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Mammalian cells are suitable hosts for producing recombinant therapeutic proteins, with Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells being the most commonly used cell lines. Mammalian cell expression system includes stable and transient gene expression (TGE) system, with the TGE system having the advantages of short cycles and simple operation. By optimizing the TGE system, the expression of recombinant proteins has been significantly improved. Here, the TGE system and the detailed and up-to-date improvement strategies of mammalian cells, including cell line, expression vector, culture media, culture processes, transfection conditions, and co-expression of helper genes, are reviewed. Key points: • Detailed improvement strategies of transient gene expression system of mammalian cells are reviewed • The composition of transient expression system of mammalian cell are summarized • Proposed optimization prospects for transient gene expression systems [ABSTRACT FROM AUTHOR]

Biochemical reactions in cells serve as the endogenous source of heat, maintaining a constant body temperature. This process requires proper control; otherwise, serious consequences can arise due to the unwanted but unavoidable responses of biological systems to heat. This review aims to present a range of responses to heat in biological systems across various spatial scales. We begin by examining the impaired thermogenesis of malignant hyperthermia in model mice and skeletal muscle cells, demonstrating that the progression of this disease is caused by a positive feedback loop between thermally driven Ca2+ signaling and thermogenesis at the subcellular scale. After we explore thermally driven force generation in both muscle and non-muscle cells, we illustrate how in vitro assays using purified proteins can reveal the heat-responsive properties of proteins and protein assemblies. Building on these experimental findings, we propose the concept of 'trans-scale thermal signaling'. [ABSTRACT FROM AUTHOR]

Abstract Prolactin (PRL) is a hormone produced by the pituitary gland with innumerable functions, such as lactation, reproduction, osmotic and immune regulation. The present work describes the synthesis of hPRL in human embryonic kidney (HEK293) cells, transiently transfected with the pcDNA-3.4-TOPO® vector carrying the hPRL cDNA. A concentration of ~ 20 mg/L, including glycosylated (G-hPRL) and non-glycosylated (NG-hPRL) human prolactin, was obtained, with ~ 19% of G-hPRL, which is higher than that observed in CHO-derived hPRL (~ 10%) and falling within the wide range of 5–30% reported for pituitary-derived hPRL. N-Glycoprofiling analysis of G-hPRL provided: (i) identification of each N-glycan structure and relative intensity; (ii) average N-glycan mass; (iii) molecular mass of the whole glycoprotein and relative carbohydrate mass fraction; (iv) mass fraction of each monosaccharide. The data obtained were compared to pituitary- and CHO-derived G-hPRL. The whole MM of HEK-derived G-hPRL, determined via MALDI–TOF-MS, was 25,123 Da, which is 0.88% higher than pit- and 0.61% higher than CHO-derived G-hPRL. The main difference with the latter was due to sialylation, which was ~ sevenfold lower, but slightly higher than that observed in native G-hPRL. The “in vitro” bioactivity of HEK-G-hPRL was ~ fourfold lower than that of native G-hPRL, with which it had in common also the number of N-glycan structures.

Vanadium dioxide (VO 2) is a class of thermochromic material with potential applications in various fields. Massive production and wide application of VO 2 raise the concern of its potential toxicity to human, which has not been fully understood. Herein, a commercial VO 2 nanomaterial (S-VO 2) was studied for its potential toxicity to human embryonic kidney cell line HEK293, and two most common vanadium ions, V(IV) and V(V), were used for comparison to reveal the related mechanism. Our results indicate that S-VO 2 induces dose-dependent cellular viability loss mainly through the dissolved V ions of S-VO 2 outside the cell rather than S-VO 2 particles inside the cell. The dissolved V ions of S-VO 2 overproduce reactive oxygen species to trigger apoptosis and proliferation inhibition via several signaling pathways of cell physiology, such as MAPK and PI3K-Akt, among others. All bioassays indicate that the differences in toxicity between S-VO 2 , V(IV), and V(V) in HEK293 cells are very small, supporting that the toxicity is mainly due to the dissolved V ions, in the form of V(V) and/or V(IV), but the V(V)'s behavior is more similar to S-VO 2 according to the gene expression analysis. This study reveals the toxicity mechanism of nanosized VO 2 at the molecular level and the role of dissolution of VO 2 , providing valuable information for safe applications of vanadium oxides. [Display omitted] • VO 2 NPs induced a dose-dependent toxicity to HEK293 cells. • VO 2 NPs overproduce ROS to trigger apoptosis and proliferation inhibition. • Dissolved V ions of VO 2 NPs outside cells contribute to cellular viability loss. • Differences in toxicity between S-VO 2 , V(IV), and V(V) are very small. [ABSTRACT FROM AUTHOR]

