Chemical mapping of cytosines enzymatically flipped out of the DNA helix

Haloacetaldehydes can be employed for probing unpaired DNA structures involving cytosine and adenine residues. Using an enzyme that was structurally proven to flip its target cytosine out of the DNA helix, the HhaI DNA methyltransferase (M.HhaI), we demonstrate the suitability of the chloroacetaldehyde modification for mapping extrahelical (flipped-out) cytosine bases in protein–DNA complexes. The generality of this method was verified with two other DNA cytosine-5 methyltransferases, M.AluI and M.SssI, as well as with two restriction endonucleases, R.Ecl18kI and R.PspGI, which represent a novel class of base-flipping enzymes. Our results thus offer a simple and convenient laboratory tool for detection and mapping of flipped-out cytosines in protein–DNA complexes

Chemical mapping of cytosines enzymatically flipped out of the DNA helix

Haloacetaldehydes can be employed for probing unpaired DNA structures involving cytosine and adenine residues. Using an enzyme that was structurally proven to flip its target cytosine out of the DNA helix, the HhaI DNA methyltransferase (M.HhaI), we demonstrate the suitability of the chloroacetaldehyde modification for mapping extrahelical (flipped-out) cytosine bases in protein–DNA complexes. The generality of this method was verified with two other DNA cytosine-5 methyltransferases, M.AluI and M.SssI, as well as with two restriction endonucleases, R.Ecl18kI and R.PspGI, which represent a novel class of base-flipping enzymes. Our results thus offer a simple and convenient laboratory tool for detection and mapping of flipped-out cytosines in protein–DNA complexes

