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Quavo’s Stellar Stra Group

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Excision - The Paradox !!BETTER!!

Expression of suppressor genes 53 and bcl-2 as well as of protein p21 (partly induced by p53 gene) was analyzed in a group of 77 resection specimens and bronchial excision of lung carcinomas (of all basic histological types--squamous cell, neuroendocrine, adenocarcinoma, undifferentiated). Simultaneously the relation of tumor immunophenotype and level of differentiation, cell death and 2-year-survival of patients was evaluated. Gene p53 showed non-only an expected strong expression in squamous cell carcinomas but especially in adenocarcinomas, which were newly characterized by exceptional hyper-expression of p53 in lowly differentiated variants. Expression level of protein p21 and gene p53 was parallel only in adenocarcinomas and undifferentiated carcinomas but discordant in squamous cell and neuroendocrine carcinomas. Positivity of p21 slightly prevailed in well-differentiated variants of the histological types but an exceptional positivity was found even in all the undifferentiated carcinomas. Gene bcl-2 revealed a paradox of strong expression in lowly differentiated neuroendocrine and undifferentiated carcinomas. The level of bcl-2 expression in squamous cell carcinomas was found higher than in references. Among tumors with cell death there was an inverted relation of bcl-2 and p53 expression (high/low) in neuroendocrine carcinomas but both of them were mostly negative in squamous cell carcinomas. A more frequent 2-year-survival of squamous cell carcinomas was verified for bcl-2 positive tumors and newly for p53 positive squamous cell carcinomas. Evaluation of the expression of p53, p21 and bcl-2 in lung carcinomas is so equivocal that its prognostic usage was found to be only complementary to the direct immunohistochemical investigation of the growth activities.

Excision - The Paradox


Maintenance of genomic integrity is fundamental to life. DNA damage occurs spontaneously and ubiquitously from endogenous (e.g., reactive oxygen species) and environmental sources (e.g., ultraviolet (UV) light), inflicting mutagenic and cytotoxic lesions upon the genome that drive the progression of cancer and aging. Cellular excision repair (ER) pathways, including base excision repair (BER) and nucleotide excision repair (NER), are a critical 'first line of defense' responsible for recognizing and removing DNA lesions. The overall objective of this proposal is to understand how ER pathways access DNA lesions that are 'buried' in different types of genomic chromatin. Previous studies have shown that histone post-translational modifications (PTMs) and ATP-dependent chromatin remodelers (ACR) are important for ER of DNA lesions in chromatin. However, it is not known how these chromatin remodeling activities and histone PTMs operate during repair on the diverse spectrum of distinct chromatin types in eukaryotic cells. To address this question, we have developed genome-wide methods to map the formation and repair of UV-induced cyclobutane pyrimidine dimers (CPDs) and methyl methanesulfonate (MMS)-induced N-methylpurine (NMP) base lesions. Our published study and preliminary data indicate that the CPD-seq and NMP-seq methods can be used to map DNA lesions across the yeast and human genomes at single nucleotide resolution. To better understand the genomic roles of ACRs in ER, we will use the CPD-seq and NMP-seq methods to measure repair in yeast and human cells depleted of different classes of ACRs (Aim I). Histone acetylation is an important PTM associated with DNA damage responses. Our preliminary data suggest that the Esa1/TIP60 histone acetyltransferase (HAT) complex plays a novel role in ER. To test this hypothesis, we will characterize the roles of Esa1/TIP60 and other HATs in regulating ER in both yeast and human cells (Aim II). Our preliminary data indicate that histone acetylation activity in cell-free repair extracts is important for repair of base lesions occluded in nucleosomes. These findings provide the foundation of Aim III, which will identify histone PTMs associated with BER, and characterize their functional role in BER of nucleosomes. Finally, we will investigate the detailed molecular mechanisms by which histone acetylation and ACRs regulate the activity of purified BER enzymes on mononucleosome and oligonucleosome substrates in vitro containing 'designed' DNA base lesions (Aim IV). This proposal is an ongoing investigation of the effects of DNA packaging in chromatin on the two major ER pathways (NER and BER) found in cells. As all eukaryotes, including humans, must deal with this `packaging paradox' for surveillance of the genome, results from these studies are relevant to the broad spectrum of cancer etiology, prevention and treatment.

According to a 2002 study by the National Research Council, thegreatest science-based concern facing animal biotechnology is the ecologicaland environmental impact from the escape or release of transgenic animals.Transgenic fish and insects were of high concern given their ability toescape, disperse, and become feral. Current physical and biological methodsof containment for domestically raised fish, such as net pens/sea cages andtriploidy-induced sterility, are inadequate for the 100% containment requiredof transgenic fish. Recently, genetic containment approaches that utilizegerm-cell-driven site-specific recombinases, such as the Cre/loxP system, toexcise transgenes from the germline have been described. However, the paradoxof these approaches is that while the transgenic gene of interest wasremoved, the recombinase genes themselves, which are also transgenes, werenot excised. We designed and tested a unique genetic containment approachthat utilized two germ-cell-driven site-specific recombination systems thatexcised not only the transgenic gene of interest, but also the recombinasetransgenes themselves, leaving behind two loxP footprints in the germline oftransgenic fish. Our approach utilized two lines of transgenic zebrafish, amale line and a female line. In the male line, a germ-cell-specific promoterdrives flpe recombinase, flanked by loxP sites. In the female line, the geneof interest and its promoter are flanked by FRT sites and situated betweenthe germ-cell-specific promoter and the cre recombinase coding sequence suchthat cre expression is blocked. This entire construct is also flanked by loxPsites. We theorized that when these male and female lines were crossed, theprogeny would express FLPe in germ cells and trigger excision of theFRT-flanked gene of interest. This, in turn, would bring the cre recombinasegene downstream of the germ-cell-specific promoter and its expression wouldcause excision of the loxP-flanked male line construct as well as its ownself-excision from the germ cells. Using this approach, it was anticipatedthat if these fish were outcrossed to wild-type fish, the gametes would notcarry any coding transgene and that only two loxP sites would be transmittedto the F 2 generation. flpe RNA microinjections into embryos containing thefemale line construct produced a high proportion of EGFP-progeny, were foundto express cre transcript in gonads, and produced Cre-mediated excisionevents in sperm samples. However, when flpe transcript was providedendogenously in the form of a cross with a transgenic male line constructfish, we did not observe excision of the floxed EGFP cassette in F 1 progenyas we did in the flpe RNA microinjections. When F 1 progeny were outcrossedto wild-type fish, we observed a 1:1 ratio of EGFP+ to EGFP- embryos,suggesting that germline excision by FLPe had not been achieved. A PCR screenon F 2 progeny revealed only two out of 136 fish that were positive for aCre-mediated excision event. The low efficiency of germline transgeneexcision could have been affected by several factors, including prematureself-excision of Cre such that not all floxed sequences were removed,multiple insertions of the female line construct, and poor Cre recombinationefficiency related to low temperature. 041b061a72

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