Human Monocytic Leukemia THP-1 Cells Research Articles

Pharmacophore modeling for the identification of small-molecule inhibitors of TACE

Tumor necrosis factor α-converting enzyme (TACE) plays a critical role in diverse physiological processes such as inflammation, hematopoiesis, and development. In this study, a pharmacophore model constructed from a training set of TACE inhibitors was used to screen an in-house database of organic compounds, from which compound 1 emerged as a top candidate. In a cell-free assay, compound 1 inhibited TACE enzymatic activity in a dose-dependent manner. Moreover, compound 1 inhibited the production of soluble TNF-α in human acute monocytic leukemia THP-1 cells without impacting nitric oxide production, and exhibited anti-proliferative activity against THP-1 cells. We envisage that compound 1 may be employed as a useful scaffold for the development of more potent TACE inhibitors. This study also validates the use of pharmacophore modeling to identify enzyme inhibitors.

Preparation and biological properties of ring-substituted naphthalene-1-carboxanilides.

In this study, a series of twenty-two ring-substituted naphthalene-1-carboxanilides were prepared and characterized. Primary in vitro screening of the synthesized carboxanilides was performed against Mycobacterium avium subsp. paratuberculosis. N-(2-Methoxyphenyl)naphthalene-1-carboxamide, N-(3-methoxy-phenyl)naphthalene-1-carboxamide, N-(3-methylphenyl)naphthalene-1-carboxamide, N-(4-methylphenyl)naphthalene-1-carboxamide and N-(3-fluorophenyl)naphthalene-1-carboxamide showed against M. avium subsp. paratuberculosis two-fold higher activity than rifampicin and three-fold higher activity than ciprofloxacin. The most effective antimycobacterial compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. The testing of biological activity of the compounds was completed with the study of photosynthetic electron transport (PET) inhibition in isolated spinach (Spinacia oleracea L.) chloroplasts. The PET-inhibiting activity expressed by IC50 value of the most active compound N-[4-(trifluoromethyl)phenyl]naphthalene-1-carboxamide was 59 μmol/L. The structure-activity relationships are discussed.

Involvement of L-type Ca²⁺ channel and toll-like receptor-4 in nickel-induced interleukin-8 gene expression.

The metal nickel (Ni(2+)) is found everywhere in our daily lives, including coins, costume jewelry, and even nuts and chocolates. Nickel poisoning can cause inflammatory reactions, respiratory diseases, and allergic contact dermatitis. To clarify the mechanism by which nickel induces mediators of inflammation, we used the human acute monocytic leukemia THP-1 cell line as a model. Interleukin (IL)-8 promoter activity as well as gene expression were tested by luciferase assay and real-time polymerase chain reaction. The underlying mechanisms of nickel-induced IL-8 were investigated. We found that nickel induced IL-8 gene expression via the L-type Ca(2+) channel, Toll-like receptor-4 (TRL-4) and nuclear factor NF-κB signal transduction pathways. Nickel activated NF-κB expression through extracellular signal-regulated kinase 1/2 phosphorylation and then increased IL-8 expression. Thus, the L-type Ca(2+) channel and TRL-4 play important roles in nickel-induced inflammatory gene expressions.

Reaction between lawsone and aminophenol derivatives: Synthesis, characterization, molecular structures and antiproliferative activity

Reaction between two bioreductive reactants lawsone (2-hydroxy-1,4-napthoquinone) and derivatives 2-aminophenol without catalyst is reported. The reaction between lawsone and 4-chloro-2-aminophenol leads to formation of red colored major product 1A:[2-[(5-chloro-hydroxyphenyl)amino]naphthalene-1,4-dione] and fluorescent orange colored minor compound 1B:[10-chloro-benzo[α]phenoxazine-5-one]. Molecular structure of 1A and 1B were determined by single crystal X-ray diffraction. Two mechanisms were proposed to the formation of red 1A and 1B. ‘Ortho–para’ tautomeric equilibrium was observed in DMSO-d6 solution in 1A, which was revealed by 1H, 13C NMR and LC-MS studies. Molecules of 1A formed dimers via NH⋯O interaction and polymeric chain of dimers was formed by OH⋯O interactions. Cl⋯Cl interactions were observed between the polymeric chains of dimers in 1A. Molecules of 1B show Cl⋯N interaction. Antiproliferative properties is studied for 1A–5A compounds (obtained by the reaction of lawsone with 2-amino-4-methylphenol;2A, 2-aminophenol;3A, 3-aminophenol;4A and 4-aminophenol;5A) and evaluated against two cancer cell lines, THP1 (human monocytic leukemia cells) and COLO205 (colorectal adenocarcinoma) and one normal cell line, HEK293T (human embryonic kidney). The values of 50% inhibitory concentration (IC50) of compounds 1A–5A was determined using XTT assay. The cytotoxic effects of compounds 2A and 3A were observed against COLO205 and compounds 4A and 5A on THP1 were observed to be higher in comparison to their effect on HEK293T cell lines.

