Fluorescence-based proteasome activity profiling

With the proteasome emerging as a therapeutic target for cancer treatment, accurate tools for monitoring proteasome (inhibitor) activity are in demand. In this chapter, we describe the synthesis and use of a fluorescent proteasome activity probe that allows for accurate profiling of proteasomal activity in cell lysates, intact cells, and murine and human patient-derived material, with high sensitivity using SDS-PAGE. The probe allows for direct scanning of the gel for fluorescent emission of the distinct proteasomal subunits and circumvents the use of Western blot analysis. Due to its suitable biochemical and biophysical properties, the fluorescent probe can also be used for confocal laser scanning microscopy and flow cytometry-based experiments

Development of a hypersensitive periodate-cleavable amino acid that is methionine- and disulfide-compatible and its application in MHC exchange reagents for T cell Characterisation

Incorporation of cleavable linkers into peptides and proteins is of particular value in the study of biological processes. Here we describe the synthesis of a cleavable linker that is hypersensitive to oxidative cleavage as the result of the periodate reactivity of a vicinal amino alcohol moiety. Two strategies directed towards the synthesis of a building block suitable for solid-phase peptide synthesis were developed: a chemoenzymatic route, involving l-threonine aldolase, and an enantioselective chemical route; these led to α,γ-diamino-β-hydroxybutanoic acids in diastereoisomerically mixed and enantiopure forms, respectively. Incorporation of the 1,2-amino alcohol linker into the backbone of a peptide generated a conditional peptide that was rapidly cleaved at very low concentrations of sodium periodate. This cleavable peptide ligand was applied in the generation of MHC exchange reagents for the detection of antigen-specific T cells in peripheral blood cells. The extremely low concentration of periodate required to trigger MHC peptide exchange allowed the co-oxidation of methionine and disulfide residues to be avoided. Conditional MHC reagents hypersensitive to periodate can now be applied without limitations when UV irradiation is undesired or less practical

Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes

Epitope-tagged active-site-directed probes are widely used to visualize the activity of deubiquitinases (DUBs) in cell extracts, to investigate the specificity and potency of small-molecule DUB inhibitors, and to isolate and identify DUBs by mass spectrometry. With DUBs arising as novel potential drug targets, probes are required that can be produced in sufficient amounts and to meet the specific needs of a given experiment. The established method for the generation of DUB probes makes use of labor-intensive intein-based methods that have inherent limitations concerning the incorporation of unnatural amino acids and the amount of material that can be obtained. Here, we describe the total chemical synthesis of active-site-directed probes and their application to activity-based profiling and identification of functional DUBs. This synthetic methodology allowed the easy incorporation of desired tags for specific applications, for example, fluorescent reporters, handles for immunoprecipitation or affinity pull-down, and cleavable linkers. Additionally, the synthetic method can be scaled up to provide significant amounts of probe. Fluorescent ubiquitin probes allowed faster, in-gel detection of active DUBs, as compared to (immuno)blotting procedures. A biotinylated probe holding a photocleavable linker enabled the affinity pull-down and subsequent mild, photorelease of DUBs. Also, DUB activity levels were monitored in response to overexpression or knockdown, and to inhibition by small molecules. Furthermore, fluorescent probes revealed differential DUB activity profiles in a panel of lung and prostate cancer cells

Традиции и обычаи туркменского народа в прошлом и настоящем

Основными отличительными чертами этноса являются присущие ему традиции и обычаи. Сформированные на ранних стадиях развития человеческого общества, они сохраняют свою историческую значимость, являются частью современной жизни, выполняя воспитательную, эстетическую функции, оказывая влияние на мировоззрение и поведение представителей того или иного народа. Туркменистан – страна, в которой особенно бережно относятся к дошедшим до настоящего времени традициям и обычаям, многие из которых сложились под влиянием религии. И это не случайно: в Туркменистане достаточно строго соблюдаются законы шариата, служащие своеобразными правилами, обязательными для всех. Исламская религия играла важнейшую роль в становлении туркменских традиций на протяжении веков. Она и сейчас составляет основу жизни населения, в том числе и молодежи. Мусульманские обряды и обычаи стали общенациональными и соблюдаются практически всем коренным населением Туркменистана

Сопоставительный анализ украинских и узбекских пословиц о Родине

Пословицы и поговорки живут в народной речи и сопровождают человека на протяжении всей его жизни. Меткие образные высказывания отражают все стороны человеческого бытия, запечатлевают исторические события, отображают мировосприятие того или иного народа. Под пословицами понимаются краткие изречения, имеющие поучительный смысл и передающиеся народом из поколения в поколение. При цитировании документа, используйте ссылку http://essuir.sumdu.edu.ua/handle/123456789/3283