In the nervous system, the roles of Kv channels are well established as being critical for regulating action potential frequency, membrane potential, and neurotransmitter release. However, their role in glial cells, a non-excitable cell type, is yet to be fully understood. Whole-cell current kinetics, pharmacology, immunocytochemistry and RT-PCR were used to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that ~70%, 10% and less than 5% of total A current is associated with Kv4, Kv3 and Kv1 channels, respectively. In addition, pharmacology and kinetics provide novel evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Since astrocytes are known to be subjected to neuronal firing frequencies of up to 200 Hz in the hippocampus, the role of A currents in membrane voltage oscillations was assessed. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. Astrocytes and HEK293 cells were used to investigate the mechanism of the previously found GABAA induced anion-mediated reduction of Kv channels in more detail. Astrocytes demonstrate an anion concentration specific depolarizing effect on inactivating A-type (also termed transient voltage-gated) K+ channel activation kinetics whereas a hyperpolarizing effect was seen upon expression of Kv4.2 or Kv1.4 in HEK293 cells, but only after disruption of the cytoskeleton using cytochalasin D. It is hypothesized that cytoskeletal interactions and Cl -mediated effects are mediated through N-terminal conformational stabilities. <p>In summary, the results indicate that hippocampal astrocytes in vitro express multiple A type Kv channel á subunits with accessory, Ca2+-sensitive cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Functions of these channels remain to be determined in a physiological setting, but suggest that A-type Kv channels enable astrocytes to respond rapidly with membrane voltage oscillations to high frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity. Furthermore, studies of anion and cytoskeletal effects on Kv channels demonstrate channel function to be highly localized/targeted and susceptible to changes in ionic environment.

In the nervous system, the roles of Kv channels are well established as being critical for regulating action potential frequency, membrane potential, and neurotransmitter release. However, their role in glial cells, a non-excitable cell type, is yet to be fully understood. Whole-cell current kinetics, pharmacology, immunocytochemistry and RT-PCR were used to characterize A-type current in hippocampal astrocyte cultures to better understand its function. Pharmacological analysis suggests that ~70%, 10% and less than 5% of total A current is associated with Kv4, Kv3 and Kv1 channels, respectively. In addition, pharmacology and kinetics provide novel evidence for a significant contribution of KChIP accessory proteins to astrocytic A-channel composition. Localization of the Shaw Kv3.4 channel to astrocytic processes and the Shal Kv4.3 channel to soma suggest that these channels serve a specific function. Since astrocytes are known to be subjected to neuronal firing frequencies of up to 200 Hz in the hippocampus, the role of A currents in membrane voltage oscillations was assessed. Although TEA-sensitive delayed-rectifying currents are involved in the extent of repolarization, 4-AP-sensitive A currents serve to increase the rate. Astrocytes and HEK293 cells were used to investigate the mechanism of the previously found GABAA induced anion-mediated reduction of Kv channels in more detail. Astrocytes demonstrate an anion concentration specific depolarizing effect on inactivating A-type (also termed transient voltage-gated) K+ channel activation kinetics whereas a hyperpolarizing effect was seen upon expression of Kv4.2 or Kv1.4 in HEK293 cells, but only after disruption of the cytoskeleton using cytochalasin D. It is hypothesized that cytoskeletal interactions and Cl -mediated effects are mediated through N-terminal conformational stabilities. <p>In summary, the results indicate that hippocampal astrocytes in vitro express multiple A type Kv channel á subunits with accessory, Ca2+-sensitive cytoplasmic subunits that appear to be specifically localized to subcellular membrane compartments. Functions of these channels remain to be determined in a physiological setting, but suggest that A-type Kv channels enable astrocytes to respond rapidly with membrane voltage oscillations to high frequency incoming signals, possibly synchronizing astrocyte function to neuronal activity. Furthermore, studies of anion and cytoskeletal effects on Kv channels demonstrate channel function to be highly localized/targeted and susceptible to changes in ionic environment.