762: Curcumin induces the apoptosis of human monocytic leukemia THP-1 cells via the activation of JNK/ERK Pathways

Background Curcumin is a principal compound of turmeric, commonly used to treat tumors and other diseases. However, its anti-cancer activity in human acute monocytic leukemia THP-1 cells is not clear. This study aimed to study the anti-cancer effect and action of curcumin on THP-1 cells.

Patterns of genome-wide VDR locations.

The genome-wide analysis of the binding sites of the transcription factor vitamin D receptor (VDR) is essential for a global appreciation the physiological impact of the nuclear hormone 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Genome-wide analysis of lipopolysaccharide (LPS)-polarized THP-1 human monocytic leukemia cells via chromatin immunoprecipitation sequencing (ChIP-seq) resulted in 1,318 high-confidence VDR binding sites, of which 789 and 364 occurred uniquely with and without 1,25(OH)2D3 stimulation, while only 165 were common. We re-analyzed five public VDR ChIP-seq datasets with identical peak calling settings (MACS, version 2) and found, using a novel consensus summit identification strategy, in total 23,409 non-overlapping VDR binding sites, 75% of which are unique within the six analyzed cellular models. LPS-differentiated THP-1 cells have 22% more genomic VDR locations than undifferentiated cells and both cell types display more overlap in their VDR locations than the other investigated cell types. In general, the intersection of VDR binding profiles of ligand-stimulated cells is higher than those of unstimulated cells. De novo binding site searches and HOMER screening for binding motifs formed by direct repeats spaced by three nucleotides (DR3) suggest for all six VDR ChIP-seq datasets that these sequences are found preferentially at highly ligand responsive VDR loci. Importantly, all VDR ChIP-seq datasets display the same relationship between the VDR occupancy and the percentage of DR3-type sequences below the peak summits. The comparative analysis of six VDR ChIP-seq datasets demonstrated that the mechanistic basis for the action of the VDR is independent of the cell type. Only the minority of genome-wide VDR binding sites contains a DR3-type sequence. Moreover, the total number of identified VDR binding sites in each ligand-stimulated cell line inversely correlates with the percentage of peak summits with DR3 sites.

Characterization of Genomic Vitamin D Receptor Binding Sites through Chromatin Looping and Opening

The vitamin D receptor (VDR) is a transcription factor that mediates the genomic effects of 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). Genome-wide there are several thousand binding sites and hundreds of primary 1,25(OH)2D3 target genes, but their functional relation is largely elusive. In this study, we used ChIA-PET data of the transcription factor CTCF in combination with VDR ChIP-seq data, in order to map chromatin domains containing VDR binding sites. In total, we found 1,599 such VDR containing chromatin domains and studied in THP-1 human monocytic leukemia cells four representatives of them. Our combined ChIP-seq and FAIRE-seq time course data showed that each of these four domains contained a master VDR binding site, where an increase of VDR binding pairs with 1,25(OH)2D3-promoted chromatin opening and the presence of a highly significant DR3-type sequence below the peak summit. These sites differed in their relative VDR binding but not in their kinetics, while other loci either had a weaker and delayed VDR association or could not be confirmed at all. All studied chromatin domains contained at least one primary 1,25(OH)2D3 target gene demonstrating a characteristic slope of mRNA increase, while neighboring genes responded delayed, if at all. In conclusion, the observation of ligand-inducible VDR binding and chromatin opening combined with a DR3-type sequence highlighted genome-wide 160 VDR loci that have within their chromatin domain a more than 4-fold increased likelihood to identify a primary 1,25(OH)2D3 target gene than in the vicinity of other genomic VDR binding sites.

