129. Zaburzenia liczby kopii genów erbB-1 i erbB-2 i ich związek z danymi klinicznymi u leczonych operacyjnie chorych na niedrobnokomórkowego raka płuca

Szlak sygnałowy nabłonkowego czynnika wzrostu (ang. epidermal growth factor, EGF) i jego receptory (najważniejsze z nich to erbB-1 i erbB-2) odgrywają ważną rolę w patogenezie i progresji raka płuca. Kliniczne znaczenie nadekspresji tych receptorów pozostaje nadal przedmiotem kontrowersji a dotychczasowe badania przynosiły sprzeczne wyniki. Z uwagi na opracowane ostatnio leki o wybiórczym działaniu przeciwnowotworowym, działające poprzez powyższe receptory, poznanie rokowniczego znaczenia tych zaburzeń może odegrać ważną rolę w ustalaniu optymalnego sposobu leczenia. Celem pracy była ocena rokowniczego znaczenia anomalii średniej liczby kopii genów (ang. avarage gene copy numer, AGCN) u chorych na niedrobnokomórkowego raka płuca poddanych resekcji miąższu płucnego.Materiał i metodyDo badania włączono 112 chorych leczonych operacyjnie w latach 1996–1999. U 61 chorych (55%) rozpoznano postać raka płaskonabłonkowego, u 30 (27%) gruczolakoraka, u 10 (9%) raka wielokomórkowego i u 10 (9%) chorych postacie mieszane. Stopień zaawansowania nowotworu kształtował się następująco: I=54 (48%), II=11 (10%), III=40 (36%), IV=7 (6%). Materiał do analizy molekularnej stanowiło DNA izolowane ze świeżo mrożonych wycinków guza pobranych podczas zabiegu operacyjnego. AGCN określano za pomocą podwójnie różnicowej reakcji polimerazy i elektroforezy agarozowej.WynikiŚrednia wartość AGCN dla erbB-1 wynosiła 2,0 (zakres od 0,01 do 28,07), a dla erbB-2 1,34 (zakres od 0,11 do 9,9). Stwierdzono znamienną zależność pomiędzy AGCN dla obu genów. Nie stwierdzono znamiennego związku z jakimkolwiek głównym parametrem klinicznym. Do czasu przeprowadzenia analizy zmarło 73 chorych (65%). Po czasie obserwacji o medianie 5,1 roku nie stwierdzono wpływu AGCN na czas przeżycia (log-rank, P=0,9; 0,83; 0,8 odpowiednio dla erbB-1, erbB-2 i dla obu genów łącznie).WnioskiUzyskane wyniki wskazują na brak rokowniczego znaczenia AGCN erbB-1 i erbB-2 u chorych na raka płuca poddanych resekcji miąższu płucnego

Bifunctional TaqII restriction endonuclease: redefining the prototype DNA recognition site and establishing the Fidelity Index for partial cleaving

<p>Abstract</p> <p>Background</p> <p>The TaqII enzyme is a member of the <it>Thermus </it>sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various <it>Thermus </it>sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III.</p> <p>Results</p> <p>Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC).</p> <p>Conclusions</p> <p>The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from <it>Thermus aquaticus </it>has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions (pH and salt concentrations) were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity (pH = 6.0 and no salt). Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical applications. The extension of the Fidelity Index will be helpful for DNA manipulation with enzymes only partially cleaving DNA.</p

Radiative electron capture in the first forbidden unique decay of 81Kr

The photon spectrum accompanying the orbital K-electron capture in the first forbidden unique decay of 81Kr was measured. The total radiation intensity for the photon energies larger than 50 keV was found to be 1.47(6) x 10^{-4} per K-capture. Both the shape of the spectrum and its intensity relative to the ordinary, non-radiative capture rate, are compared to theoretical predictions. The best agreement is found for the recently developed model which employs the length gauge for the electromagnetic field.Comment: 7 pages, 6 figure

Construction of bionanoparticles with the use of a recombinant DNA vector-enzymatic system, containing artificial poliepitopic proteins, for the delivery of new generation vaccines

DNA/RNA amplification technologies, such as the Polymerase Chain Reaction have revolutionized modern biology, medical diagnostics and forensic analyses, among others. A number of alternative nucleic acids amplification methods have been developed, tailored to specific applications. Here we present a refined version of a DNA fragment amplification technology, which enables the construction of ordered concatemers in a head-to-tail-orientation. A very high number of DNA segments, at least 500 copies, can be consecutively linked. Other key features include: (i) the application of a dedicated vector-enzymatic system, including selected subtype IIS restriction endonucleases, which has been designed to automatically generate long Open Reading Frames and (ii) an amplification-expression vector with a built-in strong transcription promoter along with optimal translation initiation signals, which allow for a high level of expression of the constructed artificial poliepitopic protein. This highly advanced technology makes it possible to obtain ordered polymers of monomeric, synthetic or natural, DNA far beyond the capabilities of current chemical synthesis methods. The constructed poliepitopic proteins are further used for construction of several types of nanoparticles, including inclusion bodies and bacteriophages, containing multiple genetic fusion with poliepitopic proteins.The technology offers significant advances in a number of scientific, industrial and medical applications, including new vaccines and tissue pro-regenerative methods. The technology is protected by an international patent application and is available for licensing. Acknowledgments: project was supported by National Center for Research and Development, Warsaw, Poland, grant no STRATEGMED1/235077/9/NCBR/2014 and POIG.01.04.00-22-140/12; Jagiellonian Center for Innovation, Krakow, Poland; SATUS VC, Warsaw, Poland and BioVentures Institute Ltd, Poznan, Poland

Time- and Dose-Dependent Induction of HSP70 in Lemna minor Exposed to Different Environmental Stressors

The objective of this study was to examine the influence of different stressors, including cadmium (heavy metal), anthracene (polycyclic aromatic hydrocarbon—PAH) and chloridazon (herbicide), on population growth and biosynthesis of cytoplasmic HSP70 in Lemna minor (duckweed) in short (4 h)- and long (7 days)-term tests. A heat shock response was confirmed in Lemna exposed to high temperature: 35, 37.5, 40, or 42.5°C in short-term (4 h) treatments. The chemicals tested stimulated the biosynthesis of the cytoplasmic HSP70 protein in a concentration-dependent way (0.5–5 μM), higher in fronds exposed to lower doses of stressors. Additionally, production of HSP70 was greater after 4 h of incubation than after 7 days. The results suggest that HSP70 could be applied as a non-specific and sensitive detector of stress induced by different chemicals at concentrations below those that produce the type of response observed in classical cytotoxicity tests, such as growth inhibition

Hindered E4 decay of the 12+ yrast trap in 52Fe

AbstractThe γ decay of the 12+ yrast trap in 52Fe has been measured for the first time. The two E4 γ-branches to the 8+ states are hindered with respect to other B(E4) reduced transition probabilities measured in the f7/2 shell. The interpretation of the data is given in the full pf shell model framework, comparing the results obtained with different residual interactions. It is shown that measurements of hexadecapole transition probabilities constitute a powerful tool in discriminating the correct configuration of the involved wavefunctions

Hindered E4 decay of the 12+ yrast trap in 52Fe

Abstract The γ decay of the 12 + yrast trap in 52Fe has been measured for the first time. The two E4 γ-branches to the 8 + states are hindered with respect to other B ( E 4 ) reduced transition probabilities measured in the f 7 / 2 shell. The interpretation of the data is given in the full pf shell model framework, comparing the results obtained with different residual interactions. It is shown that measurements of hexadecapole transition probabilities constitute a powerful tool in discriminating the correct configuration of the involved wavefunctions