Chronic inflammation is considered strongly influence the morbidity and mortality of patients with end-stage renal disease (ESRD) through its multiple pathogenic roles, in association with oxidative stress, accelerated aging, endothelial dysfunction and atherosclerosis, malnutrition, dialysis-related amyloidosis, anaemia, and immune dysfunction . Hemodialysis ¡]HD¡^ is widely used for kidney failure patients,it is a method for removing waste products such as creatinine and urea. However, at present it is well known the course of hemodialysis can create obvious inflammation condition and oxidation pressure. The oxidation stress of HD can arise from the osmosis pressure and oxidative environment of dialysis tube. The oxidative stresses will finally modify proteins which turn out initiate the short term and long term complications related to renal diseases of HD patients. We identified oxidated proteins in the hemodialysis tube of 16 HD patients. The protein oxidation level was determined by Oxyblot assay. The oxidation proteins were further identified by LC/MS detection. Many serum proteins were detected to be oxidized including albumin, apoA, immunoglobin,beta-globin, hemoglobin, etc. It has been well documented that albumin is quite vulnerable to ROS and elevated levels of carbonyl groups of albumin have been reported in plasma of dialysis patients. Inflammatory effects were further tested. The oxidation proteins of HD patients induce pro-inflammatory factor TNF-alpha expression of HEK293T and HEK293 cells. These results indicate HD induce protein oxidation, and inflammatory response which may responsible for complications of End-stage kidney disease (ESRD). Keywords¡GHemodialysis, oxidative stress, ESRD, HEK293, inflammatory

NMDA receptors are the most studied receptors in the field of neuroscience and are known to play an important role in development and plasticity. These receptors exhibit different kinetics depending on their subunit composition. NR2A and NR2B are the predominating NMDAR subunits in the brain. These receptors localize to synapses where they interact with other proteins including CaMKIIα, an abundant kinase which plays an important role in synaptic plasticity. Although CaMKIIα is known to bind to all types of NMDARs, it exhibits a higher affinity to NR2B compared to NR2A subunits. Studies have shown that lactate acts as a signaling molecule promoting the expression of genes related to synaptic plasticity via NMDARs activation. However, the mechanism describing how lactate exerts these effects is not well understood. We hypothesize that the redox state change, resulting from the metabolic conversion of lactate to pyruvate, may promote the interaction between CaMKIIα and NMDARs, thereby potentiating NMDARs activity. To tackle this question, we used a pharmacogenetics model consisting of NMDARs expressing HEK293 cells in the presence or absence of CaMKIIα. To monitor NMDARs activity, we use the ratio-metric calcium dye Fura-2 in calcium imaging experiments. We report that L-lactate decreases the peak responses of the NR2A and NR2B NMDARs in the absence of CaMKII expression. Upon CaMKII presence, we found that lactate prolongs the activation period of GluN2B as observed during the washout period and modestly increase the peak response of GluN2A NMDARs. Interestingly, we confirm that expressing CaMKIIα in control (no lactate) HEK cells significantly augmented NR2B but not NR2A NMDARs. We also report that pyruvate was able to increase peak responses of both NR2A and NR2B NMDARs in the absence of CaMKII, while it only increased the NR2A-NMDAR peak responses in the presence of CaMKII. These results suggest that lactate exerts a neuroprotective effect in the absence of CaMKII and it slightly boosts NR2B NMDARs activity when CaMKII is expressed, possibly favoring plasticity. Moreover, data obtained with pyruvate indicates that in our HEK cell model pyruvate affects the NMDARs in a manner independent of the presence of CaMKII through an alternative mechanism.

Inorganic phosphate (Pi) is an essential component for structure and metabolism. PiT1 (SLC20A1) and PiT2 (SLC20A2) are members of the mammalian type-III inorganic phosphate transporters. SLC20A2 missense variants are associated with primary brain calcification. MicroRNAs (miRNAs) are endogenous noncoding regulatory RNAs, which play important roles in post-transcriptional gene regulation. MicroRNA-9 (miR-9) acts at different stages of neurogenesis, is deeply rooted in gene networks controlling the regulation of neural progenitor proliferation, and is also linked with cancers outside the nervous system. We evaluated possible interactions between miR-9 and the phosphate transporters (PiT1 and PiT2). SLC20A2, platelet-derived growth factor receptor beta (PDGFRB) and Fibrillin-2 (FBN2) showed binding sites with high affinity for mir-9, in silico. miR-9 mimic was transfected into HEK293 cells and expression confirmed by RT-qPCR. Overexpression of miR-9 in these cells caused a significant reduction in PiT2 and FBN2. PDGFRB appeared to be decreased, but was not significantly down-regulated in our hands. PiT1 showed no significant difference relative to controls. The down-regulation of PiT2 protein by miR-9 was confirmed by western blotting. In conclusion, we showed miR-9 can down-regulate PiT2, in HEK293 cells.