Abstract Background Endogenous circadian oscillators distributed across the mammalian body are synchronised among themselves and with external time via a variety of signalling molecules, some of which interact with G-protein-coupled receptors (GPCRs). GPCRs can regulate cell physiology via pathways originating with heterotrimeric G-proteins or β-arrestins. We applied an optogenetic approach to determine the contribution of these two signalling modes on circadian phase. Results We employed a photopigment (JellyOp) that activates Gαs signalling with better selectivity and higher sensitivity than available alternatives, and a point mutant of this pigment (F112A) biased towards β-arrestin signalling. When expressed in fibroblasts, both native JellyOp and the F112A arrestin-biased mutant drove light-dependent phase resetting in the circadian clock. Shifts induced by the two opsins differed in their circadian phase dependence and the degree to which they were associated with clock gene induction. Conclusions Our data imply separable G-protein and arrestin inputs to the mammalian circadian clock and establish a pair of optogenetic tools suitable for manipulating Gαs- and β-arrestin-biased signalling in live cells.

Cancer is the second leading cause of death in the world with a high annual incidence level. Researchers have been working on developing treatments for cancer. Targeted therapy is an emerging treatment modality that is more novel than surgery, radiotherapy and chemotherapy. In targeted therapy, exogenous nanoscale microparticles are applied as carriers for drugs or genes. However, conventional particles have certain limitations attributed to non-specific cytotoxicity, biocompatibility and low delivery efficacy in individual therapeutic vector systems. Exosomes are small vesicles secreted by various cells that consist of lipid bilayer membranes without organelles. Due to their excellent biocompatibility, exosomes have received increased attention in recent years for targeted therapy applications. This review briefly introduces the current status of targeted therapy, and exosomes are introduced by their structural characteristics, physiological effects and separation methods. This review also discusses the applications of engineered exosomes derived from different cells in the field of targeted therapies and compares the two-way regulation of mesenchymal stromal cell–derived exosomes in tumor therapy. [ABSTRACT FROM AUTHOR]

Here, we present a simple pipetting-based approach for generating multi-layered core-shell hydrogel droplets composed of alginate for outer shell and collagen for culturing cells inside. By using a multi-hole plastic substrate and by pipetting, multi-layered hydrogel droplets were generated in a simple and rapid manner. HEK293 cells, which are human embryonic kidney cells, were cultured for 14 days in double-layered hydrogel droplet with high viability. Cancer cells were co-cultured with epithelial cells in multi-layered hydrogel droplets and applied for drug tests with curcumin. As epithelial cells protect cancer cells from anti-cancer drugs, co-cultured cells showed lower sensitivity to curcumin. We developed a simple and easy method for creating complex hydrogel particles for 3D multicellular co-culture and developed an alternative method for drug testing in vivo. [ABSTRACT FROM AUTHOR]

Thunbergia laurifolia or Rang Jued has been used as an herbal tea and in folk medicine as a detoxifying agent. Cd contamination is globally widespread and a serious public health problem. The aim of this study was to determine the endogenous antioxidant enzyme activities and malondialdehyde (MDA) production of the crude dried extract (CDE) of T. laurifolia leaves, using human embryonic kidney (HEK293) and human liver (HepG2) cells as in vitro models. Moreover, the cytotoxicity including anti-cadmium (Cd) toxicity in both cells were measured. The experimental design had 3 treatment groups with combined, pre-, and post-treatments for investigating the anti-Cd toxicity, and cell viability was determined with MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The CDE showed low cytotoxicity and increased catalase (CAT) and glutathione peroxidase (GPx) activities with decreased malondialdehyde (MDA) levels in both cell types. It was found that the CDE protected against Cd-induced toxicity in both cell types, and a synergistic combination therapy effect was seen when CaNa2EDTA, a chelating agent, was applied. Therefore, CDE can protect against Cd-induced oxidative stress in cells, possibly due to its antioxidant properties. Moreover, using the extract or drinking the herbal tea together with chelating agent should have an efficacy advantage over using the CDE or the chelating agent singly.

3-Chloropropane-1,2-diol (3-MCPD) is a heat-produced contaminant formed during the preparation of soy sauce worldwide. The present investigation was conducted to determine the molecular aspects of 3-MCPD toxicity on human embryonic kidney cells (HEK293). Cell viability and apoptosis were assessed in response to exposure to 3-MCPD using the MTT assay and high-content screening (HCS). DNA damage, intracellular reactive oxygen species (ROS) and apoptosis-related proteins were evaluated. Genes related with apoptosis were detected by qPCR-array for further understanding the 3-MCPD induced cell apoptosis signaling pathway. Our results clearly showed that 3-MCPD treatment inhibits cell proliferation and reactive oxygen species generation. qPCR-array indicated that nine apoptotic genes were up-regulated more than 2-fold and six down-regulated more than 2-fold. Genes associated with the mitochondrial apoptotic pathway, especially BCL2 family genes, changed significantly, indicating that the mitochondrial apoptotic pathway is activated. Death receptor pathway-related genes, TNFRSF11B and TNFRSF1A, changed significantly, indicating that the death receptor pathway is also activated, resulting in the inhibition of cell growth and proliferation as well as induction of apoptosis. To sum up, the experiment results indicated that 3-MCPD induced HEK293 cell toxicity through the death receptor pathway and mitochondrial pathway. [ABSTRACT FROM AUTHOR]

