[Can Analysis of Cellular Lipidome Contribute to Discrimination of Tumour and Non-tumour Colon Cells?] / Muze analýza bunecného lipidomu prispet k rozlisení nádorových a nenádorových bunek tlustého streva?

BACKGROUNDS: Colon cancer development is often characterized by abnormalities in lipid synthesis and metabolism, which may influence energetic balance, structure and function of biological membranes, or production of specific mediators and cell signalling. The changes in lipid profile and metabolism (lipidome) may significantly affect cell behaviour and response to therapy. Permanent epithelial cell lines at various stages of cancer development are used for better understanding of this topic on cellular and molecular levels. In our study, we hypothesized that detailed analyses of colon cancer cell line lipidomes may help to identify major alterations in the amount and profile of specific lipid classes/species, which can contribute to their different response to various stimuli. MATERIAL AND METHODS: Cellular lipids were isolated from six human epithelial cell lines derived from tissues at various stages of tumour development. Liquid chromatography coupled with tandem mass spectometry analyses were performed in order to determine amount and mass profiles of all phospholipid (PL), lysophospholipid (lysoPL) and sphingolipid classes. The data was statistically evaluated (cluster and discrimination analyses) with respect to mutual comparison of cell lines and to significantly discriminating lipid types. RESULTS: The results of cluster analysis arranged cell lines in order corresponding to their level of transformation (normal cells, adenoma, carcinoma and lymph node metastasis). The results of discrimination analyses revealed the most discriminating lipid types and distinction in PL: lysoPL ratios. Particularly, significant correlation of the amount and profiles of both specific lysoPL and sphingolipid classes with cell transformation level were observed. Similar approaches are now applied to compare lipidomes of colon epithelial cells isolated from tumour vs. non-tumour samples of colon cancer patients. CONCLUSION: Our results indicate that a) selected cancer cell lines are suitable model for lipidomic studies that can serve as a basis for subsequent clinical research, b) cellular lipidome analyses may help to discriminate tumour and non-tumour cells in clinical samples, where specific types of lipids could serve as biomarkers.Key words: colon cancer - cell lines - liquid chromatography - mass spektrometry - phospholipids - sphingolipids - bioinformatics The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This work was supported by Czech Health Research Council, grant No. AZV 15-30585A.Submitted: 19. 3. 2018Accepted: 18. 4. 2018.

Non-dioxin-like organic toxicant PCB153 modulates sphingolipid metabolism in liver progenitor cells: its role in Cx43-formed gap junction impairment.

The non-dioxin-like environmental toxicant 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), member of a group of persistent organic pollutants wide-spread throughout the environment, reduces gap junction intercellular communication (GJIC), an event possibly associated with tumor promotion. Since very few studies have investigated the signaling effectors and mode(s) of action of PCB153, and it is known that the gap junction (GJ) protein Cx43 can be regulated by the bioactive sphingolipid (SL) sphingosine 1-phosphate (S1P), this in vitro study mainly addresses whether SL metabolism is affected by PCB153 in rat liver epithelial WB-F344 cells. PCB153 treatment obtained significant changes in the S1P/ceramide (Cer) ratio, known to be crucial in determining cell fate. In particular, an increase in S1P at 30 min and a decrease of the bioactive lipid at 3 h were observed, whereas Cer level increased at 1 h and 24 h. Notably, a time-dependent modulation of sphingosine kinase (SphK), the enzyme responsible for S1P synthesis, and of its regulators, ERK1/2 and protein phosphatase PP2A, supports the involvement of these signaling effectors in PCB153 toxicity. Electrophysiological analyses, furthermore, indicated that the lipophilic environmental toxicant significantly reduced GJ biophysical properties, affecting both voltage-dependent (such as those formed by Cx43 and/or Cx32) and voltage-independent channels, thereby demonstrating that PCB153 may act differently on GJs formed by distinct Cx isoforms. SphK down-regulation alone induced GJIC impairment, and, when combined with PCB153, the acute effect on GJ suppression was additive. Moreover, after enzyme-specific gene silencing, the SphK1 isoform appears to be responsible for down-regulating Cx43 expression, while being the target of PCB153 at short-term exposure. In conclusion, we provide the first evidence of novel effectors in PCB153 toxic action in rat liver stem-like cells, leading us to consider SLs as potential markers for preventing GJIC deregulation and, thus, the tumorigenic action elicited by this environmental toxicant.

