, derived from human lung carcinoma metastasis, which have a partial deletion of the TP53 gene. Protein complexes were isolated from cell lysates using the co-immunoprecipitation method, and analysed by western blotting. Complexes have been found between TAp63α and p53 mutants L194F and R282W, but not between TAp63α and R175H, R280K and I332A. It was established that mut p53 72R binds more efficiently to TAp63α
Interactions between protein Itch, p63/p73 and mutated p53 in tumor cells
Get PDFTumor-supresorski gen TP53 mutiran je u više od 50% tumora čovjeka. Zbog promjena u konformaciji, mutirani protein p53mut veže se za p53 divljeg tipa te p63 i p73, članove porodice p53 i tako ih inaktivira. Nisku razinu proteina p63 i p73 u stanicama održava ubikvitinska ligaza ITCH koja omogućuje njihovu razgradnju putem proteasoma. Metodom koimunoprecipitacije i analizom metodom Western blot određivala sam interakcije između proteina ITCH, p63/p73 i p53mut u tumorskim stanicama. Potvrdila sam da dolazi do stvaranja interakcija između egzogenog ITCH (divljeg tipa i ITCHmut) i izoformi TAp63α, ΔNp63α, TAp73β i ΔNp73α u stanicama koje ne eksprimiraju p53 kao i u stanicama koje eksprimiraju p53mut (p53R248W). Plazmidne vektore s ugrađenim genima za TAp63α, ΔNp63α, TAp73β i ΔNp73α kotransfecirala sam s vektorom za ITCHmut u stanice koje eksprimiraju p53R248W kako bih utvrdila utječe li ubikvitinska ligaza ITCH na stvaranje heterokompleksa između p53mut i drugih članova porodice p53. Pokazala sam da ITCHmut ne utječe na stvaranje heterokompleksa između p53mut i izoformi TAp63α, ΔNp63α i TAp73β, dok je intenzitet vezanja za ΔNp73α bio jači u prisutnosti ITCHmut. Iako je ITCH glavni regulator razine p63 i p73 u stanicama, moguće je da ima zanemarivo djelovanje na interakcije između p53mut i p63/p73.Tumor suppressor gene TP53 is mutated in over 50% of human tumors. Altered conformation enables mutated p53, p53mut, to form heterocomplex with wild type p53 as well as with p63 and p73, the members of p53 family, making them inactive. Low levels of p63 and p73 are maintained by ubiquitin ligase ITCH and subsequent proteasomal degradation. Performing coimmunoprecipitation assays followed by Western blot analysis I have determined protein interactions between ITCH, p63/p73 and p53mut in tumor cells. I confirmed the interactions between exogenous protein ligase ITCH (both wild type and functional mutant) and TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms in p53 null and p53 mutant cell line (p53R248W). Expression vectors encoding for TAp63α, ΔNp63α, TAp73β and ΔNp73α isoforms were cotransfected with mutated protein ITCHmut into the cells expressing p53R248W in order to analyze the effect of ubiquitin ligase ITCH on interactions between p53mut and p63/p73. I have shown that ITCHmut does not affect heterocomplex formation between p53mut and TAp63α, ΔNp63α and TAp73β, while ΔNp73α forms stronger interactions when ITCHmut was present. Although ubiquitin ligase ITCH is the main regulator of p63/p73 protein levels in cells, it has negligible effect on p53mut and p63/p73 interactions
The expression of p53 and p73 isoforms in human soft tissue sarcoma cell lines
No full textSarkomi mekih tkiva su zloćudni tumori mezenhimalnog porijekla. Mehanizam tumorigeneze sarkoma mekih tkiva još uvijek je neobjašnjen. Mutacije u genu TP53 su najčešće genetske promjene u tumorima. Porodica p53 obuhvaća gene TP53, TP63 i TP73 koji reguliraju mnoge vitalne biološke procese, uključujući diferencijaciju stanica, proliferaciju i apoptozu. Svaki gen proizvodi proteinske izoforme kroz više mehanizama - alternativnim prekrajanjem, korištenjem alternativnih promotora te različitih mjesta početka translacije. Za razliku od p53, protein p73 prekomjerno je eksprimiran u različitim tumorima i rijetko mutiran. Cilj ovog diplomskog rada je odrediti gensku ekspresiju izoformi gena TP53 i TP73 te proteina p53 i p73 u staničnim linijama sarkoma mekih tkiva čovjeka. Rezultati su pokazali jaču gensku ekspresiju izoformi p53α i Δ133p53α, dok je analiza proteinske ekspresije pokazala višu razinu p53α i Δ40p53α u staničnim linijama u kojima je TP53 mutiran. U staničnim linijama u kojima TP53 nije bio mutiran detektirala sam slabu ekspresiju izoformi p53α, Δ133p53α i p53β. Izoforme gena TP73 slabo su eksprimirane u svim staničnim linijama, a jedino je protein TAp73α značajno eksprimiran u svim staničnim linijama.Soft tissue sarcomas are malignant tumors of mesenchymal origin. The tumorigenic mechanism of soft tissue sarcoma is still unexplained. Mutations in the TP53 gene are the most common genetic changes in tumors. The p53 family includes the TP53, TP63 and TP73 genes that regulate many vital biological processes including cell differentiation, proliferation, and apoptosis. Each gene produces protein isoforms through multiple mechanisms – alternative splicing, use of alternative promoters and alternative translation initiation sites. Unlike p53, protein p73 is rarely mutated but is overexpressed in various tumors. The aim of this graduate thesis is to determine the expression profile of the TP53 and TP73 genes and p53 and p73 proteins in human soft tissue sarcoma cell lines. The results showed stronger gene expression of p53α and Δ133p53α isoforms, while protein analysis revealed higher levels of p53α and Δ40p53α proteins in cell lines with mutated TP53. In TP53 mutant cell lines, I detected poor expression of isoforms p53α, Δ133p53α and p53β. TP73 gene isoforms were poorly expressed in all cell lines, and only TAp73α protein was significantly expressed in all cell lines
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Get PDFWe 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
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe 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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