Mesoporous silica nanoparticles (MSNs) have been widely evaluated for their potential use as carriers for cancer diagnosis and therapy. Understanding the toxicity of MSNs is crucial to their biomedical applications. Although several groups have reported the cytotoxicity of MSNs, the genotoxicity (inducing genetic aberrations) of MSNs in normal human cells has not been extensively investigated. Gene amplification and mutation may initiate and promote carcinogenesis, and changes in mRNA expression can affect normal human physical functions. In this study, human embryonic kidney 293 (HEK293) cells were treated overnight with MSNs at a concentration of 120 µg/mL. The cells were assayed with fluorescent in situ hybridization to check for chromosome changes and gene amplification. Mutations in the epidermal growth factor receptor 1 ( EGFR1) and KRAS genes were checked with DNA sequencing. The effects of MSNs on mRNA expression were investigated with an Agilent human mRNA microarray. No chromosomal alterations or gene mutations in EGFR or KRAS were observed in the control HEK293 cells or HEK293 cells exposed to MSNs. The microarray analysis showed that MSNs significantly altered gene expression. The expression of 579 genes was upregulated and that of 1263 genes was downregulated in HEK293 cells treated with MSNs compared with the control HEK293 cells. Our findings suggest that exposure to MSNs is genotoxic to normal human cells, leading to changes in the expression of some genes. This genotoxicity may cause cellular dysfunction and certain benign diseases. We have not shown that MSN exposure induces serious genotoxicity involving carcinogenesis. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal hydrolase degrading various N-acylethanolamines at acidic pH. NAAA prefers anti-inflammatory and analgesic palmitoylethanolamide to other N-acylethanolamines as a substrate, and its specific inhibitors are shown to exert anti-inflammatory and analgesic actions in animal models. Therefore, these inhibitors are expected as a new class of therapeutic agents. Here, we introduce an NAAA assay system, using [ 14 C]palmitoylethanolamide and thin-layer chromatography. The preparation of NAAA enzyme from native and recombinant sources as well as the chemical synthesis of N-[1'- 14 C]palmitoyl-ethanolamine is also described., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

CLC-K chloride channels play a crucial role in kidney physiology and genetic mutations, affecting their function are responsible for severe renal salt loss in humans. Thus, compounds that selectively bind to CLC-Ka and/or CLC-Kb channels and modulate their activity may have a significant therapeutic potential. Here, we compare the biophysical and pharmacological behaviors of human CLC-K channels expressed either in HEK293 cells or in Xenopus oocytes and we show that CLC-K channel properties are greatly influenced by the biochemical environment surrounding the channels. Indeed, in HEK293 cells the potentiating effect of niflumic acid (NFA) on CLC-Ka/barttin and CLC-Kb/barttin channels seems to be absent while the blocking efficacy of niflumic acid and benzofuran derivatives observed in oocytes is preserved. The NFA block does not seem to involve the accessory subunit barttin on CLC-K1 channels. In addition, the sensitivity of CLC-Ks to external Ca2+ is reduced in HEK293 cells. Based on our findings, we propose that mammalian cell lines are a suitable expression system for the pharmacological profiling of CLC-Ks. [ABSTRACT FROM AUTHOR]

Human embryonic kidney cells 293 (HEK293) are widely used as cellular heterologous expression systems to study transfected ion channels. This work characterizes the endogenous expression of TRPM4 channels in HEK293 cells. TRPM4 is an intracellular Ca2+-activated non-selective cationic channel expressed in many cell types. Western blot analyses have revealed the endogenous expression of TRPM4. Single channel 22pS conductance with a linear current–voltage relationship was observed using the inside-out patch clamp configuration in the presence of intracellular Ca2+. The channels were permeable to the monovalent cations Na+ and K+, but not to Ca2+. The open probability was voltage-dependent, being higher at positive potentials. Using the whole-cell patch clamp “ruptured patch” configuration, the amplitude of the intracellular Ca2+-activated macroscopic current was dependent on time after patch rupture. Initial transient activation followed by a steady-increase reaching a plateau phase was observed. Biophysical analyses of the macroscopic current showed common properties with those from HEK293 cells stably transfected with human TRPM4b, with the exception of current time course and Ca2+ sensitivity. The endogenous macroscopic current reached the plateau faster and required 61.9±3.5μM Ca2+ to be half-maximally activated versus 84.2±1.5μM for the transfected current. The pharmacological properties, however, were similar in both conditions. One hundred μM of flufenamic acid and 9-phenanthrol strongly inhibited the endogenous current. Altogether, the data demonstrate the expression of endogenous TRMP4 channels in HEK293 cells. This observation should be taken into account when using this cell line to study TRPM4 or other types of Ca2+-activated channels. [ABSTRACT FROM AUTHOR]