[SILS appendectomy for acute appendicitis - two-year experience]. / SILS apendektómia pre akútnu apendicitídu - dvojrocné skúsenosti.

INTRODUCTION: The study aims to present our two-year experience with SILS (Single Incision Laparoscopic Surgery) appendectomy in patients operated on for acute appendicitis symptoms. The results obtained were analysed and then compared with patients operated by standard laparoscopy, as well as with data available from the published literature with emphasis on safety and advantages of a new operating technique. MATERIAL AND METHODS: A retrospective analysis of patients operated on at the Third Department of Surgery, Slovak Medical University, the Kosice-Saca Hospital, a.s., in the past two years was performed. Age, sex, BMI, length of operation, post-operative hospitalization period, occurrence of pre- and post-operative complications were evaluated. RESULTS: In the period from 1 November 2009 to 31 October 2011, 116 patients were operated on by the authors, 47 of them by the SILS technique. The group included 26 males and 21 females with average age of 37.13 years (18-80) and average BMI 26.3 kg/m2 (18-47.1). Average length of the operation was 54.81 minutes (30-100). The length of post-operative hospital stay was 3.83 days (2-6). An abscess in the surgical wound was found in three patients. One patient had to be reoperated due to a pericaecal abscessed hematoma. Incisional hernia was not observed in our group. CONCLUSION: Our results are comparable with the data from international published literature and confirm that the SILS appendectomy is a safe method and represents an appropriate alternative to the standard laparoscopic technique. It is suitable for surgeons with advanced experience in laparoscopy. Apart from the excellent cosmetic effect, other advantages or disadvantages in comparison with standard laparoscopy will require confirmation by prospective randomized studies.

Some biological actions of PEG-conjugated RNase A oligomers.

Previously we have shown that monomeric RNase A has no significant biological activity, whereas its oligomers (dimer to tetramer) prepared by lyophilizing from 50% acetic acid solutions, show remarkable aspermatogenic and antitumor activities. Furthermore, conjugates prepared by chemical binding of native RNase A to polyethylene glycol (PEG) have shown a significant aspermatogenic and antitumor activities. In this work we show that the chemical conjugation of PEG to the RNase A C-dimer, and to the two RNase A trimers (NC-trimer and C- trimer) decreases the aspermatogenic activity of the oligomers while increasing their inhibitory activity on the growth of the human UB900518 amelanotic melanoma transplanted in athymic nude mice. Moreover, the PEG-conjugated RNaseA oligomers are devoid, like the free oligomers, of any embryotoxic activity.

Blood phagocyte activation during open heart surgery with cardiopulmonary bypass.

Open heart surgery with a cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response which significantly contributes to adverse postoperative complications. The purpose of this study was to characterize the activation of blood phagocytes during open heart surgery with CPB. Blood samples were collected during and up to 24 h after surgery. The production of reactive oxygen species (ROS) in whole blood, the expression of surface molecules by blood phagocytes and complement activity in the plasma were determined. A cDNA microarray analysis of leukocyte RNA profile of genes was performed related to the inflammatory response. Activation of the complement was already observed at the beginning of CPB. This was followed by an increase in the neutrophil number and in both spontaneous and opsonized zymosan-activated ROS production after the onset of reperfusion. The activation of blood phagocytes was affirmed by changes in surface receptors involved in the adhesion and migration of leukocytes (CD11b, CD62L and CD31). Gene arrays also confirmed the activation of leukocytes 4 h after reperfusion. In conclusion, open heart surgery with a cardiopulmonary bypass was found to be associated with a rapid and pronounced activation of blood phagocytes and complement activation which was partly independent at the onset of CPB.

Protopine hydrochloride.

Protopine hydrochloride (5,6,14,14a-tetrahydro-14a-hydroxy-7-methyl-8H-bis[1,3]benzodioxolo[5,6-a:4,5-g]quinolizinium chloride, C20H20NO5(+)-Cl(-)) is the salt of the isoquinoline alkaloid protopine. It is formed by the action of dilute hydrochloric acid on the protopine free base. The N-methyl and hydroxyl groups are in a trans configuration in the quinolizine ring and the central quinolizine N-C bond is unusually long [1.579 (2) A]. The crystal is a racemate.

