Tag Archives: lymphocytes

DNA loop domain rearrangements in blast transformed human lymphocytes and lymphoid leukaemic Jurkat T cells

K. Afanasieva1, V. Olefirenko1, A. Martyniak1,
L. Lukash2, A. Sivolob1*

1Taras Shevchenko National University of Kyiv, Ukraine;
2Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: sivolob@univ.kiev.ua

Received: 06 April 2020; Accepted: 25 June 2020

Chromatin loops are important elements of both chromatin higher-order structure and transcription regulation system. Our previous works have shown that several features of the loop domain organization could be investigated by single cell gel electrophoresis (the comet assay) using the kinetic approach. In this study we applied this technique to study DNA loop domain organization in lymphoid cells: human lymphocytes, lymphoblasts cultivated during 24 h and 44 h, and T cells of Jurkat cell line. Two features of the loop domain organization were found to depend on the cell functional state. First, DNA fraction in the loops of large sizes (more than ~200 kb) was essentially increased in proliferating (de-differentiated) cells in comparison with terminally differentiated lymphocytes. Second, the linear density of the loops not larger than ~200 kb was decreased in transcriptionally active cells and was increased upon their inactivation.

Redistribution of DNA loop domains in human lymphocytes under blast transformation with interleukin 2

K. S. Afanasieva, M. I. Chopei, A. V. Lozovik,
S. R. Rushkovsky, A. V. Sivolob

Educational and Scientific Centre Institute of Biology and Medicine,
Taras Shevchenko National University of Kyiv, Ukraine;
e-mail: aphon@ukr.net

At higher order levels chromatin fibers in interphase nuclei are organized into loop domains. Gene regulatory elements (promoters and enhancers) are often located near the sites of loop attachments. Therefore, loop domains play a key role in regulation of cell transcriptional activity. We investigated the kinetics of DNA loop exit during single cell gel electrophoresis (the comet assay) of nucleoids obtained from two cell types that differ in their synthetic activity – human lymphocytes and lymphoblasts. Lymphocyte activation and transformation into lymphoblasts (blast transformation) was performed with interleukin 2. The results obtained suggest that a rearrangement of the loops occurs after lymphocyte activation. After blast transformation we observed an increase of the amount of loop domains on the surface of nucleoids against a decrease of the inner loop fraction. Therefore, the comet assay can be used for detection of large-scale changes in the cell nucleus that follow changes in cell functional state.

Characteristics of Ca(2+), Mg(2+)-ATPases of peripheral blood lymphocytes of patients with rheumatic pathology

R. V. Fafula, U. P. Efremova, Z. D. Vorobets

Danylo Halytski Lviv National Medical University, Ukraine;
e-mail: roman_fafula@ukr.net, vorobets@meduniv.lviv.ua

The analysis of the kinetic properties of Ca2+, Mg2+-ATPase of saponin-perforated peripheral blood lymphocytes of donors and patients with rheumatoid arthritis and ankylosing spondylitis was carried out. When analyzing the alterations in hydrolase activity of Ca2+, Mg2+-ATPase it was shown that affinity of Ca2+, Mg2+-ATPase of plasma membrane and membranes of endoplasmic reticulum for ATP do not significantly differ. It was found that the inhibition of examined enzyme systems occurs by mixed type both due to the reduction of maximum reaction rate and to the decrease of Ca2+, Mg2+-ATPase affinity for ATP in conditions of rheumatic pathology in the immunocompetent cells. It was identified that Ca2+, Mg2+-ATPase had significantly lower affinity for Ca2+ in lymphocytes of persons with rheumatic disorders than in donors.

