An impedimetric assay for the identification of abnormal mitochondrial dynamics in living cells

Mitochondrial dynamics (fission and fusion) plays an important role in cell functions. Disruption in mitochondrial dynamics has been associated with diseases such as neurobiological disorders and cardiovascular diseases. Analysis of mitochondrial fission/fusion has been mostly achieved through direct visualization of the fission/fusion events in live-cell imaging of fluorescently labeled mitochondria. In this study, we demonstrated a label-free, non-invasive Electrical Impedance Spectroscopy (EIS) approach to analyze mitochondrial dynamics in a genetically modified human neuroblastoma SH-SY5Y cell line with no huntingtin protein expression. Huntingtin protein has been shown to regulate mitochondria dynamics. We performed EIS studies on normal SH-SY5Y cells and two independent clones of huntingtin-null cells. The impedance data was used to determine the suspension conductivity and further cytoplasmic conductivity and relate to the abnormal mitochondrial dynamics. For instance, the cytoplasm conductivity value was increased by 11% from huntingtin-null cells to normal cells. Results of this study demonstrated that EIS is sensitive to characterize the abnormal mitochondrial dynamics that can be difficult to quantify by the conventional microscopic method.     

Evaluation of the bioactivity of novel spiroisoxazoline typecompounds against normal and cancer cell lines

The aim of this study was to investigate the in vitro cellular activity of novel spiroisoxazoline type compounds against normal and cancer cell lines from lung tissue (Hs888Lu), neuron-phenotypic cells (SH-SY5Y), neuroblastoma (SH-SY5Y), human histiocytic lymphoma (U937), lung cancer (A549), and leukaemia (HL-60). Our bioassay program revealed that the spiroisoxazoline type compounds show cytotoxicity only in lymphoma cell lines, which is in contrast with the pyrrolidine precursor of these spiroisoxazoline compounds, where significant cytotoxicity is seen in all normal and cancer cell lines. These data suggest a tumour-specific mechanism of action. In addition these data also show that spiroisoxazoline compounds are non-toxic in the human neuronphenotypic neuroblastoma SH-SY5Y cell line, and furthermore that they might protect cells from neurodegenerative disease.     

Dioscin-Mediated Autophagy Alleviates MPP+-Induced Neuronal Degeneration: An In Vitro Parkinson’s Disease Model

Autophagy is a cellular homeostatic process by which cells degrade and recycle their malfunctioned contents, and impairment in this process could lead to Parkinson’s disease (PD) pathogenesis. Dioscin, a steroidal saponin, has induced autophagy in several cell lines and animal models. The role of dioscin-mediated autophagy in PD remains to be investigated. Therefore, this study aims to investigate the hypothesis that dioscin-regulated autophagy and autophagy-related (ATG) proteins could protect neuronal cells in PD via reducing apoptosis and enhancing neurogenesis. In this study, the 1-methyl-4-phenylpyridinium ion (MPP⁺) was used to induce neurotoxicity and impair autophagic flux in a human neuroblastoma cell line (SH-SY5Y). The result showed that dioscin pre-treatment counters MPP⁺-mediated autophagic flux impairment and alleviates MPP⁺-induced apoptosis by downregulating activated caspase-3 and BCL2 associated X, apoptosis regulator (Bax) expression while increasing B-cell lymphoma 2 (Bcl-2) expression. In addition, dioscin pre-treatment was found to increase neurotrophic factors and tyrosine hydroxylase expression, suggesting that dioscin could ameliorate MPP⁺-induced degeneration in dopaminergic neurons and benefit the PD model. To conclude, we showed dioscin’s neuroprotective activity in neuronal SH-SY5Y cells might be partly related to its autophagy induction and suppression of the mitochondrial apoptosis pathway.     

