Number Of Cell Aggregates Research Articles

Reducing the danger of phytopathogen toxins by using an organomineral composition

Relevance. Contamination by toxins of phytopathogens or mycotoxins of food and feed raw materials of plant origin poses a serious threat to the production of high-quality and safe agricultural products. Reducing the risks of contamination of plant raw materials with mycotoxins depends on the effective use of biological products to reduce the toxicity of mycotoxins.Methods. Experiments were carried out on primary rat liver cells. Primary liver cells were cultured in DMEM medium in the presence of 10% fetal calf serum at 37OC and 5% CO2. Zearalenone and T-2 toxin were dissolved in a mixture of DMSO and 96% alcohol in a ratio 1:1. Zearalenone T-2 toxin and protective compositions were mixed and aged together for 6 hours and after exposure added to a medium with a cellular monolayer. The concentration of bacterial-based compositions KMBI-3 and KMCI-3 was used for research in three variants: 0.4 mg/ml, 2 mg/ml, 4 mg/ml. After 24 hours of cultivation, the cell layer was evaluated using an inverted microscope according to the following parameters: percentage of surface coverage, cell shape, number of cell aggregates, number of floating cells.Results. A dose-dependent decrease in cell viability was revealed when exposed to zearalenone and T-2 toxin, the most toxic effect was observed at doses from 0.5 х 10-4 and 8.6 х 10-8 M and 2.14 х 10-7 M, respectively. When using the protective composition KMBI-3 at a dose of 4 mg/ml, the least negative effect of zearalenone and T-2 toxin on cell culture was observed. The use of compositions of organomineral origin KMBI-3 helps to increase cell viability when exposed to the toxins zearalenone and T-2 toxin, which indicates the activation of proliferative processes in comparison with the group without the use of drugs. The use of the biopreparation KMBI-3 reduces the pathogenic effect of zearalenone and T-2 toxin on cell culture, increases the resistance of liver cells to the effects of phytopathogen toxins.

Discovery of Cell Aggregate-Inducing Peptides

Most cells within the human body interact with neighboring cells and extracellular matrix (ECM) components to establish a unique 3D organization. These cell–cell and cell–ECM interactions form a complex communication network of biochemical and mechanical signals critical for normal cell physiology. The behavior of cells in a 3D environment is fundamentally different from that of cells in monolayer culture. Aggregation can affect cell–cell interactions, being more representative of the normal tissue microenvironment. Therefore, 3D cell culture technologies have been developed. The general method for cell aggregate is a physical method; it is difficult to control the size and number of cell aggregates. In any case, no chemical method has been discovered yet, so a new method to solve these problems is needed. In this paper, we describe the induction of a cell aggregate of the newly discovered (Lys-Pro)12(KP24) peptide. Since it was revealed that KP24 had cell aggregate-inducing activity, its derivatives were molecularly designed to clarify the importance of the KP24 sequence. We report that cell aggregations were induced by KP24 to form aggregates of fibroblast cells. We evaluated KP24 derivative periodic peptides such as (Lys-Pro-Pro)8(KPP24) and (Lys-Lys-Pro)8(KKP24). The relationship between the structure of the peptide chain and the activity induced by the cell aggregations was investigated from the viewpoint of basic research and the biomedical engineering field.

Lithium, a classic drug in psychiatry, improves nilotinib-mediated antileukemic effects

Although Tyrosine kinase inhibitors (TKIs) that target Bcr-Abl play a key role in Chronic Myeloid Leukemia (CML) therapy, they do not eradicate CML-initiating cells, which lead to the emergence of drug resistance. Here we used the lithium, a GSK-3 inhibitor, to attempt to potentiate the effects of nilotinib against leukemia cells. For this purpose, a K562 leukemia cell line and bone marrow cells from untreated Chronic Myeloid Leukemia (CML) patients, prior to any exposure to TKIs, were used as a model. Our results demonstrated that the combination of lithium + nilotinib (L + N) induced K562-cell death and cleaved caspase-3 when compared to lithium or nilotinib alone, accompanied by GSK-3β phosphorylation and Bcr-Abl oncoprotein levels reduction. Interestingly, these events were related to autophagy induction, expressed by increased LC3II protein levels in the group treated with L + N. Furthermore, the clonogenic capacity of progenitor cells from CML patients was drastically reduced by L + N, as well as lithium and nilotinib when used separately. The number of cell aggregates (clusters), were increased by all treatments (L + N, lithium, and nilotinib). This pioneering research has demonstrated that lithium might be of therapeutic value when targeting Bcr-Abl cells with nilotinib because it triggers cell death in addition to exerting classical antiproliferative effects, opening new perspectives for novel target and therapeutic approaches to eradicate CML.

Pili-Induced Clustering of N. gonorrhoeae Bacteria.

Type IV pili (Tfp) are prokaryotic retractable appendages known to mediate surface attachment, motility, and subsequent clustering of cells. Tfp are the main means of motility for Neisseria gonorrhoeae, the causative agent of gonorrhea. Tfp are also involved in formation of the microcolonies, which play a crucial role in the progression of the disease. While motility of individual cells is relatively well understood, little is known about the dynamics of N. gonorrhoeae aggregation. We investigate how individual N. gonorrhoeae cells, initially uniformly dispersed on flat plastic or glass surfaces, agglomerate into spherical microcolonies within hours. We quantify the clustering process by measuring the area fraction covered by the cells, number of cell aggregates, and their average size as a function of time. We observe that the microcolonies are also able to move but their mobility rapidly vanishes as the size of the colony increases. After a certain critical size they become immobile. We propose a simple theoretical model which assumes a pili-pili interaction of cells as the main clustering mechanism. Numerical simulations of the model quantitatively reproduce the experimental data on clustering and thus suggest that the agglomeration process can be entirely explained by the Tfp-mediated interactions. In agreement with this hypothesis mutants lacking pili are not able to form colonies. Moreover, cells with deficient quorum sensing mechanism show similar aggregation as the wild-type bacteria. Therefore, our results demonstrate that pili provide an essential mechanism for colony formation, while additional chemical cues, for example quorum sensing, might be of secondary importance.