Conotoxins (conopeptides) are small disulfide bonded peptides from the venom of marine cone snails. These peptides target a wide variety of membrane receptors, ion channels and transporters, and have enormous potential for a range of pharmaceutical applications. Structurally related ω-conotoxins bind directly to and selectively inhibit neuronal (N)-type voltage-gated calcium channels (VGCCs) of nociceptive primary afferent neurones. Among these, ω-conotoxin MVIIA (Prialt) is approved by the Food and Drug Administration (FDA) as an alternative intrathecal analgesic for the management of chronic intractable pain, particularly in patients refractory to opioids. A series of newly discovered ω-conotoxins from Conus catus, including CVID-F, are potent and selective antagonists of N-type VGCCs. In spinal cord slices, these peptides reversibly inhibit excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurones, and in the rat partial sciatic nerve ligation model of neuropathic pain, significantly reduce allodynic behaviour. Another family of conotoxins, the α-conotoxins, are competitive antagonists of mammalian nicotinic acetylcholine receptors (nAChRs). α-Conotoxins Vc1.1 and RgIA possess two disulfide bonds and are currently in development as a treatment for neuropathic pain. It was initially proposed that the primary target of these peptides is the α9α10 neuronal nAChR. Surprisingly, however, α-conotoxins Vc1.1, RgIA and PeIA more potently inhibit N-type VGCC currents via a GABAB GPCR mechanism in rat sensory neurones. This inhibition is largely voltage-independent and involves complex intracellular signalling. Understanding the molecular mechanisms of conotoxin action will lead to new ways to regulate VGCC block and modulation in normal and diseased states of the nervous system. [ABSTRACT FROM AUTHOR]

Abstract: Rabies virus is an important causative agent of disease resulting in an acute infection of the nervous system and death. Although curable if treated in a timely manner, rabies remains a serious public health issue in developing countries, and the indigenous threat of rabies continues in developed countries because of wildlife reservoirs. Control of rabies in wildlife is still an important challenge for governmental authorities. There are a number of rabies vaccines commercially available for control of wildlife rabies infection. However, the vaccines currently distributed to wildlife do not effectively immunize all at-risk species, particularly skunks. A replication competent recombinant adenovirus expressing rabies glycoprotein (AdRG1.3) has shown the most promising results in laboratory trials. The adenovirus vectored vaccine is manufactured using HEK 293 cells. This study describes the successful scale-up of AdRG1.3 adenovirus production from 1 to 500L and the manufacturing of large quantities of bulk material required for field trials to demonstrate efficacy of this new candidate vaccine. The production process was streamlined by eliminating a medium replacement step prior to infection and the culture titer was increased by over 2 fold through optimization of cell culture medium. These improvements produced a more robust and cost-effective process that facilitates industrialization and commercialization. Over 17,000L of AdRG1.3 adenovirus cultures were manufactured to support extensive field trials. AdRG1.3 adenovirus is formulated and packaged into baits by Artemis Technologies Inc. using proprietary technology. Field trials of AdRG1.3 rabies vaccine baits have been conducted in several Canadian provinces including Ontario, Quebec and New Brunswick. The results from field trials over the period 2006–2009 demonstrated superiority of the new vaccine over other licensed vaccines in immunizing wild animals that were previously difficult to vaccinate. [Copyright &y& Elsevier]

Abstract: Gamma-linolenic acid (GLA, 18:3 n6) is an essential polyunsaturated fatty acid of the omega-6 family and is found to be effective in prevention and/or treatment of various health problems. In this study, we evaluated the possibility of increasing γ-linolenic acid contents in mammalian cells using the delta-6 gene from Borago officinalis. The borage Δ6-desaturase gene (sDelta-6) was codon-optimized and introduced into HEK293 cells by lipofectin transfection. Co-expression of GFP with sDelta-6 and RT-PCR analysis indicated that sDelta-6 could be expressed in mammalian cells. Subsequently, the heterologous expression of borage Δ6-desaturase was evaluated by fatty acid analysis. Total cellular lipid analysis of transformed cells fed with linoleic acid (LA 18:2 n6) as a substrate showed that the expression of sDelta-6 resulted in an 228–483% (p <0.05) increase of GLA when compared with that in the control cells. The highest conversion efficiency of LA into GLA in sDelta-6+ cells was 6.9 times higher than that in the control group (11.59% vs. 1.69%; p <0.05). Our present work demonstrated that the sDelta-6 gene from borage could be functionally expressed in mammalian cells, and could convert LA into GLA. Furthermore, this study may pave the way to generate transgenic livestock that can synthesise GLA. [Copyright &y& Elsevier]

