Effect of Rho-associated kinase inhibition on actin cytoskeleton structure and calcium response in glioma C6 cells

The role of actin cytoskeleton functional state in glioma C6 cell morphology and calcium signaling was investigated through modification of myosin II activity by blocking Rho-associated kinase with the specific inhibitor Y-27632. Treatment of glioma C6 cells with ROCK inhibitor resulted in actin cytoskeleton reorganization and also in the changed shape and distribution of mitochondria. Changes in the distribution of ER, the main calcium store in glioma C6 cells, were not visible. The inhibition of myosin II activity influences the first phase of calcium signaling evoked by agonist, and both phases of thapsigargin-evoked calcium response. We suggest that the observed increase in Ca2+ release from intracellular stores induced by IP3 formation as well as inhibition of SERCA ATPase is at least in part related to severely affected mitochondria. Enhancement of capacitative calcium entry evoked by thapsigargin is probably associated with the reorganization of the acto-myosin II system. ATP-induced calcium response presents no changes in the second phase. We observed that ATP stimulation of Y-27632 pretreated cells leads to immediate morphological rearrangement of glioma C6 cells. It is a consequence of actin cytoskeleton reorganization: formation of stress fibers and relocation of phosphorylated myosin II to actin filaments. It seems that the agonist-evoked strong calcium signal may be sufficient for myosin II activation and the stress fiber organization. This is the first work showing the dependence between the functional state of the acto-myosin II system and calcium signaling stressing the reversible character of this relationship.     

The effect of new non-cross resistant antitumour agents on the energy state of human erythrocytes

Multidrug resistance (MDR) of tumour cells is related to the overexpression of ATP-dependent pumps responsible for the active efflux of antitumour agents out of resistant cells. Benzoperimidine and anthrapyridone compounds exhibit comparable cytotoxic activity against sensitive and MDR tumour cells. They diffuse extremely rapidly across the plasma membrane and render the ATP-dependent efflux inefficient. Such uptake could disturb an energy metabolism of normal cells possessing an elevated level of ATP-dependent proteins, especially erythrocytes having a high level of the MRP1, MRP4 and MRP5 proteins. In this study the effect of five antitumour agents: benzoperimidine (BP1), anthrapyridones (CO1, CO7) and reference drugs used in the clinic: doxorubicin (DOX) and pirarubicin (PIRA), on the energetic state in human erythrocytes has been examined. These compounds have various types of structure and kinetics of cellular uptake (slow--DOX, CO7, moderate--PIRA, fast--BP1, CO1) resulting in their different ability to saturate ATP-dependent transporters. The energetic state of erythrocytes was examined by determination of purine nucleotide contents (ATP, ADP, AMP), NAD(+) and values of adenylate energy charge (AEC) using an HPLC method. It was found that the level of nucleotides as well as the AEC value of erythrocytes were not changed during 24 h of incubation with these agents independently of their structure and ability to saturate ATP-dependent pumps. This is a very promising result in view of their potential use in the clinic as antitumour drugs against multidrug resistant cancers.     

Biochemical characterization and structural insight into aliphatic β‐amino acid adenylation enzymes IdnL1 and CmiS6

Macrolactam antibiotics such as incednine and cremimycin possess an aliphatic β‐amino acid as a starter unit of their polyketide chain. In the biosynthesis of incednine and cremimycin, unique stand‐alone adenylation enzymes IdnL1 and CmiS6 select and activate the proper aliphatic β‐amino acid as a starter unit. In this study, we describe the enzymatic characterization and the structural basis of substrate specificity of IdnL1 and CmiS6. Functional analysis revealed that IdnL1 and CmiS6 recognize 3‐aminobutanoic acid and 3‐aminononanoic acid, respectively. We solved the X‐ray crystal structures of IdnL1 and CmiS6 to understand the recognition mechanism of these aliphatic β‐amino acids. These structures revealed that IdnL1 and CmiS6 share a common recognition motif that interacts with the β‐amino group of the substrates. However, the hydrophobic side‐chains of the substrates are accommodated differently in the two enzymes. IdnL1 has a bulky Leu220 located close to the terminal methyl group of 3‐aminobutanoate of the trapped acyl‐adenylate intermediate to construct a shallow substrate‐binding pocket. In contrast, CmiS6 possesses Gly220 at the corresponding position to accommodate 3‐aminononanoic acid. This structural observation was supported by a mutational study. Thus, the size of amino acid residue at the 220 position is critical for the selection of an aliphatic β‐amino acid substrate in these adenylation enzymes. Proteins 2017; 85:1238–1247. © 2017 Wiley Periodicals, Inc.     

2′, 3′-<Emphasis Type="Italic">O</Emphasis>-(4-benzoylbenzoyl)–ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y<Subscript>2</Subscript> nucleotide receptor

In this study, we examined the response of glioma C6 cells to 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X₇ receptor activity. We show here that in the absence of extracellular Ca²⁺, BzATP-generated increase in [Ca²⁺]_i via Ca²⁺ release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca²⁺ response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X₇ receptor, did not reduce any of the two components of the Ca²⁺ response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca²⁺ elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y₂ but not the P2X₇ receptors.     

Nitric oxide complexes in the interaction between primary and secondary tumor of L5178Y lymphoma.