Proteomic Analysis of Protective Effects of Epimedium Flavonoids against Ethanol-Induced Toxicity in Retinoic Acid-Treated SH-SY5Y Cells

Alcohol (ethanol) is one of the most common addictive psychoactive substances in the world, and alcoholism may result in harmful effects on human health, especially on the nervous system. Flavonoids are regarded as the main active constituent in Epimedium, which has been used to cure some nervous system diseases such as amnesia for over 1000 years. Here, the protective effects of Epimedium flavonoids against ethanol-induced toxicity in retinoic acid (RA)-treated SH-SY5Y cells were investigated. Their mechanism was explored by a label-free proteomic approach combined with bioinformatic analysis for the first time. The results showed that ethanol treatment decreased cell viability by 18%, whereas the viability increased significantly after intervention with Epimedium flavonoids (p < 0.01). According to proteomic and bioinformatic analyses, hundreds of differentially expressed proteins (DEPs) were identified and classified as biological process (GO_BP), cellular component (GO_CC) and molecular function (GO_MF). Among them, GO_MF of DEPs, especially molecular function relevant to G proteins, greatly changed in SH-SY5Y cells pretreated by Epimedium flavonoids. In the alcoholism pathway, the expression of the Gi protein was up-regulated under the influence of ethanol, whereas Epimedium flavonoids could reverse the expression profile, both of which were validated by Western blot assay. In conclusion, Gi protein seemed to be an important factor in the alcoholism pathway to suppress the ethanol-induced toxicity of SH-SY5Y cells. These findings suggest a protective potential of Epimedium flavonoids against ethanol-induced toxicity to neurons via the regulation of Gi protein function.     

Interfacing SH-SY5Y human neuroblastoma cells with SU-8 microstructures

Microwell structures were fabricated using SU-8 photoresist for engineering a quasi-three-dimensional (quasi-3D) microenvironment for cultured neuronal cells. SH-SY5Y human neuroblastoma cells were successfully integrated into microwells of a nominal diameter of 100 microm, with or without 10-microm wide microchannels connecting neighboring microwells, in an aspect ratio (ratio of structure depth over width) of approximately 1. With the help of polyethylene glycol stamping and laminin coating, a neuronal-like network was achieved by integrating populations of SH-SY5Y cells with a microwell network pattern. Resting membrane potential establishment was evaluated with confocal microscopy and the potentiometric fluorescent dye tetramethylrhodamine methyl ester. It was found that the intra/extracellular fluorescent intensity ratio (R) was 2.4+/-1.4 [n (number of cells measured)=112] for SH-SY5Y cells on flat SU-8 substrates on day 5 into differentiation, which was not significantly different from the ratio on day 13 into differentiation, 2.0+/-1.8 (n=104) (P>0.05). For cells in the microwell network structures, R was 4.8+/-4.7 (n=51) and 3.9+/-3.2 (n=62) on days 5 and 13 into differentiation, respectively (P>0.5). Cells within the network structures had higher R ratios than on flat substrates, for either day 5 or 13 into differentiation (P<0.01). These results demonstrated that the well network structures, or topographically patterned substrates, were more suitable formats for promoting SH-SY5Y cell resting membrane potential establishment than flat substrates, suggesting the potential to control cellular function through substrate topography engineering.     

Luteolin isolated from the medicinal plant Elsholtzia rugulosa (Labiatae) prevents copper-mediated toxicity in β-amyloid precursor protein Swedish mutation overexpressing SH-SY5Y cells

Luteolin, a 3',4',5,7-tetrahydroxyflavone, is a plant flavonoid and pharmacologically active agent that has been isolated from several plant species. In the present study, the effects of luteolin obtained from the medicinal plant Elsholtzia rugulosa and the related mechanisms were examined in an Alzheimer's disease (AD) cell model. In this model, copper was used to exacerbate the neurotoxicity in β-amyloid precursor protein Swedish mutation stably overexpressed SH-SY5Y cells (named "APPsw cells" for short). Based on this model, we demonstrated that luteolin increased cell viability, reduced intracellular ROS generation, enhanced the activity of SOD and reversed mitochondrial membrane potential dissipation. Inhibition of caspase-related apoptosis was consistently involved in the neuroprotection afforded by luteolin. Furthermore, it down-regulated the expression of AβPP and lowered the secretion of Aβ????. These results indicated that luteolin from the Elsholtzia rugulosa exerted neroprotective effects through mechanisms that decrease AβPP expression, lower Aβ secretion, regulate the redox imbalance, preserve mitochondrial function, and depress the caspase family-related apoptosis.     