Abstract: Cyclovirobuxine D (CVB-D) has been widely used for treatment of cardiac insufficiency and arrhythmias in China. The antiarrhythmic and proarrhythmic potential of this drug might be concerned with prolongation of action potential duration and QT interval. Human-ether-a-go-go-related gene (HERG) has an important role in the repolarization of the cardiac action potential. This study investigated for the first time the effect of CVB-D on HERG channels stably expressed in HEK293 cells using the whole-cell patch-clamp technique. CVB-D inhibited HERG current (IHERG) in a concentration-dependent manner with an IC50 of 19.7μM. IHERG blockade required channel activation and was time-dependent, suggesting an open channel block. Moreover, IHERG inhibition by CVB-D was relieved by depolarization to a highly positive membrane potential (+80mV) that favored HERG channel inactivation. These findings suggested that CVB-D inhibit HERG channels in the open states. CVB-D had no effect on HERG current kinetics. Thus, we conclude that CVB-D inhibits HERG encoded potassium channels and this action might be a molecular mechanism for the previously reported APD prolongation and QT interval prolongation with this drug. [Copyright &y& Elsevier]

Background/Aims: Human ether-à-go-go-related gene (hERG) has an important role in the repolarization of the cardiac action potential. Our studies were to investigate the effects of oxymatrine (one of the natural constituents extracted from Chinese herb Sophora flavescens Ait) on hERG-encoded K+ channels at different temperatures and its underlying mechanism. Methods: The effects of oxymatrine were examined on hERG channels stably expressed in HEK293 cells using a whole-cell patch clamp technique. Results: At the temperature 30°C, oxymatrine inhibited hERG current in a concentration-dependent manner and the IC50 was ∼665 μM. However at the temperature of 20°C, low concentration oxymatrine C≤100 μM increased hERG current density. However, high concentration oxymatrine C>100 μM inhibited the hERG current density significantly. Oxymatrine only affected the activation kinetic of hERG channels at all temperatures and had a high binding affinity for open state of hERG channels except the 300 μM-20°C group which had a high binding affinity for inactive state of hERG channels. Conclusion: Oxymatrine is a low potency blocker of hERG K+ channels at 30°C, low concentration oxymatrine affect the hERG activation gating with accelerating hERG tail current at 20°C, oxymatrine is a potential hERG activator at low temperatures. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Recent evidence suggests that some atypical antipsychotic drugs may protect against oxidative stress and consequent neurodegeneration by mechanisms that remain unclear. Using the neuron-like rat pheochromocytoma (PC-12) cell line, Clozapine and N-desmethylclozapine were tested for their ability to protect against cell death due to oxidative stress induced by hydrogen peroxide (H2O2). These drugs demonstrated significant protection of PC-12 cells, as measured by both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT) and Alamar Blue cell viability assays. However, neither viability assay detected a protective effect of Clozapine on human embryonic kidney (HEK293), rat primary cortical neurons, or human neuroblastoma (SH-SY5Y) exposed to H2O2 treatment. The mechanism of protection involves a PC-12 cell-specific differential response to H2O2 treatment vs. the other cell lines. Pre-treatment with 250 µM or 125 µM diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, unexpectedly showed protection of the PC-12 cells from H2O2 treatment. Western blots revealed that Clozapine, N-desmethylclozapine, and DETC reduce the phosphorylation of extracellular signal-regulated kinase (ERK) that is caused by H2O2 exposure in PC-12 cells. In both HEK293 and SH-SY5Y cells, H2O2 exposure did not increase ERK phosphorylation over control, demonstrating a different response to H2O2 vs. PC-12 cells, and explaining why Clozapine could not protect these cells. Also, U0126, a specific MEK inhibitor, was able to protect PC-12 cells from H2O2 exposure, showing that inhibiting ERK phosphorylation is sufficient to provide protection. Cumulatively, these results indicate that Clozapine, N-desmethylclozapine, DETC, and U0126 protect PC-12 cells by blocking the cell-type specific H2O2 induced increase in ERK phosphorylation. [Copyright &y& Elsevier]

Abstract: Small- to medium-sized neurons in the dorsal root ganglion (DRG) convey nociceptive information to the spinal cord. The co-expression of TRPV1 receptors (sensitive to vanilloids, heat and acidic pH) with P2X3 receptors (sensitive to extracellular ATP) has been found in many DRG neurons. We investigated whether the co-activation of these two receptor classes in small-diameter cells leads to a modulation of the resulting current responses shaping the intensity of pain sensation. The whole-cell patch clamp method was used to record agonist-induced currents in cultured rat DRG neurons and in HEK293 cells transfected with the respective wild-type recombinant receptors or their mutants. Co-immunoprecipitation studies were used to demonstrate the physical association of TRPV1 and P2X3 receptors. At a negative holding potential, the P2X3 receptor agonist α,β-meATP induced less current in the presence of the TRPV1 agonist capsaicin than that in its absence. This inhibitory interaction was not changed at a positive holding potential, in a Ba2+-containing superfusion medium, or when the buffering of intrapipette Ca2+ was altered. The C-terminal truncation at Glu362 of P2X3 receptors abolished the TRPV1/P2X3 cross-talk in the HEK293 expression system. Co-immunoprecipitation studies with polyclonal antibodies generated against TRPV1 and P2X3 showed a visible signal in HEK293 cells transfected with both receptors. It is concluded that the two pain-relevant receptors TRPV1 and P2X3 interact with each other in an inhibitory manner probably by a physical association established by a motif located at the C-terminal end of the P2X3 receptor distal to Glu362. [Copyright &y& Elsevier]