Išplitusio sensibilizuoto krūties vėžio diagnostika ir gydymas

Laima Bloznelytė-Plėšnienė1, 2, Daiva sendiulienė1, Jurgita Liutkevičiūtė-Navickienė1, Laimutė Rutkovskienė1, Valerijus Ostapenko1, Narimantas Evaldas Samalavičius1 1 Vilniaus universiteto Onkologijos institutas, Santariškių g. 1, LT-08406 Vilnius 2 Klaipėdos universitetas, Herkaus Manto g. 84, LT-92294, Klaipėda El. paštas: [email protected] Tikslas Nustatyti sensibilizuotų navikų terapijos galimybes gydant išplitusį krūties vėžį. Ligoniai ir metodai Nuo 2001 m. 54 ligonėms išplitusiam krūties vėžiui gydyti taikyta sensibilizuotų navikų terapija (SNT). 53 ligonėms rastos dauginės metastazės. Metastazės smegenyse diagnozuotos 19 ligonių, kauluose – 29, kepenyse – 15, plaučiuose – 12, limfmazgiuose – 16, minkštuosiuose audiniuose – 13 ir odoje – 15 ligonių. Taikydami SNT į veną suleisdavome 2,5 mg/kg hematoporfirino darinio (HpD) ir jis po 24 val. selektyviai susikaupdavo navikiniame audinyje. Praėjus 24 val. po HpD suleidimo jam aktyvinti navikus apšviesdavome raudona 635 nm šviesa – tai vadinamoji fotosensibilizuotų navikų terapija (FDT), ir (arba) taikydavome radiosensibilizuotų navikų terapiją (RST) – praėjus 24, 48 bei 72 val. po HpD suleidimo naviką apšvitindavome 60Co skleidžiamais gama spinduliais 2 Gy doze. Suminė dozė – 6 Gy. RST taikėme visoms 54 ligonėms. FDT taikėme: 16 ligonių vietiškai krūtinės odoje ir poodyje išplitusiam vėžiui gydyti bei 1 ligonei metastazei akies tinklainėje gydyti. Visų odos ir poodžio navikų riboms patikslinti taikėme ir fotodinaminę diagnostiką. Praėjus 24–78 val. po HpD suleidimo, įtartinus odos plotus apšviesdavome violetine 405 nm šviesa. Navikinis audinys švytėdavo avietine spalva. Rezultatai Pritaikius SNT 54 ligonėms išplitusiam krūties vėžiui gydyti, 33 pacientėms per 7–10 dienų po RST labai pakilo Karnovskio indeksas, šešioms pacientėms visi navikai visiškai regresavo, 14 moterų visi gydyti navikai regresavo daugiau nei 50 %, ligos remisija truko ilgiau nei 6 mėn. Šešiolika ligonių navikų regresija buvo dalinė, 18 ligonių gydymas buvo neveiksmingas. SNT buvo ypač efektyvi krūties vėžio metastazėms smegenyse ir kauluose gydyti. Vienai ligonei po dviejų RST kursų visiškai išnyko visos trys metastazės galvos smegenyse. Šešioms ligonėms visos metastazės smegenyse regresavo daugiau nei 50 %, ligos remisija truko daugiau nei 6 mėnesius. Dauginių krūties vėžio metastazių smegenyse turinčių 19 ligonių vidutinis išgyvenamumas nuo metastazių smegenyse diagnozavimo buvo 12 mėnesių. Pritaikius RST 29 ligonėms dauginėms krūties vėžio metastazėms kauluose gydyti, 7 ligonėms gauta visiška visų dauginių kaulinių metastazių regresija. Ligos remisija (moterys buvo kliniškai sveikos) truko 95; 48; 21; 19; 12; 11 ir 4 mėn. nuo RST. Išvados Sensibilizuotų navikų terapija yra perspektyvus išplitusio krūties vėžio gydymo metodas. Šis metodas ypač veiksmingas gydant krūties vėžio metastazes galvos smegenyse ir kauluose. Tikslinga toliau tyrinėti šį gydymo metodą, siekiant jį modifikuoti pagal ligos išplitimą ir metastazių lokalizaciją. Reikšminiai žodžiai: išplitęs krūties vėžys, sensibilizuotų navikų terapija, hematoporfirino darinys. Sensitized treatment and diagnostics of advanced breast cancer Laima Bloznelytė-Plėšnienė1, 2, Daiva sendiulienė1, Jurgita Liutkevičiūtė-Navickienė1, Laimutė Rutkovskienė1, Valerijus Ostapenko1, Narimantas Evaldas Samalavičius1 1 Vilnius University, Institute of Oncology, Santariškių Str. 1, LT-08406 Vilnius, Lithuania 2 Klaipėda University, Herkaus Manto Str. 84, LT-92294, Klaipėda, Lithuania E-mail: [email protected] Background / objective The current methodologies used in oncology are of quite limited possibilities. Therefore, there is a constant search for the new perspective treatment methods that could prolong the life of cancer patients and improve its qualitaty. One of such methods is sensitized tumour therapy based on quite selective porphyrin accumulation in tumours. This study presents our primary results in radiosensitized advanced breast cancer therapy using hematoporphyrin derivatives as photo-and radiosensitizers. Patients and methods In 2001–2010, a total of 54 female patients with advanced breast cancer underwent radiosensitized treatment (RST). All patients had undergone chemotherapy and / or radiotherapy and surgical treatment before RST. In all cases, any radical method of treatment was impossible. Multiplex metastatic lesions were established in 53 patients. Brain multiplex metastases were diagnosed in 19 patients and multiplex bone metastases in 29 patients. Lung metastatic lesions were found in 12 patients, liver in 15 patients, lymph node metastases in 16 patients. The hematoporphyrin derivative was injected intravenously; 24, 48 and 72 hours after injection of the sensitizer, the tumorus were irradiated with gamma rays from radioactive 60Co, 2 Gy at a time (6 Gy per course). In all cases when it was possible, patients undervent also photodynamic therapy. Tumours were irradiated with red 630 nm laser light. Results As a result of RST, complete regression of all treated tumours was observed in 6 patients after two or more RST courses. A significant response – regression of more than 50% of all brain metastases and remission of the disease for over 6 months – was established in 14 patients. Partial response was observed in 16 patients with malignant brain tumours. For the rest of 18 patients the treatment was ineffective. The Karnofsky performance scale index increased immediately in 33 patients following RST treatment. RST was especially effective in the treatment of brain and bone metastatic lesions. As regards brain metastazes, in one patient all three brain metastatic lesions completely disappeared and there were no evidence of any recurrence in brain for 8 months. In 6 patients, regression of more than 50% of all brain metastases and remission of the disease for over 6 months was established. The median survival of 19 patients with multiplex brain metastases was 12 months from the moment of brain metastases detection. As regards bone metastazes, as a result of RST, all metastatic bone lesions completely disappeared in 7 patients. Conclusion Radiosensitized advanced metastatic breast cancer treatment is a hopeful method, especially when lesions involve the brain and bones. Keywords: advanced breast cancer, radiosensitized treatment, hematoporphyrin derivative