Design,synthesis, cytotoxic activity,and apoptosis inducing effects of 4- and N-substituted benzoyltaurinamide derivatives

In this study, a group of 4-substituted benzoyltaurinamide derivatives were designed, synthesized, and investigated for their anticancer activity against three cancer cell lines and one nontumorigenic cell line by MTT assay. Among the final compounds, methoxyphenyl derivatives 14, 15, 16 were found to be effective against all the tested cancerous cell lines with promising selectivity. The most active compounds were further evaluated to determine the molecular mechanism of their anticancer activity by using western blot assay and the Annexin V-FITC/PI test. Compound 14 (in SH-SY5Y and MDA-MB-231 cell lines) and 15 (in SH-SY5Y cell line) were found to induce intrinsic apoptotic pathway by upregulating BAX, caspase-3, and caspase-9, while downregulating Bcl-2 and Bcl-xL expression levels. According to mechanistic studies, compounds displayed their anticancer activity via three different mechanisms: a. caspase-dependent, b. caspase-independent, and c. caspase-dependent pathway that excluded caspase-9 activation. As a result, this study provides interesting data which can be used to design new taurine-based anticancer derivatives.     

Synthesis and In Vitro Characterization of Selective Cannabinoid CB2 Receptor Agonists: Biological Evaluation against Neuroblastoma Cancer Cells

1,8-naphthyridine-3-carboxamide structures were previously identified as a promising scaffold from which to obtain CB2R agonists with anticancer and anti-inflammatory activity. This work describes the synthesis and functional characterization of new 1,8-naphthyridin-2(1H)-one-3-carboxamides with high affinity and selectivity for CB2R. The new compounds were able to pharmacologically modulate the cAMP response without modulating CB2R-dependent β-arrestin2 recruitment. These structures were also evaluated for their anti-cancer activity against SH-SY5Y and SK-N-BE cells. They were able to reduce the cell viability of both neuroblastoma cancer cell lines with micromolar potency (IC₅₀ of FG158a = 11.8 μM and FG160a = 13.2 μM in SH-SY5Y cells) by a CB2R-mediated mechanism. Finally, in SH-SY5Y cells one of the newly synthesized compounds, FG158a, was able to modulate ERK1/2 expression by a CB2R-mediated effect, thus suggesting that this signaling pathway might be involved in its potential anti-cancer effect.     

Resveratrol protects SH-SY5Y neuroblastoma cells from apoptosis induced by dopamine

Dopamine (DA) is an oxidant that may contribute to the degeneration of dopaminergic neurons. The present study demonstrates that DA-induced cytotoxicity in human-derived neurotypic cells, SH-SY5Y, is prevented by resveratrol, one of the major antioxidative constituents found in the skin of grapes. SH-SY5Y cells, a neuroblastoma cell line, treated with DA at 300 and 500 microM for 24 h underwent apoptotic death as determined by characteristic morphological features, including nuclear condensation, and loss of mitochondrial membrane potential (MMP). Flow cytometric analysis using Annexin V showed that DA can induce significant and severe apoptosis. Exposure to resveratrol (5 microM) for 1 h prior to the DA treatment attenuated DA-induced cytotoxicity, and rescued the loss of MMP. To investigate the apoptotic signaling pathways relevant to the restoration of DA-induced apoptosis by resveratrol, we carried out quantitative analysis of Bcl-2, caspase-3, and cleaved poly ADP-ribose polymerase (PARP) by immunoblot analysis. Resveratrol pretreatment led to a decrease in cleavage of PARP, an increase in the Bcl-2 protein, and activation of caspase-3. These results suggest that DA may be a potential oxidant of neuronal cells at biologically relevant concentrations. Resveratrol may protect SH-SY5Y cells against this cytotoxicity, reducing intracellular oxidative stress through canonical signal pathways of apoptosis and may be of biological importance in the prevention of a dopaminergic neurodegenerative disorder such as Parkinson disease.     