Interferon‐γ induces leucine‐rich repeat kinase LRRK2 via extracellular signal‐regulated kinase ERK5 in macrophages

The gene encoding leucine-rich repeat kinase 2 (LRRK2) comprises a major risk factor for Parkinson's disease. Recently, it has emerged that LRRK2 plays important roles in the immune system. LRRK2 is induced by interferon-γ (IFN-γ) in monocytes, but the signaling pathway is not known. Here, we show that IFN-γ-mediated induction of LRRK2 was suppressed by pharmacological inhibition and RNA interference of the extracellular signal-regulated kinase 5 (ERK5). This was confirmed by LRRK2 immunostaining, which also revealed that the morphological responses to IFN-γ were suppressed by ERK5 inhibitor treatment. Both human acute monocytic leukemia THP-1 cells and human peripheral blood monocytes stimulated the ERK5-LRRK2 pathway after differentiation into macrophages. Thus, LRRK2 is induced via a novel, ERK5-dependent IFN-γ signal transduction pathway, pointing to new functions of ERK5 and LRRK2 in human macrophages. Leucine-rich repeat kinase 2 (LRRK2) is a major risk factor for the development of Parkinson's disease (PD). However, the role of LRRK2 in the affected neurons remains enigmatic. Recently, LRRK2 has been reported to be strongly expressed in the immune system. Here, we demonstrate that LRRK2 is induced by Interferon gamma via extracellular signal-regulated kinase 5 (ERK5) in macrophages, thus providing new insights in LRRK2 and ERK5 biology.

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies

BackgroundCysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC4 (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC). MethodsWe examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC4 and LTD4 by competing their binding to their receptors. ResultsmAbLTC and scFvLTC inhibited their binding of LTC4 or LTD4 to CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R) overexpressed in Chinese hamster ovary cells. The induction by LTD4 of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT1R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC4- and LTD4-induced aggregation of mouse platelets expressing CysLT2R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT2R antagonists but not to CysLT1R antagonists. ConclusionsThese results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT1R and CysLT2R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors. General significancemAbLTC can be used in the treatment of inflammatory diseases such as asthma.

Beneficial Effects Of Ionizing Radiation To Enhance Anti-Cancer Effects Of RIG-Like Receptor Stimulus