Epigenomic modifications mediating antibody maturation

Epigenetic modifications, such as histone modifications, DNA methylation status, and non-coding RNAs (ncRNA), all contribute to antibody maturation during somatic hypermutation (SHM) and class-switch recombination (CSR). Histone modifications alter the chromatin landscape and, together with DNA primary and tertiary structures, they help recruit Activation-Induced Cytidine Deaminase (AID) to the immunoglobulin (Ig) locus. AID is a potent DNA mutator, which catalyzes cytosine-to-uracil deamination on single-stranded DNA to create U:G mismatches. It has been shown that alternate chromatin modifications, in concert with ncRNAs and potentially DNA methylation, regulate AID recruitment and stabilize DNA repair factors. We, hereby, assess the combination of these distinct modifications and discuss how they contribute to initiating differential DNA repair pathways at the Ig locus, which ultimately leads to enhanced antibody-antigen binding affinity (SHM) or antibody isotype switching (CSR). We will also highlight how misregulation of epigenomic regulation during DNA repair can compromise antibody development and lead to a number of immunological syndromes and cancer

Photodynamic (fluorescence) diagnostics of skin lesions.

The diagnostic value and methodological features of photodynamic (fluorescence) diagnostics in skin lesions were analysed during the study. In addition, fluorescence induced by different photosensitizer precursors (5-aminolevulinic acid and methyl aminolevulinate) was compared. The possibilities of photodynamic diagnostics in skin lesions using light sources with different wavelength were investigated in this study. The optimal wavelength of light for inducing fluorescence in skin lesions was determined in order to diagnose tumours of different histological types (i.e. squamous cell carcinoma, basal cell carcinoma and premalignant lesions). The differences of fluorescence in malignant and premalignant skin lesions were evaluated. Photodynamic diagnostics furthermore was used as method of visualisation helping to delineate exact margins of skin tumour before and during the treatment and to achieve thoroughness of treatment in patients with malignant epithelial skin tumours

Effects of the treatment of buckwheat seeds (Fagopyrum esculemntum) with cold plasma and electromagnetic fields on biomass gain, quantity of secondary metabolites and yield

