UNASSIGNED: The exposure of HeLa cells to interleukin-1 alpha (IL-1α) in the presence of cycloheximide (CHX) leads to the release of active tumor necrosis factor alpha (TNF-α), eliciting cytocidal effect on these cells. A mass spectrometry (MS)-based analysis of the qualitative proteomic profiles of the HeLa cells treated only with IL-1α, CHX or simultaneously with IL-1α and CHX, in comparison to an untreated control, enabled to distinguish protein candidates possibly involved in this process. Among them protein disulphide isomerase (PDI) seemed to be particularly interesting for further research. Therefore, we focused on quantitative changes of PDI levels in HeLa cells subjected to IL-1α and CHX. Enzyme-linked immunosorbent assay (ELISA) was employed for determination of PDI concentrations in the investigated, differently treated HeLa cells. The obtained results confirmed up-regulation of PDI only in the cells stimulated with IL-1α alone. In contrary, the PDI levels in HeLa cells exposed to both IL-1α and CHX, where apoptotic process was intensive, did not increase significantly. Finally, we discuss how different expression levels of PDI together with other proteins, which were detected in this study, may influence the induction of cytotoxic effect and modulate sensitivity to cytotoxic action of IL1.
UNASSIGNED:

Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by a hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in Arabidopsis thaliana Here, we analyzed the effects of various inhibitors on SE fusion in an in vitro culture system. Different from other cell fusions, neither disruption of actin polymerization nor protein secretion impaired SE fusion. However, transcriptional and translational inhibitors decreased the SE fusion success rate and also inhibited endosperm division. Failures of SE fusion and endosperm nuclear proliferation were also induced by roscovitine, an inhibitor of cyclin-dependent kinases (CDK). These data indicate unique aspects of SE fusion such as independence of filamentous actin support and the importance of CDK-mediated mitotic control.

Maternal-to-zygotic transition is an event that developmental control of early embryos is switched from oocyte-derived factors to the zygotic genome. Ability to inhibit DNA replication, transcription, and translation is an important tool in studying events, such as zygotic genome activation, during embyogenesis. Here, we describe approaches to block DNA replication, transcription, and translation using chemical inhibitors. Then we also demonstrate how the transcript level of a maternally inherited gene, ten-eleven translocation methylcytosine dioxygenase 3, responses to the chemical treatments.

BACKGROUND: Endodontic therapy is one of the commonly used procedures for treating the teeth affected by various pathologies. One of the major problems for endodontists despite the advancements in the root canal procedures is the posttreatment endodontic flare-ups. Much debate exists regarding the completion of endodontic therapy in a single sitting or multiple sittings. Hence, we assessed the incidence of endodontic flare-ups in patients undergoing single-sitting root canal therapies.
METHODS: The present study included 200 patients who underwent single-sitting endodontic therapy. Clinical details and conditions of each and every tooth of every patient were recorded before and after the completion of endodontic therapy. Irrigation during the root canal procedures was done by 2.5% NaOCl solution in most of the cases while others were irrigated with various combinations of ethylenediaminetetraacetic acid (EDTA) and cycloheximide (CHX) solutions. Follow-up records and readings of the patents were noted and were subjected to statistical analysis.
RESULTS: Four groups were formed which divided the patients equally on the basis of their age. Out of 50 patients in the age group of 21 to 30 years, only 4 showed posttreatment endodontic flare-ups, while no endodontic flare-up was recorded in patients with age group of 31 to 50 years. Only two male and four females showed flare-ups postoperatively. A nonsignificant correlation was obtained when flare-up cases were compared on the basis of type of irrigation solution used during canal preparation.
CONCLUSIONS: Single-sitting endodontic therapy appears to be a successful procedure with good prognosis and minimal posttreatment flare-up results, even in patients with periapical pathologies.
CONCLUSIONS: Single-sitting root canal procedures can be successfully carried in patients with vital or nonvital pulp tissues and also in patients with periapical lesions.