Introduction The immune system is composed of innate and adaptive immunity. Antigen presenting cells (APCs), such as macrophages and dendritic cells, serve as a link between innate and adaptive immunity. Furthermore, APCs express pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns. Retinoic acid-inducible gene-I (RIG-I)-like receptors [RLRs; RIG-I and melanoma differentiation-associated gene 5 (MDA5)] are a type of PRRs and sense virus-derived RNA or a synthetic analog of dsRNA polyinosinic-polycytidylic acid [poly(I:C)]. Although macrophages are resistant to ionizing radiation, it remains unclear whether radiation affects RLR expression in the macrophages. Therefore, the effects of ionizing radiation on the RLR expression in macrophages and the response against poly(I:C) were herein investigated. Additionally, the anti-cancer effects of poly(I:C) and the combination treatment of poly(I:C) with ionizing radiation was examined in human lung cancer A549 cells. Methods For preparation of human macrophage-like cells, the human acute monocytic leukemia THP1 cells were treated with phorbol 12-myristate 13-acetate and then differentiated into macrophage-like cells. To stimulate RLRs, poly(I:C)/LyoVecTM (InvivoGen), which is a complex between poly(I:C) and the transfection reagent LyoVecTM, was used on macrophage-like differentiated THP-1 cells. X-irradiation was performed with an X-ray generator at a dose rate of 102.0–104.0 cGy/min. The viable cells were counted by trypan blue exclusion assay. The expression of RLRs was analyzed by reverse transcription polymerase chain reaction (RT-PCR) or western blotting. The interferon (IFN)-β expression and tumor necrosis factor (TNF)-α concentration present in culture supernatants were analyzed by RT-PCR and enzyme-linked immunosorbent assay (ELISA), respectively. The cell death analysis was performed by fluorescein isothiocyanate labeled annexin V and propidium iodide (PI) staining and analyzed by flow cytometry. Results The effects of ionizing radiation on RLR expression were first investigated. Both non-irradiated and X-irradiated (1–10 Gy) macrophage-like cells expressed RIG-I and MDA-5, with no significant difference in expression levels. Next the response of macrophage-like cells to poly(I:C)/LyoVecTM (500 ng/ml) was examined. Although the expression of IFN-β was not observed in non-stimulated macrophage-like cells, the poly(I:C)/LyoVecTM-stimulated macrophage-like cells expressed IFN-β. In X-irradiated macrophage-like cells, IFN-β expression after poly(I:C)/LyoVecTM stimulation was comparable with that of non-irradiated cells. Similar to the IFN-β expression, no significant difference in concentration of TNF-α were observed after poly(I:C)/LyoVecTMstimulation in non-irradiated and irradiated cells. These results suggest that ionizing radiation did not affect RLR expression or the response against poly(I:C) in macrophages. We next investigated the anti-cancer effects of poly(I:C)/LyoVecTM against human lung cancer A549 cells. Treatment with poly(I:C)/LyoVecTM (500 and 1000 ng/ml) suppressed the A549 cell growth (approximately 70% and 80% inhibition, respectively). Furthermore, the treatment with poly(I:C)/LyoVecTM induced both annexin V(+)/PI(−) (early apoptotic cells) and annexin V(+)/PI(+) cells (late apoptotic/necrotic cells). Finally, the combination treatment of poly(I:C) with 2 Gy was tested. The cell growth suppressive effects of 2 Gy resulted in 30% inhibition, whereas the combination of 2 Gy with poly(I:C)/LyoVecTM (500 and 1000 ng/ml) resulted in 85% and 90% inhibition, respectively. Correspondingly, the proportion of early apoptotic cells and late apoptotic/necrotic cells were higher in the combination of 2 Gy with poly(I:C)/LyoVecTM compared with 2 Gy alone or poly(I:C)/LyoVecTMalone. These results suggest that poly(I:C) and ionizing radiation synergistically exhibit anti-cancer effects against A549 cells. Conclusion This study demonstrated that ionizing radiation synergistically acted with RLR stimulation in suppressing the growth of human lung cancer cells without affecting the expression of RLRs in macrophages. Therefore, the combination of radiation therapy with RLR stimulus is expected to be an effective cancer therapy. Disclosures: No relevant conflicts of interest to declare.

Dynamics of 1α,25-dihydroxyvitamin D3-dependent chromatin accessibility of early vitamin D receptor target genes

The signaling cascade of the transcription factor vitamin D receptor (VDR) is triggered by its specific ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study we demonstrate that in THP-1 human monocytic leukemia cells 87.4% of the 1034 most prominent genome-wide VDR binding sites co-localize with loci of open chromatin. At 165 of them 1α,25(OH)2D3 strongly increases chromatin accessibility and has at further 217 sites weaker effects. Interestingly, VDR binding sites in 1α,25(OH)2D3-responsive chromatin regions are far more often composed of direct repeats with 3 intervening nucleotides (DR3s) than those in ligand insensitive regions. DR3-containing VDR sites are enriched in the neighborhood of genes that are involved in controling cellular growth, while non-DR3 VDR binding is often found close to genes related to immunity. At the example of six early VDR target genes we show that the slope of their 1α,25(OH)2D3-induced transcription correlates with the basal chromatin accessibility of their major VDR binding regions. However, the chromatin loci controlling these genes are indistinguishable in their VDR association kinetics. Taken together, ligand responsive chromatin loci represent dynamically regulated contact points of VDR with the genome, from where it controls early 1α,25(OH)2D3 target genes.

Effects of clodronate and alendronate on osteoclast and osteoblast co-cultures on silk–hydroxyapatite films