Adaptation of cAMP signaling system in SH-SY5Y neuroblastoma cells following expression of a constitutively active stimulatory G protein alpha, Q227L Gsalpha

Heterotrimeric GTP-binding proteins (G protein) are known to participate in the transduction of signals from ligand activated receptors to effector molecules to elicit cellular responses. Sustained activation of cAMP-G protein signaling system by agonist results in desensitization of the pathway at receptor levels, however it is not clear whether such receptor responses induce other changes in post-receptor signaling path that are associated with maintenance of AMP levels, i.e. cAMP-forming adenylate cyclase (AC), cAMP-degrading cyclic nucleotide phosphodiesterase (PDE) and cAMP-dependent protein kinase (PKA). Experiments were performed to determine the expression of AC, PDE, and PKA isoforms in SH-SY5Y neuroblastoma cells, in which cAMP system was activated by expressing a constitutively activated mutant of stimulatory G protein (Q227L Gsalpha). Expression of ACI mRNA was increased, but levels of ACVIII and ACIX mRNA were decreased. All of the 4 expressed isoforms of PDE (PDE1C, PDE2, PDE 4A, and PDE4B) were increased in mRNA expression; the levels of PKA RIalpha, RIbeta, and RIIbeta were increased moderately, however, those of RIIalpha and Calpha were increased remarkably. The activities of AC, PDE and PKA were also increased in the SH-SY5Y cells expressing Q227L Gsalpha. The similar changes in expression and activity of AC, PDE and PKA were observed in the SH-SY5Y cells treated with dbcAMP for 6 days. Consequently, it is concluded that the cAMP system adapts at the post-receptor level to a sustained activation of the system by differential expression of the isoforms of AC, PDE, and PKA in SH-SY5Y neuroblastoma. We also showed that an increase in cellular cAMP concentration might mediate the observed changes in the cAMP system.     

Inhibition of gamma ray-induced apoptosis by stimulatory heterotrimeric GTP binding protein involves Bcl-xL down-regulation in SH-SY5Y human neuroblastoma cells

Heterotrimeric GTP-binding proteins (G proteins) transduce extracellular signals into intracellular signals by activating effector molecules including adenylate cyclases that catalyze cAMP formation, and thus regulate various cellular responses such as metabolism, proliferation, and apoptosis. cAMP signaling pathways have been reported to protect cells from ionizing radiation-induced apoptosis, but however, the protective mechanism is not clear. Therefore, this study aimed to investigate the signaling molecules and the mechanism mediating the anti-apoptotic action of cAMP signaling system in radiation-induced apoptosis. Stable expression of a constitutively active mutant of Gas (GalphasQL) protected gamma ray-induced apoptosis which was assessed by analysis of the cleavages of PARP, caspase-9, and caspase-3 and cytochrome C release in SH-SY5Y human neuroblastoma cells. GasQL repressed the gamma ray-induced down-regulation of Bcl-xL protein, but transfection of Bcl-xL siRNA increased the gamma ray-induced apoptosis and abolished the anti-apoptotic effect of GasQL. GasQL decreased the degradation rate of Bcl-xL protein, and it also restrained the decrease in Bcl-xL mRNA by increasing the stability following ionizing irradiation. Furthermore, prostaglandin E2 that activates Gas was found to protect gamma ray-induced apoptosis, and the protective effect was abolished by treatment with prostanoid receptor antagonist specific to EP2/4R subtype. Moreover, specific agonists for adenosine A1 receptor that inhibits cAMP signaling pathway augmented gamma ray-induced apoptosis. From this study, it is concluded that Galphas-cAMP signaling system can protect SH-SY5Y cells from gamma ray-induced apoptosis partly by restraining down-regulation of Bcl-xL expression, suggesting that radiation-induced apoptosis can be modulated by GPCR ligands to improve the efficiency of radiation therapy.     

Evaluation of Anticancer and Antibacterial Activity of Four 4-Thiazolidinone-Based Derivatives