Extracellular adenosine-5'-triphosphate (ATP), released from damaged cells or coreleased as a cotransmitter from synaptic vesicles, acts on its plasma membrane receptors termed purinergic. Purinergic P2X receptors are ATP-gated cation channels. To date seven P2X isoforms designated P2X1-7 have been cloned that are organized as trimeric homomers or heteromers. All P2X subunits share a similar structure consisting of a large extracellular loop, two transmembrane domains and intracellular N- and C- termini. An additional structural feature is conserved aminoacids, these include ten conserved cysteine residues in the extracellular loop. All ectodomain cysteines form disulfide bonds which are organized in two areas: three disulfide bridges are localized in the N-termini half and two in the C-termini half at P2X receptor. ATP binding pocket is apparently localized between two neighbouring subunits. The aim of this Diploma Thesis was to examine the relevance of ectodomain cysteine residue and/or disulfide bonds for the expression, function and ATP binding properties of the P2X receptor. All ten, one by one, ectodomain cysteines were substituted by alanines and ATP-induced currents was recorded in HEK293 cells expressing wild-type P2X4 receptor and its mutants. Low responsible or nonfunctional mutants...

Auxin is one of the most important plant hormones, which provides development of a plant. PIN1 and PIN7 proteins belong to the PIN family of transporters which is among the most important auxin efflux carriers. This thesis deals with the of AtPIN1 and AtPIN7 auxin efflux carriers (from Arabidopsis thaliana) in human embryonic kidney 293 cell line. Biological activity of these proteins was tested by using radiolabeled auxins accumulation. Further inhibitors of auxin transport have been tested - NPA, CHPAA and BFA.

Natural killer cells (NK cells) are part of the immune system in human and other mammals. The task of these cells, which belong to the non-specific immunity, is to induce apoptosis in other cells of the body that may represent a threat for the body (i.e., tumour or virally infected cells). NK cells have a variety of surface receptors to recognize their target cells. A number of receptors are well-known today and they may be divided into groups based, e.g., on their structural similarities or on the type of signal which these receptors present to NK cells. Accordingly, we distinguish activation and inhibitory receptors. Inhibitory receptors inhibit NK cell response, while activation receptors elicit this response. During NK cell contact with another cell, the resulting NK cell behaviour is always the result of a certain balance of activation and inhibitory receptor responses. The NKp44 receptor is an immunoglobulin-like receptor. This receptor is very unique among other receptors in many respects, for example because it is associated with both activation and inhibitory motif. The ligand of this receptor is a proliferating cell nuclear antigen (PCNA). PCNA is a clamp protein important, inter alia, during DNA replication, in which it anchors other replisome proteins. This work is focused on...