The goal of this study was to explore the effects of osteoporosis-related therapeutics on bone remodeling in vitro. A previously established bone-tissue mimetic system consisting of silk protein biomaterials in combination with hydroxyapatite and human cells was used for the study. Silk–hydroxyapatite films were pre-complexed with the non-nitrogenous bisphosphonate clodronate or the nitrogenous bisphosphonate alendronate and cultured with THP-1 human acute monocytic leukemia cell line-derived osteoclasts, human mesenchymal stem cell derived osteoblasts or a direct co-culture of the two cell types. Metabolic activity, calcium deposition and alkaline phosphatase activity were assessed over 12weeks, and reconstructed remodeled biomaterial surfaces were also evaluated for quantitative morphological changes. Increased metabolic activity and increased roughness were found on the clodronate-complexed biomaterial substrates remodeled by osteoblasts and co-cultures of osteoblasts with osteoclasts, even at doses high enough to cause a 90% decrease in osteoclast metabolic activity. Films complexed with low doses of alendronate resulted in increased metabolic activity and calcium deposition by osteoblasts, while higher doses were similarly toxic among osteoclasts, osteoblasts and co-cultures. These results point to the utility of these well-defined bone-mimetic in vitro cultures as useful screens for therapeutics for bone-related diseases, particularly with the ability to conduct studies for extended duration (here for 12weeks) and with pre-complexed drugs to mimic conditions found in vivo.

Antimycobacterial and herbicidal activity of ring-substituted 1-hydroxynaphthalene-2-carboxanilides

In this study, a series of 22 ring-substituted 1-hydroxynaphthalene-2-carboxanilides were prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Mycobacterium marinum, Mycobacterium kansasii and Mycobacterium smegmatis. The compounds were also tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Most of tested compounds showed the antimycobacterial activity against the three strains comparable or higher than the standard isoniazid. N-(3-Fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC=28.4μmol/L) against M. marinum, N-(4-fluorophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC=14.2μmol/L) against M. kansasii, and N-(4-bromophenyl)-1-hydroxynaphthalene-2-carboxamide expressed the highest biological activity (MIC=46.7μmol/L) against M. smegmatis. This compound and 1-hydroxy-N-(3-methylphenyl)naphthalene-2-carboxamide were the most active compounds against all three tested strains. The PET inhibition expressed by IC50 value of the most active compound 1-hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide was 5.3μmol/L. The most effective compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. For all compounds, structure–activity relationships are discussed.

Antimycobacterial and Photosynthetic Electron Transport Inhibiting Activity of Ring-Substituted 4-Arylamino-7-Chloroquinolinium Chlorides

In this study, a series of twenty-five ring-substituted 4-arylamino-7-chloroquinolinium chlorides were prepared and characterized. The compounds were tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts and also primary in vitro screening of the synthesized compounds was performed against mycobacterial species. 4-[(2-Bromophenyl)amino]-7-chloroquinolinium chloride showed high biological activity against M. marinum, M. kansasii, M. smegmatis and 7-chloro-4-[(2-methylphenyl)amino]quinolinium chloride demonstrated noteworthy biological activity against M. smegmatis and M. avium subsp. paratuberculosis. The most effective compounds demonstrated quite low toxicity (LD50 > 20 μmol/L) against the human monocytic leukemia THP-1 cell line within preliminary in vitro cytotoxicity screening. The tested compounds were found to inhibit PET in photosystem II. The PET-inhibiting activity expressed by IC50 value of the most active compound 7-chloro-4-[(3-trifluoromethylphenyl)amino]quinolinium chloride was 27 μmol/L and PET-inhibiting activity of ortho-substituted compounds was significantly lower than this of meta- and para-substituted ones. The structure-activity relationships are discussed for all compounds.

Antibacterial and Herbicidal Activity of Ring-Substituted 3-Hydroxynaphthalene-2-carboxanilides

In this study, a series of twenty-two ring-substituted 3-hydroxy-N-phenylnaphthalene-2-carboxanilides were prepared and characterized. The compounds were tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Primary in vitro screening of the synthesized compounds was also performed against four Staphylococcus strains and against two mycobacterial species. 3-Hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide showed high biological activity (MIC = 55.0 µmol/L) against S. aureus as well as methicillin-resistant strains. N-(2-Fluorophenyl)-3-hydroxynaphthalene-2-carboxamide showed higher activity (MIC = 28.4 µmol/L) against M. marinum than the standard isoniazid and 3-hydroxy-N-(4-nitrophenyl)naphthalene-2-carboxamide expressed higher activity (MIC = 13.0 µmol/L) against M. kansasii than the standard isoniazid. Cytotoxicity assay of effective antimicrobial compounds was performed using the human monocytic leukemia THP-1 cell line. The PET-inhibiting activity expressed by IC50 value of the most active compound 3-hydroxy-N-(3-nitrophenyl)naphthalene-2-carboxamide was 16.9 μmol/L. The structure-activity relationships of all compounds are discussed.