Abstract: Human ether-à-go-go-related gene (hERG) has an important role in the repolarization of the cardiac action potential. Sophocarpine and sophoridine are quinolizidine alkaloids and their structures are similar. Our aim was to investigate the effects of sophocarpine or sophoridine on hERG-encoded K+ channels and the underlying structure–activity relationships. The effects of sophocarpine and sophoridine were examined on stably expressed hERG channels in HEK293 cells using a whole-cell patch clamp technique and Western blot analysis. The oil–water partition coefficients of sophocarpine and sophoridine were determined by a validated RP-HPLC method. At 300 µM, fractional block was 60.9±1.4% for sophocarpine versus 41.9±2.0% for sophoridine. Compared with sophocarpine, voltage-dependence of hERG channels inhibition by sophoridine was more notable. Sophoridine altered the activation properties, but not sophocarpine. Sophocarpine shifted the inactivation curve in a negative direction, but not sophoridine. Both drugs had no significant effect on the expression of hERG protein. The partition coefficients for the n-octanol/water system of sophocarpine and sophoridine at 37 °C were 16.03±0.42 and 1.94±0.03, respectively. In summary, sophocarpine and sophoridine are low potency blockers of hERG channels that functions by changing the channel kinetics, and sophocarpine is a more potent blocker of hERG K+ channels than sophoridine, which may be due to higher hydrophobic nature of sophocarpine compared with sophoridine. Sophocarpine may have a higher binding affinity for the inactivate state. In contrast, sophoridine has a higher binding affinity for the open state. Both drugs have no effect on the generation and trafficking of hERG protein. [Copyright &y& Elsevier]

Chemokine receptors belong to a superfamily of proteins that signal through coupled heterotrimeric G proteins. Chemokine receptor CCR5 is the major co-receptor essential for HIV entry into host cells, and now chemokine CCR5 receptor has become an important target in searching for anti-HIV drugs. Here, we describe the establishment of a human embryonic kidney (HEK) 293/CCR5-HA cell line stably expressing CCR5 receptor with influenza hemagglutinin (HA) tag at the N termini on the membrane surface of HEK293 cells. Plasmid pcDNA3.0-CCR5-HA was transfected into HEK293 cells, and monoclonal HEK293 cell lines expressing CCR5 receptor were generated under G418 selection. The expression of functional CCR5 receptor was tested by GTPγS assay, and the results showed about 5 monoclonal HEK293 cell lines expressed functional CCR5 receptor, and of which No.7 monoclonal cell line is the best. The FACS analysis results further confirmed that CCR5 receptor was positive in 96.89% of No.7 monoclonal HEK293/CCR5-HA cell line. Further results showed that RANTES significantly stimulated GTPγS binding in the dose-dependent manner, and CCR5 antagonist Sch-351125 inhibited RANTESstimulated GTPγS binding in the dose-dependent manner in No.7 monoclonal HEK293/CCR5-HA cell line. Our results suggest that the established HEK293/CCR5-HA cell line is suitable for high-throughput screening and is feasible to identify CCR5 receptor antagonists. [ABSTRACT FROM AUTHOR]

The human (h) P2X3 receptor and its mutants deficient in one out of four N-glycosylation sites were expressed in HEK293 cells. Concentration–response curves were generated by whole-cell recordings of α,β-methylene ATP (α,β-meATP)-induced currents. A gradual change of external pH from the alkaline 8.0 to the acidic 5.0 successively decreased the maximum current amplitude ( Emax) without affecting the EC50 value. The replacement of Asn-139 and -170 by Asp (N139D, N170D) abolished the pH sensitivity of the wild-type (WT) hP2X3 receptor. In the case of N194D, the Emax was again the highest at the alkaline pH value with no change from 7.4 to 6.5, whereas in the case of N290D, there was an inverse pH sensitivity, with an increase of Emax in the acidic range. However, this effect appeared to be due to enhanced protonation by the insertion of Asp into the receptor, because replacement of Asn by the neutral Thr resulted in a comparable potency of α,β-meATP at any of the pH values investigated. In accordance with the reported finding that His-206 is involved in the modulation of WT P2X3 receptors by protons, we showed that the normal change of Emax by an acidic, but not alkaline pH was abolished after substitution of this His by Ala. However, the double mutant H206A + N290D did not react to acidification or alkalinization with any change in Emax. In conclusion, only fully N-glycosylated P2X3 receptors recognize external pH with a modified sensitivity towards α,β-meATP. [ABSTRACT FROM AUTHOR]