Heme and non-heme Fe-NO complexes were observed in regard to the growth of primary and secondary solid tumors and ascites of murine L5178Y lymphoma. The complexes were detected by electron paramagnetic resonance spectroscopy at liquid nitrogen temperature. Primary solid tumors and secondary solid tumors or ascites were inoculated on the same day, or with a delay. The primary tumor inhibited growth of the secondary solid tumor only if the latter was inoculated with a delay, which did not correlate with the change of the types, nor with the increase in the level of Fe-NO complexes detected in the tissue, suggesting a "non-immunological" character of this inhibition. In some animals with solid tumors, spontaneous ascites developed. This process resulted in a marked decrease in the level of Fe-NO complexes in the solid tumor tissue. The primary solid tumor, however, did not influence the growth of secondary ascites, but intensified NO generation in the ascites of animals with partial removal of ascitic fluid. This experimental group survived 2.2 days longer than the control group without primary solid tumor. Our research revealed that the presence of Fe-NO complexes in the interaction between primary and secondary tumor strongly depends on the form of the tumor: solid or ascitic, and that murine L5178Y lymphoma may serve as a convenient model for the research on "concomitant immunity" against in vivo growing tumors. This is the first EPR study on "concomitant immunity" in regard to tumor-tumor and tumor-ascites interactions in vivo.     

ParA of Mycobacterium smegmatis co‐ordinates chromosome segregation with the cell cycle and interacts with the polar growth determinant DivIVA

Mycobacteria are among the clinically most important pathogens, but still not much is known about the mechanisms of their cell cycle control. Previous studies suggested that the genes encoding ParA and ParB (ATPase and DNA binding protein, respectively, required for active chromosome segregation) may be essential in Mycobacterium tuberculosis. Further research has demonstrated that a Mycobacterium smegmatis parB deletion mutant was viable but exhibited a chromosome segregation defect. Here, we address the question if ParA is required for the growth of M. smegmatis, and which cell cycle processes it affects. Our data show that parA may be deleted, but its deletion leads to growth inhibition and severe disturbances of chromosome segregation and septum positioning. Similar defects are also caused by ParA overproduction. EGFP–ParA localizes as pole‐associated complexes connected with a patch of fluorescence accompanying two ParB complexes. Observed aberrations in the number and positioning of ParB complexes in the parA deletion mutant indicate that ParA is required for the proper localization of the ParB complexes. Furthermore, it is shown that ParA colocalizes and interacts with the polar growth determinant Wag31 (DivIVA homologue). Our results demonstrate that mycobacterial ParA mediates chromosome segregation and co‐ordinates it with cell division and elongation.     

High genetic differentiation among wild populations of alien Medicago sativa in Lithuania

Alfalfa (Medicago sativa; =M. sativa ssp. sativa) in Lithuania is sown as albuminous forage for cattle due to favourable climatic condition. Over many generations, alfalfa plants have escaped from cultivation fields into natural ecosystems and established wild populations. We collected and analyzed individuals from seventeen wild populations of M. sativa. Using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses, 117 RAPD and 64 ISSR reproducible and highly polymorphic (90.8% for RAPD and 86.3% for ISSR) loci were established. AMOVA showed a high genetic differentiation of M. sativa populations for both types of DNA markers utilized. According to RAPD markers, the genetic variability among populations was 63.1% and 57.0% when ISSR markers were used. Taken together, these results demonstrate that wild populations of M. sativa possess a high potential of genetic variability, that could potentially result in colonization of natural ecosystems. The UPGMA cluster analysis also showed that the DNA markers discovered in this study can distinguish between M. sativa and M. falcata (=M. sativa ssp. falcata) populations and therefore may be used to study the genetic impact of M. sativa on the native populations of M. falcata.     

Varied approach of using MSCs for bovine embryo in vitro culture

The objective of the present study was to evaluate the effect of porcine Mesenchymal Stem Cells (MSCs) secreted factors on bovine in vitro embryo development by using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer and in reverse drop and by embryo culture in co-culture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in co-culture with MSCs (p?<?0.0001), and between SOF and SOF conditioned (monolayer and drop) (p?<?0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analysed by TUNEL only in two out of four experimental groups: SOF and SOF in co-culture with MSCs. There were no significant differences between any of analysed blastocysts’ groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in co-culture with MSC turned out to be effective.     

Expression and functional characterization of P2Y1 and P2Y12 nucleotide receptors in long-term serum-deprived glioma C6 cells

We characterized the expression and functional properties of the ADP-sensitive P2Y(1) and P2Y(12) nucleotide receptors in glioma C6 cells cultured in medium devoid of serum for up to 96 h. During this long-term serum starvation, cell morphology changed from fibroblast-like flat to round, the adhesion pattern changed, cell-cycle arrest was induced, extracellular signal-regulated kinase (ERK1/2) phosphorylation was reduced, Akt phosphorylation was enhanced, and expression of the P2Y(12) receptor relative to P2Y(1) was increased. These processes did not reflect differentiation into astrocytes or oligodendrocytes, as expression of glial fibrillary acidic protein and NG2 proteoglycan (standard markers of glial cell differentiation) was not increased during the serum deprivation. Transfer of the cells into fresh medium containing 10% fetal bovine serum reversed the changes. This demonstrates that serum starvation caused only temporary growth arrest of the glioma C6 cells, which were ready for rapid division as soon as the environment became more favorable. In cells starved for 72 and 96 h, expression of the P2Y(1) receptor was low, and the P2Y(12) receptor was the major player, responsible for ADP-evoked signal transduction. The P2Y(12) receptor activated ERK1/2 kinase phosphorylation (a known cell proliferation regulator) and stimulated Akt activity. These effects were reduced by AR-C69931MX, a specific antagonist of the P2Y(12) receptor. On the other hand, Akt phosphorylation increased in parallel with the low expression of the P2Y(1) receptor, indicating the inhibitory role of P2Y(1) in Akt pathway signaling. The shift in nucleotide receptor expression from P2Y(1) to P2Y(12) would appear to be a new and important self-regulating mechanism that promotes cell growth rather than differentiation and is a defense mechanism against effects of serum deprivation.