Heterocycles are commonly known for their unique features, e.g., antibacterial or anticancer properties. Although many synthetic heterocycles, such as 4-thiazolidinone (4-TZD), have been synthesized, their potential applications have not yet been fully investigated. However, many researchers have reported relevant results that can be a basis for the search for new potential drugs. Therefore, the aim of this study was to evaluate the cytotoxic, cytostatic, and antibacterial effects of certain 4-thiazolidinone-based derivatives, Les-3166, Les-5935, Les-6009, and Les-6166, on human fibroblasts (BJ), neuroblastoma (SH-SY5Y), epithelial lung carcinoma (A549), and colorectal adenocarcinoma (CACO-2) cell lines in vitro. All tested compounds applied in a concentration range from 10 to 100 µM were able to decrease metabolic activity in the BJ, A549, and SH-SY5Y cell lines. However, the action of Les-3166 was mainly based on the ROS-independent pathway, similarly to Les-6009. In turn, Les-5935 and Les-6166 were able to promote ROS production in BJ, A549, and SH-SY5Y cells, compared to the control. Les-3166, Les-6009, and Les-6166 significantly increased the caspase-3 activity, especially at the concentrations of 50 µM and 100 µM. However, Les-5935 did not induce apoptosis. Only Les-5935 showed a minor cytostatic effect on SH-SY5Y cells. Additionally, the antibacterial properties of the tested compounds against P. aeruginosa bacterial biofilm can be ranked as follows: Les-3166 > Les-5935 > Les-6009. Les-6166 did not show any anti-biofilm activity. In summary, the study showed that Les-5935, Les-6009, and Les-6166 were characterized by anticancer properties, especially in the human lung cancer cell. In cases of BJ, SH-SY5Y, and CACO-2 cells the anticancer usage of such compounds is limited due to effect visible only at 50 and 100 µM.     

Neuroprotective Activities of Crossyne flava Bulbs and Amaryllidaceae Alkaloids: Implications for Parkinson’s Disease

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases and affects approximately 6.3 million people worldwide. To date, the treatment of PD remains a challenge, as available treatment options are known to be associated with serious side effects; hence, the search for new treatment strategies is critical. Extracts from the Amaryllidaceae plant family as well as their alkaloids have been reported to have neuroprotective potentials. This study, therefore, investigated the biological activities of Crossyne flava and its isolated alkaloids in an in vitro MPP⁺ (1-methyl-4-phenylpyridinium) PD model using SH-SY5Y cells. The effects of the total extract as well as the four compounds isolated from Crossyne flava (i.e., pancratinine B (1), bufanidrine (2), buphanisine (3), and epibuphanisine (4)) were evaluated for cell viability, neuroprotection, levels of reactive oxygen species (ROS), adenosine triphosphate activity (ATP), and caspase 3/7 activity in SH-SY5Y cells. The results obtained showed that pre-treatment with both the extract and the isolated compounds was effective in protecting the SH-SY5Y cells from MPP⁺-induced neurotoxicity and inhibited ROS generation, ATP depletion as well as apoptosis induction in the SH-SY5Y cells. The results of this study show that the Amaryllidaceae plant family may be a source of novel compounds for the treatment of neurodegenerative diseases, which validates the reported traditional uses.     

The total synthesis of a ganglioside Hp-s1 analogue possessing neuritogenic activity by chemoselective activation glycosylation

The total synthesis of ganglioside 2, an analogue of the ganglioside Hp-s1 (1) which displays neuritogenic activity toward the rat pheochromocytoma cell line PC-12 cell in the presence of nerve growth factor (NGF) with an effect (34.0%) greater than that of the mammalian ganglioside GM 1 (25.4%), was accomplished by applying a chemoselective-activation glycosylation strategy. Moreover, we also demonstrate that the synthesized ganglioside 2 exhibited neuritogenic activity toward the human neuroblastoma cell line SH-SY5Y without the presence of NGF.     

A Class I HDAC Inhibitor Rescues Synaptic Damage and Neuron Loss in APP-Transfected Cells and APP/PS1 Mice through the GRIP1/AMPA Pathway