The cytotoxic effects of exposure to low concentrations of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) that are typically found in groundwater were investigated, in vitro. Most 2,4-D toxicology studies use high concentrations of the herbicide that are above those typically found in groundwater and measure overt biological endpoints. In contrast, this thesis examines the effects of low concentrations of 2,4-D and measures more subtle and sensitive endpoints such as gene expression and the generation of reactive oxygen species. This work derives from recent cDNA microarray analysis conducted in our laboratory that revealed significant alterations in the expression of 238 genes in cells exposed to nanomolar (nM) concentrations of a commercial formulation of 2,4-D. These findings are extended in this thesis to include the in vitro cytotoxic effects of low concentrations of both technical and commercial 2,4-D on two cell lines. Cells derived from liver (HepG2) and kidney (HEK293) respectively, were chosen, since liver and kidney are known to metabolize 2,4-D in vivo. Cell viability was measured using the Resazurin assay, reactive oxygen species (ROS) were measured with 2,7-dichlorofluorescin diacetate (2,7-DCFH-DA), and real timepolymerase chain reaction (RT-PCR) was used to assess changes in mRNA expression while protein expression was examined by Western blot.<p>Cell viability studies revealed that low environmental concentrations (0.1 to 100 nM) of 2,4-D induced small, but statistically significant decreases in cell viability. No concentration or time-dependent decreases in cell viability were observed in cells exposed to either forms of low environmental 2,4-D concentrations. HEK293 cells were more susceptible than HepG2 cells to the toxic effects of both forms of 2,4-D, having statistically significant lower viability at all exposure concentrations and durations. Both forms of 2,4-D reduced cell viability in both cell lines, suggesting that cytotoxicity was induced directly by 2,4-D, and not by the inert ingredients in the commercial formulation.<p>The ROS assays illustrated that 2,4-D induced statistically significant ROS production in HepG2 and HEK293 cell cultures at concentrations greater than 10 µM and 100 nM respectively. This was both a concentration and time-dependent effect in both cell lines. Although HEK293 cells were more susceptible to 2,4-D, they had 50 to 70% less ROS production than HepG2 cells, at all exposure concentrations and times.<p>The RT-PCR and Western blot analyses showed that exposure of HepG2 and HEK293 cells to low 2,4-D concentrations induced (< 2 fold) alterations in mRNA and protein levels of FTL, FTH1 and PCNA however these changes did not consistently vary with concentration.<p>Taken together, cell viability, ROS and gene expression studies show that low environmental 2,4-D concentrations induced subtle in vitro cytotoxic effects. However we have no evidence that these subtle changes pose a serious health threat to exposed humans.

Nitrate is a ubiquitous drinking water contaminant with potential adverse effects on human health. However, little is known about nitrate toxicity at the cellular and molecular level. The purpose of this study was to examine the effects of environmentally relevant concentrations of nitrate on cytotoxicity and protein expression in human cell lines. To determine if tissue-specific responses occurred, a human hepatoma cell line (HepG2) and a human embryonic kidney cell line (HEK293) were used. Both potassium and ammonium salts of nitrate were used to determine salt-specific toxicity. Test concentrations of nitrate varied from 1 μg/L to 5000 mg/L. Cells were exposed to a nitrate salt for 24, 48, or 72 hours and then examined for effects on viability (using the Neutral Red assay) or proliferation (using the BrdU ELISA assay). To determine the effects of nitrate on protein expression, levels of PCNA, Hsp70, Hsc70, and VEGF protein were monitored using Western blotting in HepG2 and HEK293 cells exposed to KNO3 or NH4NO3 for 24 hours.<p>Nitrate was cytotoxic to both cell types at high concentrations, with EC50 values between 1557 mg/L (approximately) 5852mg/L for viability, and ~2.5 mg/L 3631 mg/L for proliferation. Several EC50 values were not calculable based on the available data, but appeared to be far greater than 5000 mg/L. Ammonium nitrate was generally more toxic than potassium nitrate, and increasing exposure time generally resulted in greater toxicity. The HepG2 and HEK293 cells displayed similar responses for most assays, except the 24 hour KNO3 Neutral Red assay. Here, HEK293 viability increased with increasing KNO3 concentrations, while HepG2 viability decreased. The reason for this finding is unknown, but may involve cell-specific homeostatic mechanisms. A hormetic-like effect was observed in both cell types in several of the proliferation assays; the biological significance of this effect remains unknown.<p>No significant changes in protein expression were observed under these experimental conditions. Some subtle trends were present, such as a slight increase in Hsp70 expression with increasing nitrate concentration in both cell types. In HepG2 cells, PCNA expression increased slightly with increasing nitrate concentrations; however, the opposite effect was observed in HEK293 cells. This may be due to transcriptional or translational regulation.<p>In summary, environmentally relevant concentrations of nitrate did not appear to evoke significant cytotoxicity or changes in protein expression. Cell viability and proliferation effects were observed at higher concentrations of nitrate. Private water supplies may contain nitrate concentrations above the EC50 values in these experiments. More research is required to determine if this poses a direct threat to human health.