Augalų sėklų apdorojimas šalta plazma (ŠP) ir elektromagnetiniu lauku (EML) – novatoriškas būdas didinti augalinės produkcijos išeigą ir kokybę, nekenkiant aplinkai. Darbo tikslas buvo įvertinti dviejų grikių kultivarų ‚VB Vokiai‘ ir ‚VB Nojai‘ atsaką į stresą, sukeltą apdorojant jų sėklas šalta plazma (ŠP) ir elektromagnetiniu lauku (EML). Buvo siekiama nustatyti, kokią įtaką dviejų grikių kultivarų sėklų apdorojimas ŠP ir EML turi daigumui, augalų augimui, biomasės prieaugiui, derliui ir antrinių metabolitų kiekiui, augant lauko sąlygomis. Buvo vertinama maksimali daigumo lauko sąlygomis išeiga, periodiškai (praėjus 4, 8 ir 14 savaičių po sėjos) nustatomi augalų morfometriniai rodikliai. Antioksidacinis aktyvumas grikių lapų ekstraktuose nustatytas DPPH• radikalo sujungimo metodu. Bendras fenolinių junginių kiekis sėklose nustatytas Folin-Ciocalteu metodu, rutino ir kitų antrinių metabolitų kiekis – HPLC metodu, mineralinių medžiagų kiekis – atominės absorbcinės spektrometrijos metodu. Sėklų apdorojimas ŠP ir EML slopino (10-20 %) grikių kultivarų ‚VB Vokiai‘ ir ‚VB Nojai‘ daigumą lauko sąlygomis. ŠP veiksmingai didino abiejų kultivarų antžeminės augalo dalies svorį, stiebų ir lapų skaičių bei derlių. ŠP ženkliai didino grikių derlių: ‚VB Vokių‘ – 70 %, ‚VB Nojų‘ net 56-97 %. Praėjus 8 savaitėms po sėjos ‚VB Nojai‘ kultivaro lapų ekstraktų RJA buvo 2,9 karto didesnis, lyginant su ‚VB Vokiais‘. Stipriausias ŠP ir EML poveikis nustatytas Fe ir Zn kiekiams, tačiau šis poveikis grikių kultivarams buvo priešingas: ‚VB Vokių‘ sėklose stresoriai ženkliai padidino Fe ir mažino Zn kiekį, tačiau ‚VB Nojų‘ 7 min. ŠP mažino Fe kiekį, o EML didino Zn kiekį. Stresorių poveikis kvercetino, kvercitrino ir izoorientino kiekiui buvo stipresnis nei rutino ir D-fagomino.Treatment of plant seeds with CP and EMF is an innovative method of increasing the yield and quality of plant products without harming the environment. Purpose of work was to assess the response of two different buckwheat cultivars ‘VB Vokiai’ and ‘VB Nojai’ to the stress induced by pre-sowing seed treatment with cold plasma (CP) and electromagnetic field (EMF). The effects of CP and EMF treatments on germination, plant growth, biomass production, secondary metabolites and yields of the seeds for two buckwheat cultivars in field conditions were assessed. Maximal germination under field conditions was determined and periodic measurements (4, 8 and 14 after sowing) of morphometric parameters of buckwheat seedlings were performed. Antioxidant activity in buckwheat leaf extracts was determined by DPPH radical scavenging. The total amount of phenolic compounds in buckwheat seeds was determined by Folin-Ciocalteu method, the amount of rutin and other secondary metabolites – by the HPLC, and mineral content – by the atomic absorption spectrometry. Seed treatment with CP and EMF slightly (by 10-20%) supressed the germination of both buckwheat cultivars under field conditions. EMF treatment significantly increased the weight of the aboveground part of both cultivars, the number of stems and leaves, and the yield. The CP increased the buckwheat seed yield: for ‘VB Vokiai’ – by 70%, and for ‘VB Nojai’ by as much as 56-97%. 8 weeks after sowing, the RJA of the leaf extracts of the ‘VB Nojai’ was 2.9 times higher compared to ‘VB Vokiai’. The strongest effects of CP and EMF were found on the levels of Fe and Zn, but this effect was opposite on buckwheat cultivars: in ‘VB Vokiai’ seeds, stressors significantly increased the amount of Fe and reduced the amount of Zn, but for ‘VB Nojai’ CP 7 min. treatment reduced the levels of Fe but EMF increased the amount of Zn. The effects of stressors on the levels of quercetin, quercitrine and isorientine were more pronounced compared to the changes in the content of rutin and D-phagomin.Gamtos mokslų fakultetasBiochemijos katedr

The agency of private legal persons: theoretical and practical aspects

This thesis analyses the institution of acting on behalf of private legal persons, theoretical and practical aspects are discussed. The attention is mainly concentrated to relations of representation by different forms: representation according to the contract (contractual representation) and acting according to law (ex officio representation), as these are defined in Civil Code of the Republic of Lithuania. The article discusses the major theories of representation, concentrating on those, influencing Civil Code of the Republic of Lithuania the most. Theoretical stands on legal persons are also discussed shortly. According to legal doctrine legal person is artificial subject, which operates in legal relationships as separate legal subject, but it is not able to act by itself and it’s legal capacity is realized by natural persons. In such case private legal person is represented by natural persons who act on behalf of it. Taking into consideration such specific features of legal person, thesis analyses the governing bodies of legal person, the governing structure, the functions in acting on behalf of private legal person and the contractual representation of private legal person. The praxis of the Supreme Court of Lithuania is reviewed, the most popular opinions in legal doctrine are pointed out, and the most discussed positions due to acting on behalf of private legal person are analyzed deeper