Differential effects of selenium (Se) on normal and malignant cells treated with chemotherapy (CT) and radiation (RT).

e13583 Background: Preclinical work has demonstrated that Se compounds potentiate anticancer effects of CT and RT while reducing normal tissue toxicities. The molecular basis for the therapeutic selectivity has yet to be fully elucidated but includes modulation of intracellular glutathione (GSH) concentrations, endoplasmic reticulum (ER) stress responses, DNA repair, induction of apoptosis and cellular resistance to CT and RT. Our aim was to evaluate the dose-response relationship of the Se compound methylseleninic acid (MSA) on molecular pathways involved in the response of normal and malignant cells to CT and RT. Methods: Peripheral blood mononuclear cells (PBMC) obtained from healthy blood donors and malignant THP-1 human monocytic leukaemia cells were exposed in vitro to MSA 2.5, 5 or 15 µM in varying combinations with MSA, RT, cisplatin (Pt), doxorubicin (Dox) and cytosine arabinoside (Ara-C). GSH concentration was measured by ELISA, DNA damage and repair by COMET assay, cell viability by MTT assay and ER stress response protein expression by western blotting. Results: MSA was selectively toxic to THP-1 cells and induced a protective increase in GSH in PBMC but a decrease in high concentrations within THP-1 cells. DNA damage induced by Ara-C or Dox in the COMET assay was significantly reduced by MSA in PBMC but increased in THP-1 cells. Cell death after 2 Gy RT was increased by all doses of MSA in THP-1 cells but only by the highest dose in PBMC. The cytotoxicity of Dox and Ara-C at sublethal doses was significantly enhanced by MSA in THP-1 cells and to a lesser extent in PBMC, but MSA increased cell death from Pt only in THP-1 cells. MSA induced a protective ER stress response in PBMC exposed to Ara-C but an apoptotic response in THP-1 cells. Conclusions: MSA at clinically-relevant concentrations had a differential effect on cell survival and death responses to RT and CT with relative protection of PBMC and enhanced death of THP-1 cells. Several mechanisms mediated this therapeutic selectivity and the dose-dependence of the Se effect varied between malignant and normal cells. These assays could potentially be used in clinical trials to evaluate pharmacodynamic markers of Se effects in conjunction with CT and/or RT.

Synthesis and Biological Evaluation of 2-Hydroxy-3-[(2-aryloxyethyl)amino]propyl 4-[(Alkoxycarbonyl)amino]benzoates

A series of twenty substituted 2-hydroxy-3-[(2-aryloxyethyl)amino]propyl 4-[(alkoxycarbonyl)amino]benzoates were prepared and characterized. As similar compounds have been described as potential antimycobacterials, primary in vitro screening of the synthesized carbamates was also performed against two mycobacterial species. 2-Hydroxy-3-[2-(2,6-dimethoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride, 2-hydroxy-3-[2-(4-methoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride, and 2-hydroxy-3-[2-(2-methoxyphenoxy)ethylamino]-propyl 4-(butoxycarbonylamino)benzoate hydrochloride showed higher activity against M. avium subsp. paratuberculosis and M. intracellulare than the standards ciprofloxacin, isoniazid, or pyrazinamide. Cytotoxicity assay of effective compounds was performed using the human monocytic leukaemia THP-1 cell line. Compounds with predicted amphiphilic properties were also tested for their effects on the rate of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. All butyl derivatives significantly stimulated the rate of PET, indicating that the compounds can induce conformational changes in thylakoid membranes resulting in an increase of their permeability and so causing uncoupling of phosphorylation from electron transport.

