Differenciációs, anti-mitogénikus és túlélési jelátviteli utak vizsgálata PC12 sejtekben = Analysis of differentiation, anti-mitogenic and survival signaling in PC12 cells

A kutatás célja a PC12 patkány phaeochromocytoma sejtek neuronális differenciációját, antimitogén válaszát, túlélését, stressz- és apoptotikus reakcióit közvetítő jelátviteli jelenségek vizsgálata volt. Az NGF neuritogén hatását közvetítő TrkA receptor a Hsp90 chaperone fehérje védőhatása alatt áll, annak hiányában a TrkA proteaszomálisan degradálódik, mely a Ras/ERK-út megbénulását, a neuronális differenciáció gátlását, végül apoptózist eredményez. A proteaszóma-gátlók önmagukban is képesek a Ras/ERK-út, illetve a c-Src aktivitás serkentésére. Az NGF antimitogén hatásában a p53 tumor szuppresszor fehérje kulcsszerepet játszik, részt vesz a MAPK-kaszkádok (ERK, JNK, p38 út) modulálásában is. A PC12 sejtek túlélésében illetve stresszválaszában számos fehérje vesz részt. A RhoA és NFkappaB fehérje az antiapoptotikus jelátvitel tényezői, a protein kináz R enzim proteolítikus aktivációja pedig egy sor, a sejt apoptotikus halálához vezető stresszválasz általános jelensége. | The aim of the present study was to analyze signal transduction processes mediating the differentiation, antimitogenic, survival, stress and apoptotic responses of PC12 rat phaeochromocytoma cells. The TrkA receptor that conveys neuritogenetic signals of NGF is under the protective action of the chaperone protein Hsp90. Without this protection TrkA undergoes degradation by proteasomes leading to the blockade of the Ras/ERK signaling pathway, inhibition of neuronal differentiation and ultimately apoptotic cell death. Proteasome inhibitors by themselves are able to stimulate the Ras/ERK pathway and the phosphorylation of the proto-oncogenic c-Src protein. The tumor suppressor p53 protein plays a key role in the antimitogenic response of PC12 cells to NGF treatment presumably by modulating signaling through the MAPK cascades of ERK, JNK and p38. A number of signaling proteins participate in survival and stress responses of PC12 cells. RhoA and NFkappaB appear to be important factors of anti-apoptotic mechanisms, while proteolytic activation of protein kinase R is a general phenomenon of various forms of cellular stress leading to apoptotic cell death

A sejtek közti kommunikáció újonnan azonosított mikrovezikulum-útjának vizsgálata = Analysis of cell-derived microvesicles that represent novel players in intercellular communication

Munkánk során az extracelluláris vezikulák izolálásának és detektálásának számos meghatározó preanalitikai és analitikai paraméterére hívtuk fel a figyelmet. Elsőként mutattunk rá, hogy a mikrovezikulák és a fehérje-aggregátumok biofizikai paraméterei jelentős mértékben átfednek, és ez zavarhatja a mikrovezikulák mérését. Kidolgoztuk annak módszerét, hogy egyazon biológiai forrásból származó különböző vezikula populációkat párhuzamosan, nagy mennyiségben, intakt formában tudjunk izolálni. Összehasonlító proteomikai elemzést végeztünk thymus eredetű apoptotikus testek és mikrovezikulák esetében. Számos T sejt jelátvitelben és immunfolyamatokban szerepet játszó fehérjét és autoantigént azonosítottunk. Igazoltuk, hogy T sejt eredetű citokinek és extracelluláris vezikulák együttes hatását monociták génexpressziójára. Igazoltuk, hogy a mikrovezikulák önálló ionháztartással rendelkeznek. Kimutattuk, hogy thymocyta exoszómák nem tartalmaznak riboszómális RNS-eket, azonban feldúsulnak bennük kis RNS-ek (pl. bizonyos miRNS-ek). Polymyositises betegekben emelkedett keringő mikrovezikula számot mutattunk ki, mely korrelált a betegség bizonyos klinikai paramétereivel. Végül elsőként igazoltuk, hogy egészséges T sejt eredetű mikrovezikulák CD62P-CD161 kölcsönhatás révén specifikusan kötődnek monociták felszínéhez. | In our work we drove attention to several pre-analytical and analytical parameters affecting isolation and detection of work extracellular vesicles. We were the first to describe that microvesicles share biophysical parameters with protein aggregates which may confound microvesicle assessment by flow cytometry. We developed protocols for the isolation of large amounts of intact vesicle types secreted simultaneously by the same biological source. We carried comparative proteomic analysis of murine thymus derived apoptotic bodies and microvesicles. We identified large number of proteins involved in T cell signaling or immune functions as well as autoantigens within these structures. We provided evidence fro crosstalk between T cell derived extracellular vesicles and cytokines on the gene expression of monocytes. We have shown that microvesicles possess autonomous ion homeostasis. We found that thymocyte derived exosomes lacked the 18S and 28S ribosomal RNA molecules, while they were enriched in small RNA species (e.g. certain miRNAs). We described that patients with polyomyelitis were characterized by elevated levels of circulating microvesicle. Monocyte- and B cell-derived microvesicle numbers correlated with certain clinical parameters of the diseases. Finally, for the first time we showed that HLA-G+, trophoblast-derived microvesicles isolated from healthy pregnant blood plasma samples, bound specifically to T cells via CD62P-CD161 interaction, and induced STAT3 phosphorylation