Abstract: Clivorine is an otonecine-type pyrrolizidine alkaloid (PA) isolated from the Chinese medicinal plant Ligularia hodgsonii Hook., and our previous reports have shown its toxicity on human normal liver L-02 cells. It is generally believed that biotransformation of PAs to its metabolites is required for their toxicity; thus, there is nearly no report about the toxicity of clivorine on other non-hepatic cells in vitro. The aim of this study is to observe the toxicity of clivorine on the non-hepatic human embryonic kidney 293 (HEK293) cell that is of epithelial origin, and its primary mechanism. Our results showed that clivorine significantly reduced HEK293 cell viability, but there was no detectable apoptotic DNA ladder and cleaved fragments of caspase-3 and caspase-9 in clivorine-treated cells, which indicates the toxicity of clivorine is not due to inducing apoptosis. The results of western blot showed that clivorine induced sustained p38, c-Jun N-terminal kinase (JNK) and extracellular signal-related kinases (ERK1/2) phosphorylation in a concentration- and time-dependent manner, and the JNK inhibitor SP600125 significantly augmented the toxicity of clivorine. Our results suggest that clivorine itself has direct toxicity on HEK293 cells, and phosphorylated JNK may play some role in counteracting the toxicity of clivorine on HEK293 cells. [Copyright &y& Elsevier]

Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA−), tetraphenylborate (TPB−) and [W2(CO)10(S2CH)]−, the putative lipophilic anion $$ {\text{B}}{\left( {{\text{CF}}_{3} } \right)}^{ - }_{4} $$ and three new heterocyclic W(CO)5 derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA−, TPB− and $$ {\text{B}}{\left( {{\text{CF}}_{3} } \right)}^{ - }_{4} $$ increased and that of W(CO)5 derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA− and W(CO)5 derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB− and $$ {\text{B}}{\left( {{\text{CF}}_{3} } \right)}^{ - }_{4} $$ no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions. [ABSTRACT FROM AUTHOR]

Abstract: GPR55 is an orphan G protein-coupled receptor. In this study, we explored a possible endogenous ligand for GPR55 using HEK293 cells which expressed GPR55. We found that lysophosphatidylinositol induced rapid phosphorylation of the extracellular signal-regulated kinase in transiently or stably GPR55-expressing cells. On the other hand, lysophosphatidylinositol did not induce phosphorylation of the extracellular signal-regulated kinase in vector-transfected cells. Lysophosphatidic acid and sphingosine 1-phosphate also induced phosphorylation of the extracellular signal-regulated kinase in GPR55-expressing cells. However, these lipid phosphoric acids elicited similar responses in vector-transfected cells. Various types of other lysolipids as well as the cannabinoid receptor ligands did not induce phosphorylation of the extracellular signal-regulated kinase. We also found that lysophosphatidylinositol elicited a rapid Ca2+ transient in GPR55-expressing cells. Lysophosphatidylinositol also stimulated the binding of GTPγS to the GPR55-expressing cell membranes. These results strongly suggest that GPR55 is a specific and functional receptor for lysophosphatidylinositol. [Copyright &y& Elsevier]

Abstract: The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. In mammals, multiple subtypes of interferon-α (IFN-α) exist, most of which possess antiviral activity. Little is known about giant panda IFN-α genes and the role they may play in giant panda immunological responses to viruses. We have cloned genes encoding 12 giant panda IFN-α (AmIFN-α or AmIFNA) subtypes that share from 90 to 99% amino acid sequence identity. AmIFN-α12 has one additional amino acid at position 57, which is not present in other subtypes. Sequence identity of the AmIFN-α proteins encoded by the 12 genes compared to human IFN-α2 is approximately 58%. Unlike most of the human subtypes, each of the 12 giant panda IFN sequences has an N-glycosylation recognition site. Expression of all 12 AmIFN-α subtypes in 293 cells was confirmed by SDS-PAGE and Western blotting analysis. The antiviral activity and antiproliferative activity of each AmIFN-α subtype produced in transiently transfected 293 cell cultures were tested in vitro. All AmIFN-α subtypes were found to be stable at pH 2 or 65°C and to exhibit antiviral activity. Some IFN subtypes (AmIFN-α8 and AmIFN-α4) showed higher biological activity levels than others, whereas AmIFN-α11 exhibited lower activity. AmIFN-α had various antiproliferative activities to different target cells. To B16 cells, AmIFN-α3, AmIFN-α4, AmIFN-α8 had the highest activities, while to K562 cells, AmIFN-α3, AmIFN-α7, AmIFN-α10 had the highest activities. The various IFN-α subtypes displayed a good correlation between their antiviral and antiproliferative potencies. [Copyright &y& Elsevier]