FUNCTIONAL ACTIVITY OF CD34-POSITIVE CELLS IN CHRONIC MYELOID LEUKEMIA PATIENTS WITH DIFFERENT RESPONSE TO IMATINIB THERAPY

It is believed that the reason of the leukemic clone cell resistance to treatment with tyrosine kinase inhibitors during chronic myeloid leukemia (CML) is mutations in the genome of an early bone marrow progenitor cells that are CD34-positive. Such cells, regardless of treatment, acquire ability to proliferation and differentiation. This leads to the re-expansion of the CD34(+) cells. to determine the CD34 antigen expression in bone marrow and peripheral blood cells in CML patients with different response to imatinib therapy using the results of hematopoietic cells culturing and the data of flow cytometry. Bone marrow aspirate from 39 patients who were treated with imatinib was studied with cytogenetic, flow cytometry and culture methods in vitro. In patients with an optimal response to imatinib therapy the number of colonies was 1.8 times lower than the number of those in the group of patients with a suboptimal response to therapy. In turn, in patients with failure of imatinib therapy the number of colonies was the highest and was 2.1 times higher than the patients with optimal response. The results of cytometric studies have shown that the number of CD34(+) cells in bone marrow was significantly higher compared to the number of CD34(+) cells in peripheral blood cells and increased with the acquisition of leukemic cells the resistance to imatinib. There was a direct correlation between the number of colonies and clusters in semisolid agar in vitro and the number of CD34(+) cells in the bone marrow of patients. The correlation between the number of CD34(+) cells and the number of cell aggregates in semisolid agar in vitro indicates the prognostic value of the method for determining CD34(+) cells in the patient bone marrow. The parallel increase of their number in the peripheral blood will allow developing express methods for the detection of individual patient response to imatinib therapy.

Presenilin/γ-Secretase-mediated Cleavage of the Voltage-gated Sodium Channel β2-Subunit Regulates Cell Adhesion and Migration

The voltage-gated sodium channel beta2-subunit (beta2) is a member of the IgCAM superfamily and serves as both an adhesion molecule and an auxiliary subunit of the voltage-gated sodium channel. Here we found that beta2 undergoes ectodomain shedding followed by presenilin (PS)-dependent gamma-secretase-mediated cleavage. 12-O-Tetradecanoylphorbol-13-acetate treatment or expression of an alpha-secretase enzyme, ADAM10, resulted in ectodomain cleavage of beta2 in Chinese hamster ovary cells. Subsequent cleavage of the remaining 15-kDa C-terminal fragment (beta2-CTF) was independently inhibited by three specific gamma-secretase inhibitors, expression of the dominant negative form of PS1, and in PS1/PS2 knock-out cells. gamma-Secretase inhibitor treatment also increased endogenous beta2-CTF levels in neuroblastoma cells and mouse primary neuronal cultures. In a cell-free gamma-secretase assay, we detected gamma-secretase activity-dependent generation of a 12 kDa beta2 intracellular domain (ICD), which was loosely associated with the membrane fraction. To assess the functional role of beta2 processing by gamma-secretase, we tested whether N-[N-(3,5-difluorophenylacetyl-l-alanyl)]-S-phenylglycine t-butylester (DAPT), a specific gamma-secretase inhibitor, would alter beta2-mediated cell adhesion and migration. We found that DAPT inhibited cell-cell aggregation and migration in a wound healing assay carried out with Chinese hamster ovary cells expressing beta2. DAPT also reduced migration of neuroblastoma cells in a modified Boyden chamber assay. Since DAPT treatment resulted in increased beta2-CTF levels, we also tested whether beta2-CTFs or beta2-ICDs would directly affect cell migration by overexpressing recombinant proteins. Interestingly, elevated levels of beta2-CTFs, but not ICDs, also blocked cell migration by 81 to 93%. Together, our findings show for the first time that beta2 is a PS/gamma-secretase substrate and gamma-secretase mediated cleavage of beta2-CTF is required for cell-cell adhesion and migration of beta2-expressing cells.

Chelating compounds as cell wall-loosening agents in cell suspension cultures of Melilotus alba, Desr.

The action of 3 chelating compounds, ethylene diamine tetraacetic acid (EDTA), cyclohexane-1,2-diamine tetraacetic acid (CDTA) and diethylene triamine penta acetic acid (DTPA) on weakening the intercellular cement, the calcium pectate, and inducing cell dissociation in cell suspension culture of Melilotus has been investigated. The action was assessed by determining the percentage of free cells, size and number of cell aggregates in suspension cultures treated with different concentrations of chelating compounds. Application of low Ca level together with proper concentrations of EDTA to the cell cultures promoted cell separation. CDTA gave higher percentage of free cells when applied to the cell cultures in comparison with EDTA and DTPA. It is suggested that beside Ca other components in the intercellular cement are responsible for cell aggregation.

Studies on colony formation in vitro by mouse bone marrow cells. I. Continuous cluster formation and relation of clusters to colonies.

AbstractColony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7‐day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.