Toxicological and bioactivity evaluation of blackcurrant press cake, sea buckthorn leaves and bark from Scots pine and Norway spruce extracts under a green integrated approach

Aqueous extracts from blackcurrant press cake (BC), Norway spruce bark (NS), Scots pine bark (SP), and sea buckthorn leaves (SB) were obtained using maceration and pressurized hot water and tested for their bioactivities. Maceration provided the extraction of higher dry matter contents, including total phenolics (TPC), anthocyanins, and condensed tannins, which also impacted higher antioxidant activity. NS and SB extracts presented the highest mean values of TPC and antioxidant activity. Individually, NS extract presented high contents of proanthocyanidins, resveratrol, and some phenolic acids. In contrast, SB contained a high concentration of ellagitannins, ellagic acid, and quercetin, explaining the antioxidant activity and antibacterial effects. SP and BC extracts had the lowest TPC and antioxidant activity. However, BC had strong antiviral efficacy, whereas SP can be considered a potential ingredient to inhibit α-amylase. Except for BC, the other extracts decreased reactive oxygen species (ROS) generation in HCT8 and A549 cells. Extracts did not inhibit the production of TNF-alpha in lipopolysaccharide-stimulated THP-1 macrophages but inhibited the ROS generation during the THP-1 cell respiratory burst. The recovery of antioxidant compounds from these by-products is incentivized for high value-added applications.</p

Role of Translation Initiation Factor 2B in Control of Cell Survival by the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3β Signaling Pathway

The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway is an important mediator of growth factor-dependent survival of mammalian cells. A variety of targets of the Akt protein kinase have been implicated in cell survival, including the protein kinase glycogen synthase kinase 3β (GSK-3β). One of the targets of GSK-3β is translation initiation factor 2B (eIF2B), linking global regulation of protein synthesis to PI 3-kinase/Akt signaling. Because of the central role of protein synthesis, we have investigated the involvement of eIF2B, which is inhibited as a result of GSK-3β phosphorylation, in programmed cell death. We demonstrate that expression of eIF2B mutants lacking the GSK-3β phosphorylation or priming sites is sufficient to protect both Rat-1 and PC12 cells from apoptosis induced by overexpression of GSK-3β, inhibition of PI 3-kinase, or growth factor deprivation. Consistent with these effects on cell survival, expression of nonphosphorylatable eIF2B prevented inhibition of protein synthesis following treatment of cells with the PI 3-kinase inhibitor LY294002. Conversely, cycloheximide induced apoptosis of PC12 and Rat-1 cells, further indicating that protein synthesis was required for cell survival. Inhibition of translation resulting from treatment with cycloheximide led to the release of cytochrome c from mitochondria, similar to the effects of inhibition of PI 3-kinase. Expression of nonphosphorylatable eIF2B prevented cytochrome c release resulting from PI 3-kinase inhibition but did not affect cytochrome c release or apoptosis induced by cycloheximide. Regulation of translation resulting from phosphorylation of eIF2B by GSK-3β thus appears to contribute to the control of cell survival by the PI 3-kinase/Akt signaling pathway, acting upstream of mitochondrial cytochrome c release

Basic English Syntax with Exercises

Adaptált digitalizált tartalom. A Speciális Hallgatói Ügyeket Támogató Iroda a fogyatékossággal élő hallgatók megsegítése céljából tankönyveket adaptál. A tananyag akadálymentes, adaptált változata az arra jogosult (a fogyatékosügyi koordinátoroknál regisztrált) hallgatók számára érhető el

A Promising Way to Overcome Temozolomide Resistance through Inhibition of Protein Neddylation in Glioblastoma Cell Lines

There is no effective therapy for the lately increased incidence of glioblastoma multiforme (GBM)—the most common primary brain tumor characterized by a high degree of invasiveness and genetic heterogeneity. Currently, DNA alkylating agent temozolomide (TMZ) is the standard chemotherapy. Nevertheless, TMZ resistance is a major problem in the treatment of GBM due to numerous molecular mechanisms related to DNA damage repair, epigenetic alterations, cellular drug efflux, apoptosis-autophagy, and overactive protein neddylation. Low molecular weight inhibitors of NEDD8-activating enzyme (NAE), such as MLN4924, attenuate protein neddylation and present a promising low-toxicity anticancer agent. The aim of our study was to find an effective combination treatment with TMZ and MLN4924 in our TMZ-resistant GBM cell lines and study the effect of these combination treatments on different protein expressions such as O6-methylguanine methyltransferase (MGMT) and p53. The combination treatment successfully decreased cell viability and sensitized TMZ-resistant cells to TMZ, foreshadowing a new treatment strategy for GBM

Sucralose Targets the Insulin Signaling Pathway in the SH-SY5Y Neuroblastoma Cell Line

Sucralose is widely used as a non-nutritive sweetener (NNS). However, in order to justify its use as a non-nutritive food additive, sucralose would have to be metabolically neutral. The aim of this study was to examine whether sucralose altered the insulin signaling pathway in an in vitro cell model of Parkinson’s disease (PD)—the dopaminergic differentiated cell line SH-SY5Y. Cells were exposed to sucralose alone and in combination with either insulin or levodopa. Activation of the insulin signaling pathway was assessed by quantifying protein kinase B (AKT) and glycogen synthase kinase 3 (GSK3), as well as the phosphorylated forms of insulin-like growth factor 1 receptor (IGF1-R). Metabolic effects were assayed using MALDI-TOF MS analysis. In the cell viability test, 2 mM sucralose had a negative effect, and levodopa in all combinations had a positive effect. Sucralose treatment alone suppressed GSK3 and IGF1-R phosphorylation in a dose-dependent manner. This treatment also altered the metabolism of fatty acids and amino acids, especially when combined with insulin and levodopa. Suppression of the insulin signaling pathway and sucralose-induced changes in the metabolic profile could underlie a diet-acquired insulin resistance, previously associated with neurodegeneration, or may be an altered response to insulin or levodopa medical therapy

Integrative Epigenetic and Molecular Analysis Reveals a Novel Promoter for a New Isoform of the Transcription Factor TEAD4

TEAD4 is a transcription factor that plays a crucial role in the Hippo pathway by regulating the expression of genes related to proliferation and apoptosis. It is also involved in the maintenance and differentiation of the trophectoderm during pre- and post-implantation embryonic development. An alternative promoter for the TEAD4 gene was identified through epigenetic profile analysis, and a new transcript from the intronic region of TEAD4 was discovered using the 5’RACE method. The transcript of the novel promoter encodes a TEAD4 isoform (TEAD4-ΔN) that lacks the DNA-binding domain but retains the C-terminal protein–protein interaction domain. Gene expression studies, including end-point PCR and Western blotting, showed that full-length TEAD4 was present in all investigated tissues. However, TEAD4-ΔN was only detectable in certain cell types. The TEAD4-ΔN promoter is conserved throughout evolution and demonstrates transcriptional activity in transient-expression experiments. Our study reveals that TEAD4 interacts with the alternative promoter and increases the expression of the truncated isoform. DNA methylation plays a crucial function in the restricted expression of the TEAD4-ΔN isoform in specific tissues, including the umbilical cord and the placenta. The data presented indicate that the DNA-methylation status of the TEAD4-ΔN promoter plays a critical role in regulating organ size, cancer development, and placenta differentiation

Incorporation of Oxidized Phenylalanine Derivatives into Insulin Signaling Relevant Proteins May Link Oxidative Stress to Signaling Conditions Underlying Chronic Insulin Resistance

A link between oxidative stress and insulin resistance has been suggested. Hydroxyl free radicals are known to be able to convert phenylalanine (Phe) into the non-physiological tyrosine isoforms ortho- and meta-tyrosine (o-Tyr, m-Tyr). The aim of our study was to examine the role of o-Tyr and m-Tyr in the development of insulin resistance. We found that insulin-induced uptake of glucose was blunted in cultures of 3T3-L1 grown on media containing o- or m-Tyr. We show that these modified amino acids are incorporated into cellular proteins. We focused on insulin receptor substrate 1 (IRS-1), which plays a role in insulin signaling. The activating phosphorylation of IRS-1 was increased by insulin, the effect of which was abolished in cells grown in m-Tyr or o-Tyr media. We found that phosphorylation of m- or o-Tyr containing IRS-1 segments by insulin receptor (IR) kinase was greatly reduced, PTP-1B phosphatase was incapable of dephosphorylating phosphorylated m- or o-Tyr IRS-1 peptides, and the SH2 domains of phosphoinositide 3-kinase (PI3K) bound the o-Tyr IRS-1 peptides with greatly reduced affinity. According to our data, m- or o-Tyr incorporation into IRS-1 modifies its protein&ndash;protein interactions with regulating enzymes and effectors, thus IRS-1 eventually loses its capacity to play its role in insulin signaling, leading to insulin resistance