High spatially resolved Raman microscopy was applied to study the early apoptosis in endothelial cells and chemical and structural changes induced by this process. Application of cluster analysis enabled separation of signals due to various subcellular organelles and compartments such as the nuclei, nucleoli, endoplasmic reticulum or cytoplasm and analysis of alterations locally at the subcellular level. Different stimuli, i.e. Fas ligand, a tumor necrosis factor, and cycloheximide, an inhibitor of eukaryotic protein biosynthesis, were applied to induce apoptotic mechanisms. Due to different mechanisms of action, the changes observed in subcellular structures were different for FasL and cycloheximide. Although in both cases a statistically significant decrease of the protein level was observed in all studied cellular structures, the increase of the nucleic acids content locally in apoptotic nuclei was considerably more pronounced upon FasL-induced apoptosis compared to the cycloheximide one. Additionally, apoptosis invokes also a decrease of the proteins with the α-helix protein structure selectively for FasL in the cytoplasm and endoplasmic reticulum.

Clinical approaches for tumor treatment often rely on combination therapy where a DNA damaging agent is used in combination with a DNA repair protein inhibitor. For this reason, great efforts have been made during the last decade to identify inhibitors of DNA repair proteins or, alternatively, small molecules that specifically alter protein stability or trafficking. Unfortunately, when studying these drug candidates, classical biochemical approaches are prone to artifacts. The apurinic/apyrimidinic endonuclease (APE1) protein is an essential component of the base excision repair (BER) pathway that is responsible for repairing DNA damage caused by oxidative and alkylating agents. In this work, we combined conditional gene expression knockdown of APE1 protein by RNA interference (RNAi) technology with re-expression of an ectopic recombinant form of APE1 fused with the photoconvertible fluorescent protein (PCFP) Dendra2. Dendra2 did not alter the subcellular localization or endonuclease activity of APE1. We calculated APE1 half-life and compared these results with the classical biochemical approach, which is based on cycloheximide (CHX) treatment. In conclusion, we combined RNAi and in vivo confocal microscopy to study a DNA repair protein demonstrating the feasibility and the advantage of this approach for the study of the cellular dynamic of a DNA repair protein.

OBJECTIVE: To investigate the effect and mechanism of estrodial (E(2)) on intracellular free calcium in the endometrial-myometrial interface (EMI) smooth muscle cells from uteri with adenomyosis.
METHODS: From March 2011 to October 2011, 16 uterus specimens were collected from patients with adenomyosis undergoing hysterectomy in Beijing Obstetrics and Gynecology Hospital, which included 9 proliferative endometrium and 7 secretory endometrium. EMI smooth muscle cells from the uterus were cultured and loaded with calcium ion (Ca(2+)) fluorescent probe fluo-4/AM. The labeled cells were stimulated with the various concentration of E(2)(1×10(2), 1×10(3), 1×10(4), 1×10(5) pmol/L, respectively), then the changes of intracellular Ca(2+) fluorescence intensity were measured by laser scanning microscopy. The most suitable concentration of E(2) was selected, and the reaction difference between the EMI smooth muscle cells of two menstrual phases were also investigated; The changes of intracellular Ca(2+) fluorescence intensity were detected proliferative and secretory smooth muscle cells in E(2) conjugated to bovine serum albumin (17β-E(2)-BSA) group, cycloheximide (CHX) group, fulvestrant (ICI182780) group and pertussis toxin (PTX) group.
RESULTS: (1) The cell viability of primary cultured EMI smooth muscle cells was well at 24 hours culture. (2) 1×10(2) - 1×10(5) pmol/L E(2) can rapidly increase the intracellular Ca(2+) fluorescence intensity within 1 min (P < 0.01);The increased amplitudes caused by 1×10(4) pmol/L and 1×10(5) pmol/L E(2) were the most significant, but there was no significant difference between them (P > 0.05). 1×10(4) pmol/L was the most suitable concentration. (3) With the 1×10(4) pmol/L E(2), the Ca(2+) fluorescence intensity changes showed no significant difference between the EMI smooth muscle cells from the proliferative phase and secretory phase uterus (P > 0.05). The Ca(2+) fluorescence intensity changes were 646 ± 32 in 17β-E(2)-BSA group and 602 ± 31 in CHX group, when compared with 513 ± 26 and 617 ± 35 in respective control group, no significant difference was observed (P > 0.05). The increased amplitude of 188 ± 20 in the PTX group and 302 ± 11 in ICI182780 group exhibited significant difference with 632 ± 33 and 635 ± 24 in respective control group (P < 0.01).
CONCLUSIONS: E(2) could increase the intracellular Ca(2+) of EMI through a membrane receptor dependent and nongenomic mechanism of action.

Cytosolic background fluorescence is often observed when native low-abundance peroxisomal proteins carrying a weak peroxisomal targeting sequence are expressed as fluorescent fusion protein using a strong constitutive promoter in transiently transformed plant cells. This cytosolic fluorescence usually comes from the strong expression of the low-abundance proteins exceeding the peroxisome import efficiency. This often results in a misinterpretation of the protein subcellular localization, as there is doubt as to whether proteins are dually targeted to the cytosol and peroxisome or are exclusively localized to peroxisomes. To circumvent this experimental difficulty, the protein peroxisome import study can be optimized by de novo protein synthesis inhibition in transiently transformed cells using the translation inhibitor cycloheximide. This approach was used here successfully for the study of the subcellular localization of distinct plant isoprenoid biosynthetic enzymes, allowing us to clearly demonstrate that 5-phosphomevalonate kinase, mevalonate 5-diphosphate decarboxylase and a short isoform of farnesyl diphosphate synthase from Catharanthus roseus are exclusively localized to peroxisomes.

BACKGROUND: The taxanes paclitaxel and docetaxel are widely used in the treatment of breast, ovarian, and other cancers. Although their cytotoxicity has been attributed to cell-cycle arrest through stabilization of microtubules, the mechanisms by which tumor cells die remains unclear. Paclitaxel has been shown to induce soluble tumor necrosis factor alpha (sTNF-α) production in macrophages, but the involvement of TNF production in taxane cytotoxicity or resistance in tumor cells has not been established. Our study aimed to correlate alterations in the TNF pathway with taxane cytotoxicity and the acquisition of taxane resistance.
METHODS: MCF-7 cells or isogenic drug-resistant variants (developed by selection for surviving cells in increasing concentrations of paclitaxel or docetaxel) were assessed for sTNF-α production in the absence or presence of taxanes by enzyme-linked immunosorbent assay (ELISA) and for sensitivity to docetaxel or sTNF-α by using a clonogenic assay (in the absence or presence of TNFR1 or TNFR2 neutralizing antibodies). Nuclear factor (NF)-κB activity was also measured with ELISA, whereas gene-expression changes associated with docetaxel resistance in MCF-7 and A2780 cells were determined with microarray analysis and quantitative reverse transcription polymerase chain reaction (RTqPCR).
RESULTS: MCF-7 and A2780 cells increased production of sTNF-α in the presence of taxanes, whereas docetaxel-resistant variants of MCF-7 produced high levels of sTNF-α, although only within a particular drug-concentration threshold (between 3 and 45 nM). Increased production of sTNF-α was NF-κB dependent and correlated with decreased sensitivity to sTNF-α, decreased levels of TNFR1, and increased survival through TNFR2 and NF-κB activation. The NF-κB inhibitor SN-50 reestablished sensitivity to docetaxel in docetaxel-resistant MCF-7 cells. Gene-expression analysis of wild-type and docetaxel-resistant MCF-7, MDA-MB-231, and A2780 cells identified changes in the expression of TNF-α-related genes consistent with reduced TNF-induced cytotoxicity and activation of NF-κB survival pathways.
CONCLUSIONS: We report for the first time that taxanes can promote dose-dependent sTNF-α production in tumor cells at clinically relevant concentrations, which can contribute to their cytotoxicity. Defects in the TNF cytotoxicity pathway or activation of TNF-dependent NF-κB survival genes may, in contrast, contribute to taxane resistance in tumor cells. These findings may be of strong clinical significance.

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