Abstract: The bovine creatine transporter (CreaT) has been purified from membranes of HEK293 cells stably expressing high levels of the transporter. Membranes were solubilized with decyl maltoside and the CreaT was purified (¿90% pure) by affinity chromatography on wheat germ agglutinin (WGA)¿Sepharose and gel-filtration. The CreaT was shown to be an approximately 70kDa glycoprotein by SDS¿polyacrylamide gel electrophoresis and Western blotting. Identification of the CreaT was confirmed by sequencing tryptic peptides by mass spectrometry. Laser light scattering showed the majority of the CreaT to be present as a 224kDa species. Additional purification was obtained when the Creat was eluted from the WGA column and purified by gel-filtration in Fos-choline 12 instead of decyl maltoside, followed by a second WGA affinity step to exchange the detergent for sodium cholate. This resulted in a 30-fold purification (¿95% purity) of the ~70kDa CreaT, with a yield of 15%. From this, it is estimated that the CreaT comprises ~3% of total HEK293-CreaT membrane protein. Gel-filtration showed the transporter to migrate with an apparent molecular mass of 210kDa. Circular dichroism showed a predominantly a-helical structure, consistent with the 12 transmembrane domains predicted for the transporter. This work has enabled the purification of the CreaT in amounts (~100µg) that make it feasible to consider structural studies of a member of the Na+- and Cl--dependent neurotransmitter transporter family. [Copyright &y& Elsevier]

Abstract: The influence of single-walled carbon nanotubes (SWCNTs) on human HEK293 cells is investigated with the aim of exploring SWCNTs biocompatibility. Results showed that SWCNTs can inhibit HEK293 cell proliferation, decrease cell adhesive ability in a dose- and time-dependent manner. HEK293 cells exhibit active responses to SWCNTs such as secretion of some 20–30kd proteins to wrap SWCNTs, aggregation of cells attached by SWCNTs and formation of nodular structures. Cell cycle analysis showed that 25μg/ml SWCNTs in medium induced G1 arrest and cell apoptosis in HEK293 cells. Biochip analysis showed that SWCNTs can induce up-regulation expression of cell cycle-associated genes such as p16, bax, p57, hrk, cdc42 and cdc37, down-regulation expression of cell cycle genes such as cdk2, cdk4, cdk6 and cyclin D3, and down-regulation expression of signal transduction-associated genes such as mad2, jak1, ttk, pcdha9 and erk. Western blot analysis showed that SWCNTs can induce down-regulation expression of adhesion-associated proteins such as laminin, fibronectin, cadherin, FAK and collagen IV. These results suggest that down-regulation of G1-assoicated cdks and cyclins and upregulation of apoptosis-associated genes may contribute to SWCNTs induced G1 phase arrest and cell apoptosis. In conclusion, SWCNTs can inhibit HEK293 cells growth by inducing cell apoptosis and decreasing cellular adhesion ability. [Copyright &y& Elsevier]

Mesoridazine, a phenothiazine antipsychotic agent, prolongs the QT interval of the cardiac electrocardiogram and is associated with Torsade de pointes-type arrhythmias. In this study, we examined the effects of mesoridazine on human ether-a-go-go-related gene (HERG) K+ currents. HERG channels were stably expressed in human embryonic kidney 293 cells and studied using standard whole-cell patch-clamp technique (37 °C). Mesoridazine blocked HERG currents in a concentration-dependent manner (IC50 550 nM at 0 mV); block increased significantly over the voltage range where HERG activates and saturated at voltages eliciting maximal HERG channel activation. Tonic block of HERG current by mesoridazine (1.8 μM) was minimal (<2–4%). The rate of the onset of HERG channel block was rapid and dose dependent (τ = 54 ± 7 ms at 0 mV and 1.8 μM mesoridazine), but not significantly affected by test potentials ranging from –30 to +30 mV. The V1/2 for steady-state activation was shifted from –31.2 ± 1.0 to –39.2 ± 0.5 mV (P < 0.01). The apparent rate of HERG channel deactivation was significantly reduced (fast τ = 153 ± 8 vs. 102 ± 6 ms at –50 mV, P < 0.01; slow τ = 1113 ± 63 vs. 508 ± 27 ms, P < 0.01). The inactivation kinetics and voltage dependence of steady-state inactivation of the HERG channel were not significantly altered by mesoridazine. These findings demonstrate that mesoridazine is a potent and rapid open-channel blocker of HERG channels. This block would explain the QT prolongation seen clinically at therapeutic concentrations (0.3–3.6 μM). [Copyright &y& Elsevier]

The hand-held gene gun uses a pulse of helium to fire small gold particles coated with desiccated DNA (microcarriers) at target cells. This method of biolistic transfection is becoming increasingly popular as an effective means of rapid gene delivery into mammalian tissue. Current methods of microcarrier preparation, however, are slow (up to 2 days) and can result in variations in transfection efficiency due to a number of problems including shearing of DNA, agglomeration and adhesion of gold particles. Here we describe an improved, more rapid method of microcarrier preparation. To evaluate the new procedure we have used DNA encoding yellow fluorescent protein (EYFP), a modified version of the green fluorescent protein, which we have transfected into HEK293 cells. The data show that transfection by the new method results in high levels of transfection efficiency and low variability compared to an alternative method. [Copyright &y& Elsevier]