As a neurodegenerative disease, Alzheimer’s disease (AD) seriously affects the health of older people. Changes in synapses occur first over the course of the disease, perhaps even before the formation of Aβ plaques. Histone deacetylase (HDAC) mediates the damage of Aβ oligomers to dendritic spines. Therefore, we examined the relationship between HDAC activity and synaptic defects using an HDAC inhibitor (HDACI), BG45, in the human neuroblastoma SH-SY5Y cell line with stable overexpression of Swedish mutant APP (APPsw) and in APP/PS1 transgenic mice during this study. The cells were treated with 15 μM BG45 and the APP/PS1 mice were treated with 30 mg/kg BG45. We detected the levels of synapse-related proteins, HDACs, tau phosphorylation, and amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors using Western blotting and immunohistochemistry. We also measured the expression of cytoskeletal proteins in the cell model. The mRNA levels of the glutamate ion receptor alginate subunit 2 (GRIK2), sodium voltage-gated channel beta subunit (SCN3B), synaptophysin (SYP), Grm2 (the gene encoding glutamate receptor subunit 2 (GluR2)), Grid2IP, glutamate receptor interacting protein 1 (GRIP1), and GRIP2 were detected to explore the effects of the HDACI on regulating the expression of synaptic proteins and AMPA receptors. According to our studies, the expressions of HDAC1, HDAC2, and HDAC3 were increased, which were accompanied by the downregulation of the synapse-related proteins SYP, postsynaptic dendritic protein (PSD-95), and spinophilin as early as 24 h after transfection with the APPsw gene. BG45 upregulated the expression of synapse-related proteins and repaired cytoskeletal damage. In vivo, BG45 alleviated the apoptosis-mediated loss of hippocampal neurons, upregulated synapse-related proteins, reduced Aβ deposition and phosphorylation of tau, and increased the levels of the synapse-related genes GRIK2, SCN3B, SYP, Grm2, and Grid2IP. BG45 increased the expression of the AMPA receptor subunits GluA1, GluA2, and GluA3 on APPsw-transfected cells and increased GRIP1 and GRIP2 expression and AMPA receptor phosphorylation in vivo. Based on these results, HDACs are involved in the early process of synaptic defects in AD models, and BG45 may rescue synaptic damage and the loss of hippocampal neurons by specifically inhibiting HDAC1, HDAC2, and HDAC3, thereby modulating AMPA receptor transduction, increasing synapse-related gene expression, and finally enhancing the function of excitatory synapses. BG45 may be considered a potential drug for the treatment of early AD in further studies.     

2-DE and MALDI-TOF-MS for a comparative analysis of proteins expressed in different cellular models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder characterized by the selective loss of motor neurons from the spinal cord and brain. About 10% of ALS cases are familial (FALS), and in 20% of these cases the disease has been linked to mutations in the Cu,Zn-SOD1 gene. Although the molecular mechanisms causing these forms of ALS are still unclear, evidence has been provided that motor neurons injuries associated with mutant superoxide dismutase (SOD1)-related FALS result from a toxic gain-in-fuction of the mutated enzyme. To understand better the role of these mutations in the pathophysiology of FALS we have compared the pattern of proteins expressed in human neuroblastoma SH-SY5Y cell line with those of cell lines transfected with plasmids expressing the wild-type human SOD1 and the H46R and G93A mutants. 2-DE coupled to MALDI-TOF-MS were the proteomic tools used for identification of differentially expressed proteins. These included cytoskeletal proteins, proteins that regulate energetic metabolism and intracellular redox conditions, and the ubiquitin proteasome system. The proteomic approach allowed to expand the knowledge on the pattern of proteins, with altered expression, which we should focus on, for a better understanding of the possible mechanism involved in mutated-SOD1 toxicity. The cellular models considered in this work have also evidenced biochemical characteristics common to other SOD1-mutated cellular lines connected to the pathogenesis of ALS.     

Direct monitoring of the expression of the green fluorescent protein-extracellular signal-regulated kinase 2 fusion protein in transfected cells using capillary electrophoresis with laser-induced fluorescence detection

Proper subcellular localization of the extracellular signal-regulated kinases (ERKs) is important in regulating physiological functions such as proliferation and differentiation in the pheochromocytoma cell line (PC12 cells). Thus, a direct visualization method is necessary to observe ERK localization within the cell or in crude cellular extracts. In this paper, a determination method was established for the detection of ERK2 localization in PC12 cells using green fluorescent protein (GFP) and capillary electrophoresis with laser-induced fluorescence (LIF). GFP as a reporter or labeling tag for gene expression in biochemistry and cell biology was used for the detection of ERK2 localization in PC12 cells. PC12 cells were transfected with GFP-ERK2 plasmid construct that was inserted into a variant GFP gene (enhanced green fluorescent protein), and successfully expressed GFP-ERK2 fusion proteins. GFP-ERK2 fusion proteins were detected within 5 min by CE analysis using an uncoated fused-silica capillary with LIF. Optimum conditions for GFP-ERK2 fusion proteins detection were 100 mM 3-(cyclohexylamino)-1-propanesulfonic acid buffer containing 100 mM sodium dodecylsulfate, pH 11, running at 20 degrees C. This result offers new opportunity in screening for the determination of localization of intracellular components, protein-protein interactions and kinase activity within the cells.     