Uncoupling p70(s6) kinase activation and proliferation: rapamycin-resistant proliferation of human CD8(+) T lymphocytes.

Rapamycin is a fungal macrolide that inhibits the proliferation of T cells. Studies in both animals and humans have found that rapamycin significantly reduces graft rejection. However, though CD8(+) T cells are involved in graft infiltration and rejection, little is known regarding the effects of rapamycin on CD8(+) human T cell responses. In this study, we examined the mechanism of rapamycin-induced inhibition of Ag-driven activation of CD8(+) T cells. Surprisingly, a heterogeneous proliferative response in the presence of rapamycin was observed among different Ag-specific CD8(+) T cell clones; this was also observed in CD8(+) peripheral blood T cells activated with TCR cross-linking ex vivo. Inhibition of T cell proliferation by rapamycin was controlled by both the strength of signal delivered through the Ag receptor as well as the specific costimulatory signals received by the T cell. Rapamycin-resistant proliferation occurred despite inhibition of p70(s6) kinase activity. Moreover, rapamycin-resistant proliferation of the CD8(+) T cell clones was blocked by anti-IL-2 Abs, suggesting that while some of the parallel pathways triggered by IL-2R signaling are sensitive to the effects of rapamycin, others account for the Ag-driven rapamycin resistance. These data provide a new framework for examining the specific mechanism of action of rapamycin in human disease.

CD28/CTLA-4 and CD80/CD86 families: signaling and function.

T cell stimulation in the absence of a second, costimulatory signal can lead to anergy or the induction of cell death. CD28 is a major T cell costimulatory receptor, the coengagement of which can prevent anergy and cell death. The CD28 receptor is a member of a complex family of polypeptides that includes at least two receptors and two ligands. Cytotoxic lymphocyte-associated molecule-4 (CTLA-4, CD152) is the second member of the CD28 receptor family. The ligands or counterreceptors for these two proteins are the B7 family members, CD80 (B7-1) and CD86 (B7-2). This article reviews the CD28/CTLA4 and CD80/CD86 families, and outlines the functional outcomes and biochemical signaling pathways recruited after CD28 ligation.

CD80 and CD86 are not equivalent in their ability to induce the tyrosine phosphorylation of CD28.

Ligation of either CD80 (B7-1) or CD86 (B7-2), two principal ligands for CD28, is thought to skew the immune response toward Th1 or Th2 differentiation. We have examined early signal transduction pathways recruited following T cell stimulation with either CD80 or CD86. Purified human peripheral T cells or Jurkat T cells were stimulated with Chinese hamster ovary (CHO) cells expressing either human CD80 (CHO-CD80) or human CD86 (CHO-CD86) or with anti-CD28 monoclonal antibody (mAb). In the presence of phorbol 12-myristate 13-acetate, both CHO-CD80 and CHO-CD86, like anti-CD28 mAb, were capable of stimulating cytokine production from both human peripheral T cells and Jurkat T cells. Both CHO-CD80 and CHO-CD86, in the presence of anti-CD3 mAb, costimulated NFAT-dependent transcriptional activation. Several intracellular signaling proteins, such as CBL and VAV, were phosphorylated on tyrosine in response to CD80, CD86, and anti-CD28 mAb. Surprisingly, although stimulation of Jurkat T cells with either CHO-CD80 or anti-CD28 mAb resulted in robust tyrosine phosphorylation of CD28 itself, ligation with CHO-CD86 was unable to induce detectable CD28 tyrosyl phosphorylation over a range of stimulation conditions. In addition, the association of phosphoinositide 3-kinase with CD28 and enhanced tyrosine phosphorylation of phospholipase Cgamma were seen after anti-CD28 mAb and CHO-CD80 stimulation but to a much lesser extent after CHO-CD86 stimulation. Thus, ligation of CD28 with either CD80 or CD86 leads to shared early signal transduction events such as the tyrosine phosphorylation of CBL and VAV, to NFAT-mediated transcriptional activation, and to the costimulation of interleukin-2 and granulocyte-macrophage colony-stimulating factor production. However, CD80 and CD86 also induce distinct signal transduction pathways including the tyrosine phosphorylation of CD28 and phospholipase Cgamma1 and the SH2-dependent association of phosphoinositide 3-kinase with CD28. These quantitative, if not qualitative, differences between signaling initiated by these two ligands for CD28 may contribute to functional differences (e.g. Th1 or Th2 differentiation) in T cell responses.

Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic.

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.

2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein as a dual-emission fluorescent indicator of intracellular pH suitable for argon laser confocal microscopy.

The widely used fluorescent probe 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) serves as a pH-sensitive indicator in classical microscopy. Characteristics of BCECF were studied and a way of employing the probe in a confocal laser scanning microscope equipped with an argon laser at 488 nm was developed, based on the fact that the emission fluorescence spectra are pH-dependent with spectral maximum shift from 518 to 529 nm. Optical filters for the dual-emission ratio method were set to 506 and 529 nm. pH values measured inside a single cell of Saccharomyces cerevisiae were similar to those obtained with other pH estimation methods.

Fusion of macrophages on an implant surface is associated with down-regulated expression of ligands for galectin-1 and -3 in the rat.

Galectins have a wide range of biological activities which are elicited by binding to appropriate glycoligands. Besides regulation of the expression of the galectins the extent of the presence of suitable binding sites will be relevant to infer the cellular responsiveness to this class of sugar receptors. Thus ligand presentation requires monitoring by the tissue lectin. We demonstrate the expression of galectin-3 by macrophages and foreign-body giant multinucleate cells colonizing a cellophane implant in the rat by the A1D6 monoclonal antibody. The extents of ligand presence are visualized in the same cells by biotinylated galectin-3 and also by galectin-1 which is produced by diverse mammalian cell types and widely distributed. Labeled mistletoe (VAA) and tomato (LEA) lectins are used as tools to assess the degree of similarity of the binding profile between endogenous and exogenous proteins. The presentation of alpha-galactosides is monitored with a natural immunoglobulin G subfraction obtained by two consecutive affinity chromatography steps. The binding of labeled galectins and plant lectins was significantly lower to foreign-body giant multinucleate cells than to mononuclear macrophages. The application of the alpha-galactoside-specific probe yielded no significant staining. The potential problem of epitope accessibility could be excluded by the concomitant positivity obtained with an IgG subfraction with selectivity to beta-galactosides also obtained by affinity chromatography. These results provide no evidence for a role of alpha-galactosides for the binding of galectins in the rat macrophages colonizing the implant. The reduced level of expression of glycoligands for galectin-1 and -3 in foreign-body giant multinucleate cells in contrast with the mononuclear macrophages suggests an inhibitory influence of macrophage fusion on the expression of galectin-reactive molecules.

Uncoupling activation-dependent HS1 phosphorylation from nuclear factor of activated T cells transcriptional activation in Jurkat T cells: differential signaling through CD3 and the costimulatory receptors CD2 and CD28.

CD3, CD2, and CD28 are functionally distinct receptors on T lymphocytes. Engagement of any of these receptors induces the rapid tyrosine phosphorylation of a shared group of intracellular signaling proteins, including Vav, Cbl, p85 phosphoinositide 3-kinase, and the Src family kinases Lck and Fyn. Ligation of CD3 also induces the tyrosine phosphorylation of HS1, a 75-kDa hematopoietic cell-specific intracellular signaling protein of unknown function. We have examined changes in HS1 phosphorylation after differential stimulation of CD3, CD2, and CD28 to elucidate its role in T cells and to further delineate the signaling pathways recruited by these receptors. Unlike ligation of CD3, stimulation with anti-CD28 mAb or CHO cells expressing the CD28 ligands CD80 or CD86 did not lead to tyrosine phosphorylation of HS1 in Jurkat T cells. Additionally, no tyrosine phosphorylation of HS1 was induced by mitogenic pairs of anti-CD2 mAbs capable of activating the transcription factor NFAT (nuclear factor of activated T cells). Costimulation through CD28 and/or CD2 did not modulate the CD3-dependent phosphorylation of HS1. In vivo studies indicated that CD3-induced HSI phosphorylation was dependent upon both the Src family tyrosine kinase Lck and the tyrosine phosphatase CD45, did not require MEK1 kinase activity, and was regulated by protein kinase C activation. Thus, although CD3, CD28, and CD2 activate many of the same signaling molecules, they differed in their capacity to induce the tyrosine phosphorylation of HSI. Furthermore, activation-dependent tyrosine phosphorylation of HS1 was not required for NFAT transcriptional activation.

Bond strength of photocured composite resin facings: clinical versus laboratory procedures.

