Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives

AbstractA novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction

Assembling of G-strands into novel tetra-molecular parallel G4-DNA nanostructures using avidin–biotin recognition

We describe a method for the preparation of novel long (hundreds of nanometers), uniform, inter-molecular G4-DNA molecules composed of four parallel G-strands. The only long continuous G4-DNA reported so far are intra-molecular structures made of a single G-strand. To enable a tetra-molecular assembly of the G-strands we developed a novel approach based on avidin–biotin biological recognition. The steps of the G4-DNA production include: (i) Enzymatic synthesis of long poly(dG)-poly(dC) molecules with biotinylated poly(dG)-strand; (ii) Formation of a complex between avidin-tetramer and four biotinylated poly(dG)-poly(dC) molecules; (iii) Separation of the poly(dC) strands from the poly(dG)-strands, which are connected to the avidin; (iv) Assembly of the four G-strands attached to the avidin into tetra-molecular G4-DNA. The average contour length of the formed structures, as measured by AFM, is equal to that of the initial poly(dG)-poly(dC) molecules, suggesting a tetra-molecular mechanism of the G-strands assembly. The height of tetra-molecular G4-nanostructures is larger than that of mono-molecular G4-DNA molecules having similar contour length. The CD spectra of the tetra- and mono-molecular G4-DNA are markedly different, suggesting different structural organization of these two types of molecules. The tetra-molecular G4-DNA nanostructures showed clear electrical polarizability. This suggests that they may be useful for molecular electronics

Ribonucleotide reductases of Salmonella Typhimurium : transcriptional regulation and differential role in pathogenesis

Ribonucleotide reductases (RNRs) are essential enzymes that carry out the de novo synthesis of deoxyribonucleotides by reducing ribonucleotides. There are three different classes of RNRs (I, II and III), all having different oxygen dependency and biochemical characteristics. Salmonella enterica serovar Typhimurium (S. Typhimurium) harbors class Ia, class Ib and class III RNRs in its genome. We have studied the transcriptional regulation of these three RNR classes in S. Typhimurium as well as their differential function during infection of macrophage and epithelial cells. Deletion of both NrdR and Fur, two main transcriptional regulators, indicates that Fur specifically represses the class Ib enzyme and that NrdR acts as a global repressor of all three classes. A Fur recognition sequence within the nrdHIEF promoter has also been described and confirmed by electrophoretic mobility shift assays (EMSA). In order to elucidate the role of each RNR class during infection, S. Typhimurium single and double RNR mutants (as well as Fur and NrdR mutants) were used in infection assays with macrophage and epithelial cell lines. Our results indicate class Ia to be mainly responsible for deoxyribonucleotide production during invasion and proliferation inside macrophages and epithelial cells. Neither class Ib nor class III seem to be essential for growth under these conditions. However, class Ib is able to maintain certain growth in an nrdAB mutant during the first hours of macrophage infection. Our results suggest that, during the early stages of macrophage infection, class Ib may contribute to deoxyribonucleotide synthesis by means of both an NrdR and a Fur-dependent derepression of nrdHIEF due to hydrogen peroxide production and DNA damage associated with the oxidative burst, thus helping to overcome the host defenses

Хірургічне лікування мультирезистентного туберкульозу як фактор підвищення ефективності лікування

Effective treatment of patients takes an important place in the fight against tuberculosis.Objective. A comparative analysis of the effectiveness of treatment of patients with firstly diagnosed MDR TB with the use of different approaches to the treatment of such patients.Material and methods. Statistical data of patients with firstly diagnosed pulmonary MDR TB who were registered and treated at the Kharkiv Regional TB Dispensary No. 1 in the period from 2014 and who had completed the main course of chemotherapy to the time of the study.Results and discussion. In group I (n=93; using conservative treatment) 58 patients (61.9%) completed treatment effectively; 12 patients (13,3%) died (9 of them (9,5%) died from TB); treatment failure was diagnosed in 6 patients (6,7%); 17 patients (18,1%) interrupted treatment. In group II (n=12; using surgical methods) 9 patients (77,8%) were effectively treated; 1 patient (8,3%) died, 1 patient (8,3%) had treatment failure and 1 patient (8,3%) have interrupted treatment.Conclusion. The effectiveness of treatment of patients with MDR TB with a complex approach to their treatment (using surgical methods) is significantly increased (by 14,7%), while the rate of patients with ineffective treatment becomes less (less deaths by 4.8%, less treatment failures by 4,5%, less treatment interruptions by 5,5%).Эффективное лечение пациентов занимает важное место в борьбе с туберкулезом.Цель работы — сравнительный анализ эффективности лечения пациентов с впервые диагностированным мультирезистентным туберкулезом с использованием разных подходов к лечению таких пациентов.Материал и методы. Статистические данные пациентов с впервые диагностированным мультирезистентным туберкулезом легких, которые были зарегистрированы и лечились в Харьковском областном противотуберкулезном диспансере № 1 в период с 2014 года и на момент исследования завершили основной курс химиотерапии.Результаты и обсуждение. В группе I (n = 93, с использованием консервативного лечения) 58 пациентов (61,9%) успешно завершили лечение; 12 пациентов (13,3%) умерли (9 из них (9,5%) умерли от ТБ); неудача лечения была у 6 пациентов (6,7%); 17 пациентов (18,1%) прервали лечение. В группе II (n = 12, с использованием хирургических методов) эффективно пролечены 9 пациентов (77,8%); 1 пациент (8,3%) умер, у 1 пациента (8,3%) неудача лечения и 1 пациент (8,3%) прервал лечение. Вывод. Эффективность лечения пациентов с мультирезистентным туберкулезом при комплексном подходе к их лечению (с использованием хирургических методов) значительно повышается (на 14,7%), а количество пациентов с неэффективным лечением — уменьшается (смертей меньше на 4,8%, неудачи лечения — на 4,5%, а прерываний лечения — на 5,5%).Ефективне лікування пацієнтів посідає важливе місце в боротьбі з туберкульозом.Мета роботи — порівняльний аналіз ефективності лікуванняпацієнтів на вперше діагностований мультирезистентний туберкульоз з використанням різних підходів до лікування таких хворих.Матеріал і методи. Статистичні дані пацієнтів на впершедіагностований мультирезистентний туберкульоз легенів, щобули зареєстровані та лікувались у Харківському обласномупротитуберкульозному диспансері № 1 у період з 2014 року і на момент дослідження завершили своє лікування.Результати. У групі І (n=93, з використанням консервативного лікування) 58 пацієнтів (61,9%) успішно завершили лікування; 12 пацієнтів (13,3%) померли (9 з них від туберкульозу); невдача лікування була у 6,7%; 17 пацієнтів (18,1%) перервали лікування. У групі ІІ (n=12, з використанням хірургічних методів) ефективно пролікованих було 9 пацієнтів (77,8%); 1 пацієнт (8,3%) помер; в 1 пацієнта (8,3%) була невдача лікування, та 1 пацієнт (8,3%) перервав лікування.Висновок. Ефективність лікування пацієнтів із мультирезистентним туберкульозом при комплексному підході до їх лікування підвищується (на 14,7%), а з неефективним лікуванням — зменшується (смертей менше на 4,8%, а переривань лікування на 5,5%)