Anti-Inflammatory and Antiproliferative Properties of Sweet Cherry Phenolic-Rich Extracts

Cherries have largely been investigated due to their high content in phenolics in order to fully explore their health-promoting properties. Therefore, this work aimed to assess, for the first time, the anti-inflammatory potential of phenolic-targeted fractions of the Saco cherry, using RAW 264.7 macrophages stimulated with lipopolysaccharide. Additionally, the cytotoxic effects on gastric adenocarcinoma (AGS), neuroblastoma (SH-SY5Y) and normal human dermal fibroblast (NHDF) cells were evaluated, as well as the ability to protect these cellular models against induced oxidative stress. The obtained data revealed that cherry fractions can interfere with cellular nitric oxide (NO) levels by capturing NO radicals and decreasing inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, it was observed that all cherry fractions exhibited dose-dependent cytotoxicity against AGS cells, presenting cytotoxic selectivity for these cancer cells when compared to SH-SY5Y and NHDF cells. Regarding their capacity to protect cancer cells against oxidative injury, in most assays, the total cherry extract was the most effective. Overall, this study reinforces the idea that sweet cherries can be incorporated into new pharmaceutical products, smart foods and nutraceuticals.     

Adsorptive detagging of poly-histidine tagged protein using hexa-histidine tagged exopeptidase

The ubiquitous use of poly-histidine fusion tags has made the purification of the recombinant target proteins much simpler, although the presence of residual fusion tags can generate immunogenic products or products with changed biological activities. This work presents a generic method of removing poly-histidine fusion tags from recombinant proteins through the use of a hexa-histidine tagged exopeptidase (DAPase) when both tagged species are adsorbed to the immobilized metal affinity chromatography (IMAC) adsorbent. Adsorptive detagging was performed in the presence of 50mM imidazole in order to allow the cleavage reaction by the hexa-histidine tagged DAPase to occur. The progress of batch and adsorptive detagging by DAPase of maltose binding protein (MBP) tagged with two variants of hexa-histidine fusion tag was successfully monitored using cationic exchange chromatography. A single-step, column-based detagging strategy was then optimized to maximize the recovery of native MBP. The kinetics of batch and on-column digestion for both HT6 and HT15 fusion tags were investigated. The process involved the sequential removal of dipeptides during the digestion of full-length fusion protein down to its fully detagged native form. During the course of tag digestion, 4 and 7 different intermediates were detected for HT6 and HT15 tagged MBP respectively. The characteristics of on-column cleavage of poly-histidine fusion tags by DAPase as a function of incubation temperature and amount of protease activity used were examined. It was found that the influence of fusion tag design on the batch and column-based detagging yield and efficiency was substantial. In addition, the structural difference of fusion tags affects the binding strength of the fusion protein, which can influence the resulting product purity. Despite being a longer tag, HT15 fusion tag was the preferred sequence for shortening the time needed for on-column detagging. These results can be applied to the wider use of the proposed platform protocol for the on-column cleavage of poly-histidine tagged proteins using exopeptidases.     

Acylated mono-, bis- and tris- cinchona-based amines containing ferrocene or organic residues: synthesis, structure and in vitro antitumor activity on selected human cancer cell lines

A series of novel functionalized mono-, bis- and tris-(S)-{[(2S,4R,8R)-8-ethyl-quinuclidin-2-yl](6-methoxyquinolin-4-yl)}methanamines including ferrocene-containing derivatives was obtained by the reaction of the precursor amine with a variety of acylation agents. Their in vitro antitumor activity was investigated against human leukemia (HL-60), human neuroblastoma (SH-SY5Y), human hepatoma (HepG2) and human breast cancer (MCF-7) cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay and the 50% inhibitory concentration (IC(50)) values were determined. Our data indicate that the precursor amine has no antitumor activity in vitro, but the bis-methanamines with ureido-, thioureido and amide-type linkers display attractive in vitro cytotoxicity and cytostatic effects on HL-60, HepG2, MCF-7 and SH-SY5Y cells. Besides 1H- and 13C-NMR methods the structures of the new model compounds were also studied by DFT calculations.