The tensile strengths of laboratory versus clinical photocured composite resins have been investigated. Metal surfaces were bonded to photocured composite resin by either retentive beads, Sebond or Silicoating. The bond strengths were measured by an Instron machine and the fracture sites observed. Following repair with the adhesives Dentacolor Opaquer, Fusion and Cover up 2, the Instron measurements were repeated. The metal/facing bond strength was the highest in samples fabricated by the Silicoating technique, the bond strength exceeding the cohesive forces in the composite resin facing. The tensile strengths of metal/facing bonded by Sebond and retentive beads were similar. Fractures that include the facing and partially reveal the metal are the least resistant to tension after repair. Renewal of the Opaque layer and completion of the facing is superior to any of the repair methods used in this paper. The original fracture point is the weakest after repair. All the repair kits tested were inferior to the original restoration materials.

Measurement of membrane potential in Saccharomyces cerevisiae by the electrochromic probe di-4-ANEPPS: effect of intracellular probe distribution.

Changes in the membrane potential of Saccharomyces cerevisiae were monitored by the electrochromic probe 3-(4-(2-(6-(dibutylamino)-2-naphthyl)-trans- ethenyl)pyridinium)propanesulfonate (di-4-ANEPPS) that should incorporate into the plasma membrane. The probe had suitable spectral characteristics and exhibited an electrochromic shift upon a change in membrane potential but the magnitude of the response increased with time. The presence and properties of the cell wall affected the extent of cell staining. The time dependence of the fluorescent response indicated that the probe was not incorporated solely into the plasma membrane but spread gradually into the whole cell; this was confirmed by confocal microscopy. The probe is therefore suitable for assessing membrane potential changes only over time intervals up to 30 min. Longer monitoring will require either a modified staining protocol or a derivatization of the probe molecule. As found by using the dioctyl derivative di-8-ANEPPS, extending the aliphatic chains of the di-4-ANEPPS molecule does not prevent the dye from penetrating into the cell or liposome interior and, in addition, impairs staining.

Distribution of individual cytoplasmic pH values in a population of the yeast Saccharomyces cerevisiae.

Fluorescence ratio imaging microscopy using pH-sensitive fluorescent dyes makes it possible to evaluate statistical distribution of intracellular pH in a population of the yeast S. cerevisiae examined in a thin layer of suspension in a Petri dish. The distribution appears to fit a Gaussian curve with a half-width around the 0.4 pH unit. The curve became slightly narrower after resuspension in a strong buffer; the mean values shifted with the pH of the buffer. The shape of the distribution curves of both resting and growing cells in various phases of growth does not change significantly. Likewise, addition of 1% of glucose, 50 microM suloctidil or 100 microM diethylstilbestrol brings about no alteration. The only value which clearly changes is the average cytoplasmic pH.

[CADISO (Cardiovascular Diseases and Stability of the Organism): 10 years of integrated research on natural substances at the Charles University Pharmacy School in Hradec Králové]. / CADISO: 10 let integrovaného výzkumu prírodních látek na Farmaceutické fakulte UK v Hradci Králové.

The research project CADISO (Cardiovascular Diseases and Stability of the Organism--Phytotherapeutical Possibilities) is an alliance board associating approximately 20 professional institutions, its centre and coordinating laboratory being the Department of Pharmaceutical Botany and Ecology, Faculty of Pharmacy, Charles University, Hradec Králové. It aims at the prevention (additives in food industry) and therapy of diseases of the circulatory apparatus using phytotherapeutic agents and substances isolated from higher plants and fungi. The project is divided into three systems of investigation: ADAPRETE, i.e. the substances increasing the nonspecific resistance of the organism (adaptogenes of plant origin, immunostimulants), DIACORD, dealing with the substances acting on the cardiovascular system in a mediated way, i.e. treating the diseases the eventual action of which alters the heart and vessels (antihypercholesterolemics, antihyperlipidemics, antidiabetics, anti-oxidants and quenchers of free radicals, hepatoprotectives, anti-aggregating agents), and CORCORAN studying the taxons and substances isolated from them which act directly on the circulatory system (cardiotonic, anti-arrhythmic and vasodilating agents). To achieve a real purpose of the project, the linking-up of the phytochemical, pharmacological-toxicological, pharmaceutical-technological fields, production of raw materials and legislation is ensured.