Kinetic properties of Na(+), K(+)-activated, Mg(2+)-dependent ATP-hydrolysis of blood lymphocytes in patients with rheumatoid arthritis and ankylosing spondyloarthritis

R. V. Fafula, U. P. Efremova, N. E. Lychkovska, Z. D. Vorobets

Danylo Halytski Lviv National Medical University, Ukraine;
e-mail: roman_fafula@ukr.net; vorobets@meduniv.lviv.ua

The comparative analysis of the kinetic proper­ties of ouabain-sensitive Na+, K+-ATPase activity of saponin-perforated blood lymphocytes of donors and patients with rheumatoid arthritis (RA) and ankylosing spondyloarthritis (AS) was carried out. When analyzing the alterations in hydrolase activity of the examined enzyme it was shown that in the blood lymphocytes of patients with RA and AS the primary active transport of Na+ and K+ ions is less intensive in comparison with practically healthy donors, but it is characterized by almost the same capacity as in donors. The affinity constant of Na+, K+-ATPase for ATP in the blood lymphocytes in patients with RA and AS is greater 3.1 and 2.5 times, respectively, in comparison with healthy donor. It was found that in conditions of rheumatic pathology in immunocompetent cells the inhibition of Na+, K+-ATPase activity is not related to the reduction of maximum reaction rate, but is related to the decrease of Na+, K+-ATPase affini­ty to ATP. However, Mg2+-binding center of Na+, K+-ATPase in patients with RA and AS remains native. It was identified that the affinity constant of Na+, K+-ATPase to Na+ ions in the blood lymphocytes of patients with RA and AS is 2.75 times lower than its value in healthy donors. Na+, K+-ATPase of the blood lymphocytes of patients with RA and AS retains its native receptor properties and sensitivity to ouabain does not change.

Peculiarities of arginase and NO-synthase pathways of L-arginine metabolism in peripheral blood lymphocytes of patients with ovarian cancer

O. I. Yakubets, R. V. Fafula, D. Z. Vorobets, Z. D. Vorobets

Danylo Halytski Lviv National Medical University, Ukraine;
е-mail: vorobets@meduniv.lviv.ua

The peculiarities of arginase and NO-synthase pathways of L-arginine metabolism in peripheral blood lymphocytes of patients with ovarian cancer were studied. It was shown that the development of cancer pathology is associated with an imbalance in the NO synthesis in blood lymphocytes. The reason for such imbalance is the activation of arginase and inducible isoform of NO-synthase (iNOS) and significant inhibition of its constitutive isoform. The analysis of the kinetic properties of NOS of blood lymphocytes of patients with ovarian cancer was carried out. It was shown that the affinity constant of iNOS affinity for L-arginine is 5.4-fold lower than for eNOS of blood lymphocytes of persons in the control group. The inhibition of eNOS occurs via non-competitive type and is related to the reduction of maximum reaction rate.

Distribution of 5-fluorouracil between lymphocytes and blood plasma

M. A. Stashkevych, E. V. Khomutov, O. P. Shatova, Yu. V. Dumanskiy, I. I. Zinkovych

M. Gorky Donetsk National Medical University, Ukraine;
e-mail: matviyenko.maryna@gmail.com

In blood plasma of 8 healthy volunteers with resuspended lymphocytes incubated with 5-fluorou­racil (5-FU) the drug distribution between cells and liquid was assessed by means of HPLC. Rapid accumulation of 5-FU in lymphocytes was proved (the drug concentration on the 3-rd minute is 2.5-fold higher than in plasma) as well as the absence of temporal changes of 5-FU content both in lymphocytes and blood plasma during 30 minutes of experiment.

Laureates of the Palladin Prize of the National Academy of Sciences of Ukraine (1993-1994)

R. P. Vynogradova , V. M. Danilova

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: valdan@biochem.kiev.ua

The article presents biographical information and research activities of the O. V. Palladin prize laureates. Namely, M. P. Dmytrenko (1941-2011) was awarded the prize in 1993 for the monograph “Purine metabolism and its regulation in lymphocytes” (K. Naukova Dumka, 1991). Z. P. Vasyurenko (born 1937), A. F. Frolov (born in 1931) and V. V. Smirnov (1937-2002) were awarded in 1994 for the monograph “Fatty acid profiles of bacteria which are pathogenic to humans and animals” (K. Naukova Dumka, 1992).