Сравнительный анализ протеомного профиля кератиноцитов HaCaT с использованием 1DE-гель концентрирования

Using tandem mass spectrometry with electrospray ionization, a comparative analysis of HaCaT keratinocyte proteins was carried out before and after exposure of cells to sodium dodecyl sulfate (25 mg/ml) for 48 hours; proteins encoded by human chromosome 18 genes were chosen as the comparison proteins. A total of 2418 proteins were detected in the HaCaT immortalized human keratinocytes, 70% of these proteins were identified by two or more unique peptides. Panoramic mass spectrometry analysis identified 38 proteins encoded by chromosome 18 genes, 27 proteins were common to control HaCaT cells and HaCaT cells exposed to SDS. Using the Metascape database (https://metascape.org), an enrichment analysis of GO terms of the Biological Process category of chromosome 18 gene encoded proteins of HaCaT keratinocytes was performed before and after the SDS exposure. The SDS exposure resulted in a slight enrichment of the GO term "response to stimulus" (GO:0050896) and the related GO term "negative regulation of biological process" (GO:0048519). We found decreased expression levels of membrane proteins encoded by chromosome 18 genes related to cell-cell adhesion (GO:0098609), such as DSC1, DSC3, and DSG1. A decrease in the expression level of desmosomal cadherins is characteristic of malignant neoplasms developing from epithelial tissue cells of various internal organs, mucous membranes, and skin. The method of preparation of HaCaT keratinocyte samples used in this work increased the sensitivity of proteomic analysis of cell culture and made it possible to identify twice as many proteins in one gel strip as compared to the number of proteins (1284) in HaCaT samples subjected to osmotic shock and cleavage by trypsin in solution.Проведена оценка протокола пробоподготовки образцов клеточной культуры кератиноцитов, основанного на солюбилизации белков в присутствии 0.2% додецилсульфата натрия (SDS), процедуре 1DE-гель концентрирования (SDS-PAGE без фракционирования в разделяющем геле) и расщеплении трипсином в геле для углубленного протеомного анализа кератиноцитов НаСаТ в одной полосе белка. С помощью тандемной масс-спектрометрии с электроспрейной ионизацией (LC-MS/MS) проведен сравнительный анализ белков кератиноцитов НаСаТ до и после воздействия SDS в субтоксической дозе (25 мг/мл) в течение 48 ч. В качестве белков сравнения выбраны белки, кодируемые генами хромосомы 18 человека. Всего в иммортализованных кератиноцитах человека линии НаСаТ обнаружено 2418 белков, из них около 70% идентифицировано по двум и более уникальным пептидам. По результатам панорамного масс-спектрометрического анализа удалось идентифицировать 38 белков, кодируемых генами хромосомы 18; из них 27 белков были общими для контрольных клеток и клеток НаСаТ, подвергнутых воздействию SDS. С использованием базы данных Metascape был проведен анализ обогащения терминами онтологии генов (GO) категории биологические процессы (biological process) белков хромосомы 18 кератиноцитов НаСаТ до и после воздействия SDS. Обработка клеточной культуры SDS приводила к незначительному обогащению GO термина “ответ на стимул” (GO:0050896 - response to stimulus) и связанного с ним GO термина “негативная регуляция биологических процессов” (GO:0048519 - negative regulation of biological process). Было обнаружено снижение уровня экспрессии мембранных белков, кодируемых генами хромосомы 18, относящихся к межклеточной адгезии (GO:0098609 - cell-cell adhesion), таких как DSC1, DSC3 и DSG1. Снижение уровня экспрессии десмосомальных кадгеринов характерно для злокачественных новообразований, развивающихся из клеток эпителиальной ткани различных внутренних органов, слизистых оболочек, кожи. Примененный в работе способ подготовки образцов кератиноцитов НаСаТ позволил идентифицировать в одной полосе геля в два раза больше белков по сравнению с образцами НаСаТ, подвергнутыми осмотическому шоку и расщеплению трипсином в растворе

Direct electrochemistry of Heme Proteins on Electrodes Modified with Didodecyldimethyl Ammonium Bromide and Carbon Black

A novel matrix based on commercially available carbon black (CB) N220 and didodecyldimethyl ammonium bromide (DDAB) was shown to be a reliable support for direct electron transfer reactions between screen printed electrode (SPE) and Fe(III)-heme proteins. Cytochrome c(cytc), myoglobin (Mb), horseradish peroxidase (HRP) and cytochromes P450 (CYP 51A1, CYP 3A4, CYP 2B4) generated well-shaped cyclic voltammograms on SPE/CB/ DDAB electrodes (both in solution and in immobilized state). The attractive performance characteristics of CB modified electrodes are advantageous over single-walled carbon nanotubes (SW CNT) based ones. The achieved direct electrochemistry of heme proteins on CB/DDAB-modified electrodes provided successful elaboration of the immunosensor for cardiac Mb. The immunosensor showed applicability for diagnostics of myocardial infarction displaying significant difference in cardiac Mb content of human blood plasma samples taken from the corresponding patients

APPLICATION OF TIME-RESOLVED FLUORESСENCE TECHNIQUE FOR STABILITY ANALYSIS OF FIREFLY LUCIFERASE LUCIOLA MINGRELICA

The characteristics of intrinsic fluorescence of firefly luciferase L. mingrelica during de-naturation with urea were studied. The dependence of both steady-state and time-resolved fluorescence parameters on the urea concentration revealed a single transition with the mid-point at 3.1±0.1M

Genotoxic Effect of Chronic Exposure to DDT on Lymphocytes, Oral Mucosa and Breast Cells of Female Rats

The genotoxicity of some environmental contaminants may affect human health directly by damaging genetic material and thus plays an important role in cancer development. Xenoestrogens are one kind of environmental pollutants that may alter hormonal routes or directly affect DNA. The number of available biomarkers used to assess genetic risk and cancer is very extensive. The present study evaluated genotoxicity produced by the pesticide DDT on systemic and mammary gland cells obtained from adult female Wistar rats. Oral mucosa cells micronuclei were assessed; the comet assay in peripheral blood-isolated lymphocytes and mammary epithelial cells was also carried out. Additionally, oxidative stress was studied in mammary tissue through a lipid peroxidation assay. Our data showed an increase in lipid peroxidation, product of an increase in free oxygen radical levels, which leads to an oxidative stress status. Our results suggest that DDT is genotoxic, not only for lymphocytes but also to mammary epithelial cells

Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The enzyme was overexpressed in Escherichia coli at up to 14% of total soluble protein and purified to homogeneity in three steps. CYP154H1 activity was reconstituted using putidaredoxin reductase and putidaredoxin from Pseudomonas putida DSM 50198 as surrogate electron transfer partners. In biocatalytic reactions with different aliphatic and aromatic substrates of varying size, the enzyme converted small aromatic and arylaliphatic compounds like ethylbenzene, styrene, and indole. Furthermore, CYP154H1 also accepted different arylaliphatic sulfides as substrates chemoselectively forming the corresponding sulfoxides and sulfones. The enzyme is moderately thermostable with an apparent melting temperature of 67°C and exhibited still 90% of initial activity after incubation at 50°C

Resource saving influence in enterprise development in Latvia

Maģistra darba tēma „Ekonomisko resursu taupības ietekme uz uzņēmējdarbības attīstību Latvijā” ir aktuāla, jo uzņēmējdarbība saskaras ar augošām galveno izejvielu un minerālu cenām, to trūkums un cenu nestabilitāte graujoši ietekmē ekonomiku. Mūsu ekonomikas sistēma joprojām veicina resursu neefektīvu izmantošanu, jo par dažiem no tiem tiek prasīta pārāk zema cena. Lai gan dažu dinamisku uzņēmējdarbības veidu pārstāvji jau ir novērtējuši produktīvākas resursu izmantošanas izdevīgumu, daudzi uzņēmumi un patērētāji vēl nav izpratuši viņiem veicamo pārmaiņu vērienu un steidzamību. Efektīvas resursu izmantošanas veicināšana lielā mērā nosaka uzņēmējdarbības veiksmi, un tai būtu jāpalīdz uzlabot konkurētspēju un rentabilitāti, kas palīdz nodrošināt ilgtspējīgu ekonomiskās krīzes pārvarēšanu un var sekmēt darbvietu izveidi. Resursu izmantošanas efektivitāte ir veids, kas ļauj ekonomikai radīt vairāk ar mazāk līdzekļiem, sniedzot lielāku vērtību ar mazāku ieguldījumu, ilgtspējīgi izmantojot resursus un samazinot to ietekmi uz vidi, līdz ar to darba mērķis ir izpētīt uzņēmējdarbības attīstību ekonomisko resursu efektīvas izmantošanas kontekstā. Kā pētīšanas metodes un paņēmienus autore šajā darbā izmanto ekonomisko analīzi un statistiskās analīzes metodes (salīdzināšana). Darbs izmantojams uzņēmējdarbības attīstības tendenču raksturošanai laika periodā no 2005. gada līdz 2012. gadam, kā arī nākotnes attīstības virzienu noteikšanai un analīzei ekonomisko resursu efektīvas izmantošanas kontekstā. Darba apjoms: 105 lpp.This master’s thesis “Resource saving influence in enterprise development in Latvia” is topical based on most company need for resources and the rapid change of price and availability. In our current economic situation there are almost no practices for a better way how to use commodities and other valuable resources. Only some of world’s enterprises care for the change of practices on how to use recourses more effectively with no leftovers. On the other hand the most of enterprises don’t care for the change in their habits and doesn’t understand that soon there will be much higher prices for all resources and commodities. The use of resources effectively is based on the luck of the company and it should be supported by the government for stimulating their economic results and profitability. This practice is a huge icebreaker in long-term and can bring great success for the company as well for the environment around us. Effective use of resources not only makes business processes more cost efficient, but can help to achieve a better effect on nature. The main goal for this thesis is to investigate enterprise development in the field of resource management and saving. The enterprises data is collected for the period from year 2005 until 2012. This thesis can be used to describe the trends of enterprise resource usage during the mentioned period. This master's thesis contains 105 pages

Biology for nurse

367 p.; 22 cm

Giáo trình về cơ sở di truyền học

245 tr. ; 21 cm