Temporal dynamics and transcriptional control using single-cell gene expression analysis

BackgroundChanges in environmental conditions lead to expression variation that manifest at the level of gene regulatory networks. Despite a strong understanding of the role noise plays in synthetic biological systems, it remains unclear how propagation of expression heterogeneity in an endogenous regulatory network is distributed and utilized by cells transitioning through a key developmental event.ResultsHere we investigate the temporal dynamics of a single-cell transcriptional network of 45 transcription factors in THP-1 human myeloid monocytic leukemia cells undergoing differentiation to macrophages. We systematically measure temporal regulation of expression and variation by profiling 120 single cells at eight distinct time points, and infer highly controlled regulatory modules through which signaling operates with stochastic effects. This reveals dynamic and specific rewiring as a cellular strategy for differentiation. The integration of both positive and negative co-expression networks further identifies the proto-oncogene MYB as a network hinge to modulate both the pro- and anti-differentiation pathways.ConclusionsCompared to averaged cell populations, temporal single-cell expression profiling provides a much more powerful technique to probe for mechanistic insights underlying cellular differentiation. We believe that our approach will form the basis of novel strategies to study the regulation of transcription at a single-cell level.

Human acidic mammalian chitinase (AMCase); molecular cloning, protein expression, and production of monoclonal antibody

Event Abstract Back to Event Human acidic mammalian chitinase (AMCase); molecular cloning, protein expression, and production of monoclonal antibody Kanokwan Lowhalidanon1, Piyanat Meekrathok1, Sunisa Thongsom1, Wipa Suginta1 and Panida Khunkaewka1* 1 Suranaree University of Technology, Biochemistry-Electrochemistry Research Unit, Thailand Human acidic mammalian chitinase (AMCase), is a 50 kDa protein containing a 30 kDa N-terminal catalytic domain that can hydrolyze chitin. This enzyme belongs to family 18 glycosyl hydrolases. High abundant of this enzyme was found in gastrointestinal tract. In lung, interleukin-13 was proposed to play a key role as primary effector that induces secretion of AMCase by airway epithelial cells and macrophages. The secreted AMCase activates elevation of monocyte chemoattractant protein 1 (MCP-1) and eotaxin-1, which induce the recruitment of eosinophils, T cells, and macrophages to site of inflammation in lung epithelial cells. Nonetheless, the mechanisms underlying AMCase mediated cells recruitment and its surface receptor have not been identified. Present study, the gene encoding AMCase was isolated from the human cDNA, cloned into pQE-tri vector, and the recombinant protein was expressed in E. coli M15. The affinity-purified AMCase was further used as immunogen for monoclonal antibody (mAb) production by standard hybridroma technique. Two specific mAbs against AMCase were successfully raised and named as 4G1 and 6E5. Both monoclones belong to IgG1 isotype. Western blot analysis showed that the mAbs strongly reacted with a corresponding protein with molecular weight of about 50 kDa in human acute monocytic leukemia cell line, THP1, and peripheral blood mononuclear cells. In conclusion, the AMCase was cloned and expressed in E. Coli system. Two specific mAbs to AMCase were raised and specifically reacted to human AMCase. The generated mAbs will be used for further functional analysis of the AMCase in regulation of the immune system. Acknowledgements This work was supported by the National Research Council of Thailand and Suranaree University of Technology, Thailand. References Louise E. Donnelly and Peter J. Barnes. (2004). Acidic mammalian chitinase – a potential target for asthma therapy. TRENDS in Pharmacological Science Vol.25. Keywords: AMCase, Molecular cloning, protein expression, Monoclonal antibody, Asthma Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Lowhalidanon K, Meekrathok P, Thongsom S, Suginta W and Khunkaewka P (2013). Human acidic mammalian chitinase (AMCase); molecular cloning, protein expression, and production of monoclonal antibody. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00189 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 11 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Panida Khunkaewka, Suranaree University of Technology, Biochemistry-Electrochemistry Research Unit, Nakhonratchasima, 30000, Thailand, kpanida@sut.ac.th Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kanokwan Lowhalidanon Piyanat Meekrathok Sunisa Thongsom Wipa Suginta Panida Khunkaewka Google Kanokwan Lowhalidanon Piyanat Meekrathok Sunisa Thongsom Wipa Suginta Panida Khunkaewka Google Scholar Kanokwan Lowhalidanon Piyanat Meekrathok Sunisa Thongsom Wipa Suginta Panida Khunkaewka PubMed Kanokwan Lowhalidanon Piyanat Meekrathok Sunisa Thongsom Wipa Suginta Panida Khunkaewka Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Development of an in vitro skin sensitization test based on ROS production in THP-1 cells

Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM–H2DCFDA), for 15min, then incubated with test chemicals for 30min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals.