Search results for: kinase assays

Authors: Ravid Inbar

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CaMKII (calcium-calmodulin dependent protein kinase II) makes up 2% of the protein in our brain and has a critical role in memory formation and long-term potentiation of neurons. Despite this, research has yet to uncover the role of one of the domains on the activation of this kinase. The following proposes to express the protein without the hub domain in E. coli, leaving only the kinase and regulatory segment of the protein. Next, a series of kinase assays will be conducted to elucidate the role the hub domain plays on CaMKII sensitivity to calcium/calmodulin activation. The hub domain may be important for activation; however, it may also be a variety of domains working together to influence protein activation and not the hub alone. Characterization of a protein is critical to the future understanding of the protein's function, as well as for producing pharmacological targets in cases of patients with diseases.

Keywords: CaMKII, hub domain, kinase assays, kinase + reg seg

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Authors: Nichole Haag, Kimberly Velk, Tyler McCune, Chun Wu

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Methicillin/multiple-resistant Staphylococcus aureus (MRSA) are infectious bacteria that are resistant to common antibiotics. A previous in silico study in our group has identified a hypothetical protein SAV1226 as one of the potential drug targets. In this study, we reported the bioinformatics characterization, as well as cloning, expression, purification and kinetic assays of hypothetical protein SAV1226 from methicillin/vancomycin-resistant Staphylococcus aureus Mu50 strain. MALDI-TOF/MS analysis revealed a low degree of structural similarity with known proteins. Kinetic assays demonstrated that hypothetical protein SAV1226 is neither a domain of an ATP dependent dihydroxyacetone kinase nor of a phosphotransferase system (PTS) dihydroxyacetone kinase, suggesting that the function of hypothetical protein SAV1226 might be misannotated on public databases such as UniProt and InterProScan 5.

Keywords: Methicillin-resistant Staphylococcus aureus, dihydroxyacetone kinase, essential genes, drug target, phosphoryl group donor

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Authors: Roshika Tyagi, Shuvomoy Banerjee

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Among various diseases, cancer has become a huge threat to human beings globally. In the context of viral infection, Epstein–Barr virus (EBV) infection is ubiquitous in nature world-wide as well as in India. Recepteur d’Origine Nantais (RON) receptor tyrosine kinase is overexpressed in Lymphoblastoid cell lines (LCLs) but undetectable in primary B-cells. Biologically, RON expression was found to be essential for EBV transformed LCLs proliferation. In our study, we investigated whether EBV latent antigen EBNA3C is playing a crucial role in regulating RON receptor tyrosine kinase function in EBV-induced malignancies. Interestingly, we observed that expression pattern of RON was modulated by EBNA3C in EBV transformed LCLs compared with EBV negative BJAB cell line by PCR and western blot analysis. Moreover, in the absence of EBNA3C, RON expression was found low in western blot and immunofluorescence analysis and cell proliferation rate was significantly reduced in LCLs by cell viability assays. Therefore, our study clearly indicating the potential role of EBNA3C expressed in EBV-infected B-cells for modulating the functions of oncogenic kinases that leads to EBV induced B-cell transformation.

Keywords: apoptosis, cell proliferation, Epstein–barr virus, receptor tyrosine kinase

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Authors: Serap Pektas

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Ataxia telangiectasia mutated (ATM) is a serine-threonine kinase, which is the major regulator of the DNA damage response. ATM is activated upon the formation of DNA double-strand breaks (DSBs) in the cells. ATM phosphorylates the proteins involved in apoptotic responses, cell cycle checkpoint control, DNA repair, etc. Tumor protein p53, known as p53 is one of these proteins that phosphorylated by ATM. Phosphorylation of p53 at Ser15 residue leads to p53 stabilization in the cells. Often enzymes activity is affected by hydrogen ion concentration (pH). In order to find the optimal pH range for ATM activity, steady-state kinetic assays were performed at acidic and basic pH ranges. Ser15 phosphorylation of p53 is determined by using ELISA. The results indicated that the phosphorylation rate was better at basic pH range compared with the acidic pH range. This could be due to enzyme stability, or enzyme-substrate interaction is pH dependent.

Keywords: ataxia telangiectasia mutated, DNA double strand breaks, DNA repair, tumor protein p53

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Authors: İsmail Çeli̇k, Gülgün Ayhan-Kılcıgi̇l, Arzu Onay-Beşi̇kçi̇

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In this study, some 2-(2-phenyl/substitutedphenyl)- lH-benzo[d]'imidazol-l-yl)-N'-(alkylthiosemicarbazide were designed and prepared. Firstly, 2-phenyl/ suhstitutedphenyl-lH-Benzo[d]imidazole was prepared via oxidative condensation of o-phenylenediamine, benzaldehyde and sodium metabisulfite. Treatment of the benzimidazole compound with ethyl chloroacetate in KOH/DMSO gave the ester compound ethyl 2-(2-substitutedphenyl)-1H-benzo[d]imidazol-l-yl)acetate. Hydrazine hydrate and the ester in ethanol were refluxed for 4 h to give 2-(2-phenyl/substitutedphenyl)-1H-benzo[d]imidazol-l-yl)acetohydrazide. Thiosemicarbazides were obtained by condensing acyl hydrazide with the alkylisothiocyanate in ethanol. Following the structure elucidation, benzimidazole compounds were tested for their EGFR kinase inhibitory activities by using ADP-GloTM Kinase Assay.

Keywords: benzimidazole, EGFR kinase inhibitor, synthesis, thiosemicarbazide

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Authors: Nihar Ranjan, Derrick Watkins, Dev P. Arya

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Current methods in the study of antibiotic activity of ribosome targeted antibiotics are dependent on cell based bacterial inhibition assays or various forms of ribosomal binding assays. These assays are typically independent of each other and little direct correlation between the ribosomal binding and bacterial inhibition is established with the complementary assay. We have developed novel high-throughput capable assays for ribosome targeted drug discovery. One such assay examines the compounds ability to bind to a model ribosomal RNA A-site. We have also coupled this assay to other functional orthogonal assays. Such analysis can provide valuable understanding of the relationships between two complementary drug screening methods and could be used as standard analysis to correlate the affinity of a compound for its target and the effect the compound has on a cell.

Keywords: bacterial resistance, aminoglycosides, screening, drugs

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Authors: Ibrahim M. Labouta, Marwa H. El-Wakil, Hayam M. Ashour, Ahmed M. Hassan, Manal N. Saudi

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The receptor tyrosine kinase c-Met is an attractive target for therapeutic treatment of cancers nowadays. Among the wide variety of heterocycles that have been explored for developing c-Met kinase inhibitors, the 1,2,4-triazines have been rarely investigated, although they are well known in the literature to possess antitumor activities. Herein we describe the design and synthesis of a novel series of 1,2,4-triazine derivatives possessing N-acylarylhydrazone moiety and another series combining the 1,2,4-triazine scaffold to the well-known anticancer drug 6-MP in order to explore their “double-drug” effect. The synthesized compounds were evaluated for their in vitro antitumor activity against three c-Met addicted cancer cell lines (A549, HT-29 and MKN-45). Most compounds showed moderate to excellent antiproliferative activity and four compounds showed potent inhibitory activity more than the reference drug Foretinib against one or more cancer cell lines. The obtained results revealed that the potent compounds are highly selective to A549 (lung adenocarcinoma) cancer cell line. The c-Met kinase inhibitory activity of the potent derivatives is still under investigation. The present study clearly demonstrates that the 1,2,4-triazine core ring exhibits promising antitumor activity with potential c-Met kinase inhibitory activity.

Keywords: 1, 2, 4-triazine, antitumor, c-Met inhibitor, double-drug

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Authors: Christolyn Raj, Lewis Levitz

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Introduction: Two cases of late presentation ( > five years ) of bilateral pseudophakic macula edema related to oral tyrosine kinase inhibitors are described. These cases are the first of their type in the published literature. A review of ocular inflammatory complications of tyrosine kinase inhibitors in the current literature is explored. Case Presentations(s): Case 1 is an 83-year-old female who has been stable on Ibrutinib (Imbruvica ®) for chronic lymphocytic leukemia (CLL). She presented with bilateral blurred vision from severe cystoid macula edema seven years following routine cataract surgery. She was treated with intravitreal steroids with complete resolution without relapse. Case 2 is a 76-year-old female who was on therapy for polycythemia vera with Ruxolitinib (Jakafi®). She presented with bilateral blurred vision from mild cystoid macula edema six years following routine cataract surgery. She responded well to topical steroids without relapse. In both cases, oral tyrosine kinase inhibitor agents were presumed to be the underlying cause and were ceased. Over the last five years, there have been increasing reports in the literature of the inflammatory effects of tyrosine kinase inhibitors on the retina, uvea and optic nerve. Conclusion: Late presentation of pseudophakic macula edema following routine cataract surgery is rare. Such presentations should prompt investigation of the chronic use of systemic medications, especially oral kinase inhibitors. Patients who must remain on these agents require ongoing ophthalmologic assessment in view of their long-term inflammatory side effects.

Keywords: macula edema, oral kinase inhibitors, retinal toxicity, pseudo-phakia

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Authors: Jae Bok Heo, Yun Mi Lee, Hee Rang Yun

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Several GTPases are required for ribosome biogenesis and assembly. We recently characterized rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), belonging to the YlqF/YawG family of GTPases, as playing a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating the function of OsNug2, yeast two-hybrid screens were carried out using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a potential interacting protein candidate. In vitro pull down and bimolecular fluorescence complementation assays confirmed the interaction between OsNug2 and OsSTK1, and like green fluorescent protein-tagged OsNug2, green fluorescent protein-tagged OsSTK1 was targeted to the nucleus of Arabidopsis protoplasts. OsSTK1 was not found to affect the GTP-binding activity of OsNug2; however, when recombinant OsSTK1 was included in OsNug2 assay reaction mixtures, OsSTK1 increased the GTPase activity of OsNug2. To test whether OsSTK1 phosphorylates OsNug2 in vitro, a kinase assay was performed. OsSTK1 was found to have weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Yeast complementation testing resulted in a GAL::OsNug2(S209N) mutant-harboring yeast strain exhibiting a growth-defective phenotype on galactose medium at 39°C, divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of mutant OsNug2(S209N) was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings reported here indicate that OsSTK1 functions as a positive regulator protein of OsNug2 by enhancing the GTPase activity of OsNug2, and that the phosphorylation of serine 209 of OsNug2 is essential for the complete function of OsNug2 in ribosome biogenesis.

Keywords: OsSTK1, OsNug2, GTPase activity, GTP binding activity, phosphorylation

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Authors: Sukrat Sinha, Abhay Kumar, Shanthy Sundaram

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The Leishmania homologue of activated C kinase (LACK) is known T cell epitope from soluble Leishmania antigens (SLA) that confers protection against Leishmania challenge. This antigen has been found to be highly conserved among Leishmania strains. LACK has been shown to be protective against L. donovani challenge. A comprehensive analysis of several LACK sequences was completed. The analysis shows a high level of conservation, lower variability and higher antigenicity in specific portions of the LACK protein. This information provides insights for the potential consideration of LACK as a putative candidate in the context of visceral Leishmaniasis vaccine target.

Keywords: bioinformatics, genome assembly, leishmania activated protein kinase c (lack), next-generation sequencing

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Authors: Saha Saradindu, Das Payel, Somdeb BoseDasgupta

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Tuberculosis caused by Mycobacteria tuberculosis is a dreadful disease and more so with the advent of extreme and total drug-resistant species. Mycobacterial pathogenesis is an ever-changing paradigm from phagosome maturation block to phagosomal escape into macrophage cytosol and finally acid tolerance and survival inside the lysosome. Mycobacteria are adept at subverting the host immune response by highjacking host cell signaling and secreting virulence factors. One such virulence factor is a ser/thr kinase; Protein kinase G (PknG), which is known to prevent phagosome maturation. The host substrates of PknG, allowing successful pathogenesis still remain an enigma. Hence we carried out a comparative phosphoproteomic screen and identified a number of substrates phosphorylated by PknG. We characterized some of these substrates in vivo and in vitro and observed that PknG mediated phosphorylation of these substrates leads to reduced TNFa production as well as decreased response to TNFa induced macrophage necroptosis, thus enabling mycobacterial survival and proliferation.

Keywords: mycobacteria, Protein kinase G, phosphoproteomics, necroptosis

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Authors: R. Frikha, H. Kamoun

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Background:Chronicmyeloidleukemia (CML), a hematologicaldisease, ischaracterized by t (9; 22) and relatedoncogene BCR-ABL formation. Although Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of CML, resistanceoccurs and possibly médiates by mutation in severalgenesindependently of the bcr-abl1 kinase mechanism. it has been reportedthat JAK2V617F/BCR-ABL double positivitymaybe a potential marker of resistance in CML. Aims: This studywasinvestigated the JAK2V617F mutation in TKI-resistant CML patients. Methods: A retrospectivestudywasconducted in the Hospital University of Sfax, south of Tunisia, including all CML TKI-resistant patients. A Sanger sequencingwasperformedusing a high-fidelity DNA polymerase. Results:Nineresistant CP-CML patients wereenrolled in thisstudy. The JAK2V617F mutation wasdetectedin 3 patients with TKI resistance. Conclusion:Despite the limit of ourstudy, ourfinding highlights the high frequency of JAK2V617F/BCR-ABL double positivity as an important marker of resistance. So; the combination of JAK and TKI inhibitorsmightbe effective and potentiallybeguided by molecular monitoring of minimal residual disease1.

Keywords: chronic myeloid leukemia, tyrosine kinase inhibitors, resistance, JAK2V617F, BCR-ABL

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Authors: Pooja Kumari, Anandkumar Tengli

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Aurora kinase enzyme, a protein on overexpression, leads to metastasis and is extremely important for women’s health in terms of prevention or treatment. While creating a targeted technique, the aim of the work is to design purine molecules that inhibit in aurora kinase enzyme and helps to suppress breast cancer. Purine molecules attached to an amino acid in DNA block protein synthesis or halt the replication and metastasis caused by the aurora kinase enzyme. Various protein related to the overexpression of aurora protein was docked with purine molecule using Biovia Drug Discovery, the perpetual software. Various parameters like X-ray crystallographic structure, presence of ligand, Ramachandran plot, resolution, etc., were taken into consideration for selecting the target protein. A higher negative binding scored molecule has been taken for simulation studies. According to the available research and computational analyses, purine compounds may be powerful enough to demonstrate a greater affinity for the aurora target. Despite being clinically effective now, purines were originally meant to fight breast cancer by inhibiting the aurora kinase enzyme. In in-silico studies, it is observed that purine compounds have a moderate to high potency compared to other molecules, and our research into the literature revealed that purine molecules have a lower risk of side effects. The research involves the design, synthesis, and identification of active purine molecules against breast cancer. Purines are structurally similar to the normal metabolites of adenine and guanine; hence interfere/compete with protein synthesis and suppress the abnormal proliferation of cells/tissues. As a result, purine target metastasis cells and stop the growth of kinase; purine derivatives bind with DNA and aurora protein which may stop the growth of protein or inhibits replication and stop metastasis of overexpressed aurora kinase enzyme.

Keywords: aurora kinases, in silico studies, medicinal chemistry, combination therapies, chronic cancer, clinical translation

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Authors: Rahul Shrivastava, Manish Tripathi, Mohmmad Yasir, Shailesh Singh

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Life style changes and depletion in atmospheric ozone layer in recent decades lead to increase in skin cancer including both melanoma and nonmelanomas. Natural products which were obtained from different plant species have the potential of anti skin cancer activity. In regard of this, present study focuses the potential effect of Glycosmis pentaphylla against anti skin cancer activity. Different Phytochemical constituents which were present in the roots of Glycosmis pentaphylla were identified and were used as ligands after sketching of their structures with the help of ACD/Chemsketch. These ligands are screened for their anticancer potential with proteins which are involved in skin cancer effects with the help of pyrx software. After performing docking studies, results reveal that Noracronycine secondary metabolite of Glycosmis pentaphylla shows strong affinity of their binding energy with Ribosomal S6 Kinase 2 (2QR8) protein. Ribosomal S6 Kinase 2 (2QR8) has an important role in the cell proliferation and transformation mediated through by N-terminal kinase domain and was induced by the tumour promoters such as epidermal growth factor. It also plays a key role in the neoplastic transformation of human skin cells and in skin cancer growth. Noracronycine interact with THR-493 and MET-496 residue of Ribosomal S6 Kinase 2 protein with binding energy ΔG = -8.68 kcal/mole. Thus on the basis of this study we can say that Noracronycine which present in roots of Glycosmis pentaphylla can be used as lead compound against skin cancer.

Keywords: glycosmis pentaphylla, pyrx, ribosomal s6 kinase, skin cancer

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Authors: Ismail Celik, Gulgun Ayhan Kilcigil, Berna Guven, Zumra Kara, Arzu Onay-Besikci

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Epidermal Growth Factor Receptor is the cell-surface receptor of the ErbB (erythroblastic leukemia viral oncogene homologue receptors) family of tyrosine kinases. It plays a vital role in regulating the proliferation and differentiation of cells. However, a variety of mechanisms, such as EGFR expression, mutation, and ligand-dependent receptor dimerization, are associated with the development of various activated EGFR tumors. EGFR is highly expressed in most solid tumors, including breast, head and neck cancer, non-small cell lung cancer (NSCLC), renal, ovarian, and colon cancers. Thus, specific EGFR inhibition plays one of the key roles in cancer treatment. The compounds used in the treatment as tyrosine kinase inhibitors are known to contain the benzimidazole isosterium indole, pazopanib, and axitinibin indazole rings. In addition, benzimidazoles have been shown to exhibit protein kinase inhibitory activity in addition to their different biological activities.Based on these data, it was planned and synthesized of some oxadiazole bearing benzimidazole derivatives [N-cyclohexyl-5-((2-phenyl/substitutedphenyl-1H-benzo[d]imidazole-1-yl) methyl)-1,3,4-oxadiazole-2-amine]. EGFR kinase inhibitory efficiency of the synthesized compounds was determined by comparing them with a known kinase inhibitor erlotinib in vitro, and two of the compounds bearing phenyl (19a) and 3,4-dibenzyloxyphenyl (21a) ring exhibited significant activities.

Keywords: benzimidazole, EGFR kinase inhibitory, oxadiazole, synthesis

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Authors: Nattawit Thiapairat

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Multiple diseases have been linked to excessive levels of free radicals, which cause tissue or cell damage as a result of oxidative stress. Many plants are sources of high antioxidant activity. Derris scandens has a high amount of phenolic and flavonoid contents which demonstrated good biological activities. This study focused on the antioxidant activity of polyphenols extracted from D. scandens. This study performs total flavonoids content and various antioxidant assays, which were 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity assays. The total flavonoid content of D. scandens extract was determined and expressed as quercetin equivalents (QE)/g measured by the aluminum chloride colorimetric method. The antioxidant activity of D. scandens extract was also determined by DPPH and ABTS assays. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. The half-maximal inhibitory concentration (IC50) values for D. scandens extract from DPPH and ABTS assays were 41.79 μg/mL ± 0.783 and 29.42 μg/mL ± 0.890, respectively, in the DPPH assay. To conclude, D. scandens extract consists of a high amount of total phenolic content, which exhibits a significant antioxidant activity. However, further investigation regarding antioxidant activity such as SOD, ROS, and RNS scavenging assays and in vivo experiments should be performed.

Keywords: ABTS assay, antioxidant activity, Derris scandens, DPPH assays, total flavonoid content

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Authors: Ji Hye Im, Nguyen T. T. Phuong, Keon Wook Kang

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Estrogens are important for the development and growth of estrogen receptor (ER)-positive breast cancer, for which anti-estrogen therapy is one of the most effective treatments. However, its efficacy can be limited by either de novo or acquired resistance. Aromatase is a key enzyme for the biosynthesis of estrogens, and inhibition of this enzyme leads to profound hypoestrogenism. Here, we found that the basal expression and activity of aromatase were significantly increased in tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells compared to control MCF-7 cells. We further revealed that aromatase immunoreactivity in tumor tissues was increased in recurrence group after TAM therapy compared to non-recurrence group after TAM therapy. Phosphorylation of Akt, extracellular signal-regulated kinase (ERK), and p38 kinase were all increased in TAMR-MCF-7 cells. Inhibition of phosphoinositide 3-kinase (PI3K) suppressed the transactivation of the aromatase gene and its enzyme activity. Furthermore, we have also shown that PI3K/Akt-dependent cAMP-response element binding protein (CREB) activation was required for the enhanced expression of aromatase in TAMR-MCF-7 cells. Our findings suggest that aromatase expression is up-regulated in TAM-resistant breast cancer via PI3K/Akt-dependent CREB activation.

Keywords: TAMR-MCF-7, CREB, estrogen receptor, aromatase

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Authors: Hae-Jun Lee, Ji-Sun Shin, Kyung-Tae Lee

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Potentilla species (Rosasease) have been used in traditional medicine to treat different ailment, disease or malady. In this study, we investigated the anti-inflammatory effects of ethanol extracts of NUTT (EPP) in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages and septic mice. EPP suppressed LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-induced Raw 264.7 macrophages. Consistent with these observations, EPP reduced the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by downregulation of their promoter activities. EPP inhibited tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) at production and mRNA levels. Molecularly, EPP attenuated the LPS-induced transcriptional activity, and DNA-binding activity of nuclear factor-κB (NF-κB), and this was associated with a decrease of translocation and phosphorylation of p65 NF-κB by inhibiting the inhibitory κB-α (IκB-α) degradation and IκB kinase-α/β (IKK-α/β) phosphorylation. Furthermore, EPP suppressed the LPS-induced activation of activator protein-1 (AP-1) by reducing the expression of c-Fos and c-Jun in nuclear. EPP also reduced the phosphorylation of mitogen-activated protein kinase (MAPK), such as p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). In a sepsis model, pretreatment with EPP reduced the LPS-induced lethality. Collectively, these results suggest that the anti-inflammatory effects of EPP were associated with the suppression of NF-κB and AP-1 activation, and support its possible therapeutic role for the treatment of sepsis.

Keywords: anti-inflammation, activator protein-1, nuclear factor κB, Potentilla paradoxa Nutt

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Authors: Marcela Dvorakova, Lenka Langhansova, Premysl Landa

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A series of quinazoline derivatives bearing aromatic rings in 2- and 4-positions were prepared and tested for their biological activity. Firstly, the compounds were evaluated for their potential to inhibit various kinases, such as autophagy activating kinase ULK1, 3-Phosphoinositide-dependent kinase 1, and TANK-binding kinase 1. None of the compounds displayed any activity on these kinases. Secondly, the compounds were tested for their anti-inflammatory activity expressed as cyclooxygenase (COX) isoforms and 5-lipoxygenase (5-LOX) inhibition. Three of the compounds showed significant selectivity towards COX-1 isoform (COX-2/COX-1 SI = 20-30). They inhibited COX-1 in a single-digit µM range. There was also one compound that exhibited inhibitory activity towards all three tested enzymes in µM range (IC50COX-1 = 1.9 µM; IC50COX-2 and 5-LOX = 10.1µM. COX-1 inhibition was until recently considered undesirable due to COX-1 constitutive expression in most cell types and tissues. Thus, there are not many compounds known with selective COX-1 activity. However, it is now believed that COX-1 plays an important role in the pathophysiology of several acute and chronic disorders, including cancer or neurodegenerative diseases. Thus, the discovery of effective COX-1 selective inhibitors is desirable and important.

Keywords: cyclooxygenases, kinases, lipoxygenases, quinazolines

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Authors: Jer-Yuh Liu, Je-Chiuan Ye, Jin-Ming Hwang

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Protein kinase C alpha (PKCα) is a key signaling molecule in human cancer development. As a therapeutic strategy, targeting PKCα is difficult because the molecule is ubiquitously expressed in non-malignant cells. PKCα is regulated by the cooperative interaction of the transcription factors myeloid zinc finger 1 (MZF-1) and Ets-like protein-1 (Elk-1) in human cancer cells. By conducting tissue array analysis, herein, we determined the protein expression of MZF-1/Elk-1/PKCα in various cancers. The data show that the expression of MZF-1/Elk-1 is correlated with that of PKCα in hepatocellular carcinoma (HCC), but not in bladder and lung cancers. In addition, the PKCα down-regulation by shRNA Elk-1 was only observed in the HCC SK-Hep-1 cells. Blocking the interaction between MZF-1 and Elk-1 through the transfection of their binding domain MZF-160–72 decreased PKCα expression. This step ultimately depressed the epithelial-mesenchymal transition potential of the HCC cells. These findings could be used to develop an alternative therapeutic strategy for patients with the PKCα-derived HCC.

Keywords: protein kinase C alpha, myeloid zinc finger 1, ets-like protein-1, hepatocellular carcinoma

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Authors: Ireneusz Majsterek, Dariusz Pytel, J. Alan Diehl

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PKR-like ER kinase (PERK) is serine/threonie endoplasmic reticulum (ER) transmembrane kinase activated during ER-stress. PERK can activate signaling pathways known as unfolded protein response (UPR). Attenuation of translation is mediated by PERK via phosphorylation of eukaryotic initiation factor 2α (eIF2α), which is necessary for translation initiation. PERK activation also directly contributes to activation of Nrf2 which regulates expression of anti-oxidant enzymes. An increased phosphorylation of eIF2α has been reported in Alzheimer disease (AD) patient hippocampus, indicating that PERK is activated in this disease. Recent data have revealed activation of PERK signaling in non-Hodgkins lymphomas. Results also revealed that loss of PERK limits mammary tumor cell growth in vitro and in vivo. Consistent with these observations, activation of UPR in vitro increases levels of the amyloid precursor protein (APP), the peptide from which beta-amyloid plaques (AB) fragments are derived. Finally, proteolytic processing of APP, including the cleavages that produce AB, largely occurs in the ER, and localization coincident with PERK activity. Thus, we expect that PERK-dependent signaling is critical for progression of many types of diseases (human cancer, neurodegenerative disease and other). Therefore, modulation of PERK activity may be a useful therapeutic target in the treatment of different diseases that fail to respond to traditional chemotherapeutic strategies, including Alzheimer’s disease. Our goal will be to developed therapeutic modalities targeting PERK activity.

Keywords: PERK kinase, small molecule inhibitor, neurodegenerative disease, Alzheimer’s disease

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Authors: Maryam Sadeghi, Claude Bazin, Daniel Hodouin, Laura Perez Barnuevo

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The performance of a separation process is usually evaluated using performance indices calculated from elemental assays readily available from the chemical analysis laboratory. However, the separation process performance is essentially related to the properties of the minerals that carry the elements and not those of the elements. Since elements or metals can be carried by valuable and gangue minerals in the ore and that each mineral responds differently to a mineral processing method, the use of only elemental assays could lead to erroneous or uncertain conclusions on the process performance. This paper discusses the advantages of using performance indices calculated from minerals content, such as minerals recovery, for process performance assessments. A method is presented that uses elemental assays to estimate the minerals content of the solids in various process streams. The method combines the stoichiometric composition of the minerals and constraints of mass conservation for the minerals through the concentration process to estimate the minerals content from elemental assays. The advantage of assessing a concentration process using mineral based performance indices is illustrated for an iron ore concentration circuit.

Keywords: data reconciliation, iron ore concentration, mineral composition, process performance assessment

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Authors: Manuel A. Alonso-Tarajano, Roberto Mosca, Patrick Aloy

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Protein kinases participate in a myriad of cellular processes of major biomedical interest. The in vivo substrate specificity of these enzymes is a process determined by several factors, and despite several years of research on the topic, is still far from being totally understood. In the present work, we have quantified the contributions to the kinase substrate specificity of i) the phosphorylation sites and their surrounding residues in the sequence and of ii) the association of kinases to adaptor or scaffold proteins. We have used position-specific scoring matrices (PSSMs), to represent the stretches of sequences phosphorylated by 93 families of kinases. We have found negative correlations between the number of sequences from which a PSSM is generated and the statistical significance and the performance of that PSSM. Using a subset of 22 statistically significant PSSMs, we have identified specificity determinant residues (SDRs) for 86% of the corresponding kinase families. Our results suggest that different SDRs can function as positive or negative elements of substrate recognition by the different families of kinases. Additionally, we have found that human proteins with known function as adaptors or scaffolds (kAS) tend to interact with a significantly large fraction of the substrates of the kinases to which they associate. Based on this characteristic we have identified a set of 279 potential adaptors/scaffolds (pAS) for human kinases, which is enriched in Pfam domains and functional terms tightly related to the proposed function. Moreover, our results show that for 74.6% of the kinase– pAS association found, the pAS colocalize with the substrates of the kinases they are associated to. Finally, we have found evidence suggesting that the association of kinases to adaptors and scaffolds, may contribute significantly to diminish the in vivo substrate crossed- specificity of protein kinases. In general, our results indicate the relevance of several SDRs for both the positive and negative selection of phosphorylation sites by kinase families and also suggest that the association of kinases to pAS proteins may be an important factor for the localization of the enzymes with their set of substrates.

Keywords: kinase, phosphorylation, substrate specificity, adaptors, scaffolds, cellular colocalization

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Authors: Chanasit Chaocharoenphat

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Hibiscus sabdariffa is a herbal plant that is commonly used for home remedies in Thailand. This study aims to determine the antioxidant activity of polyphenols, as oxidative stress plays a vital role in the development of cancer, and H. sabdariffa was used in this study. The total flavonoids content was determined using the aluminium chloride colourimetric method and expressed as quercetin equivalents (QE)/g and the antioxidant capacity of the flavonoids using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging capacity assays. The IC50 values of H. sabdariffa extract were 167.14 μg/mL ± 0.843 and 77.59 μg/mL ± 0.798, respectively. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. To summarise, H. sabdariffa extract contains a high concentration of total flavonoids and exhibits potent antioxidant activity. However, additional antioxidant activity assays such as superoxide dismutase (SOD), reactive oxygen species (ROS), and reactive nitrogen species (RNS) scavenging assays and in vitro antioxidant experiments should be carried out to investigate the molecular mechanism of the compound.

Keywords: ABTS assay, antioxidant activity, Gracilaria fisheri, DPPH assays, total flavonoid content

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Authors: Yuda Purwana Roswanjaya, Wouter Kohlen, Rene Geurts

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The Trema genus represents a group of fast-growing tropical tree species within the Cannabaceae. Interestingly, five species nested in this lineage -known as Parasponia- can establish rhizobium nitrogen-fixing root nodules, similar to those found in legumes. Parasponia and legumes use a conserved genetic network to control root nodule formation, among which are genes also essential for mycorrhizal symbiosis (the so-called common symbiotic pathway). However, Trema species lost several genes that function exclusively in nodulation, suggesting a loss-of the nodulation trait in Trema. Strikingly, in a Trema orientalis population found in Malaysian Borneo we identified a truncated SYMBIOSIS RECEPTOR KINASE (SYMRK) mutant allele lacking a large portion of the c-terminal kinase domain. In legumes this gene is essential for nodulation and mycorrhization. This raises the question whether Trema orientalis can still be mycorrhized. To answer this question, we established quantitative mycorrhization assay for Parasponia andersonii and Trema orientalis. Plants were grown in closed pots on half strength Hoagland medium containing 20 µM potassium phosphate in sterilized sand and inoculated with 125 spores of Rhizopagus irregularis (Agronutrion-DAOM197198). Mycorrhization efficiency was determined by analyzing the frequency of mycorrhiza (%F), the intensity of the mycorrhizal colonization (%M) and the arbuscule abundance (%A) in the root system. Trema orientalis RG33 can be mycorrhized, though with lower efficiency compared to Parasponia andersonii. From this we conclude that a functional SYMRK kinase domain is not essential for Trema orientalis mycorrhization. In ongoing experiments, we aim to investigate the role of SYMRK in Parasponia andersonii mycorrhization and nodulation. For this two Parasponia andersonii symrk CRISPR-Cas9 mutant alleles were created. One mimicking the TorSYMRKRG33 allele by deletion of exon 13-15, and a full Parasponia andersonii SYMRK knockout.

Keywords: endomycorrhization, Parasponia andersonii, symbiosis receptor kinase (SYMRK), Trema orientalis

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Authors: Azza T. Tahera, Eman M. Ahmeda, Nadia A. Khalila, Yassin M. Nissanb

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New 3-(pyridin-4-yl)-[1,2,4] triazolo [4,3-b] pyridazine derivatives 2a-i, 4a,b and 6a,b were designed, synthesized and evaluated as cytotoxic agents. All compounds were investigated for their in vitro cytotoxicity at a single dose 10-5M concentration towards 60 cancer cell lines according to USA NCI protocol. The preliminary screening results showed that the majority of tested compounds exhibited remarkable activity against SR (leukemia) cell panel. Molecular docking for all synthesized compounds was performed on the active site of c-Met kinase. The most active compounds, 2f and 4a were further evaluated at a seven dose level screening and their IC50 as a c-Met kinase inhibitors were determined in vitro.

Keywords: triazolopyridazines, pyridazines, cytotoxic activity, cell panel

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Authors: Dong-Gyu Leem, Ji-Sun Shin, Sang Yoon Choi, Kyung-Tae Lee

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In this study, we focused on the anti-proliferative effects of panaxydol, a C17 polyacetylenic compound derived from Panax ginseng roots, against various human cancer cells. We treated with panaxydol to various cancer cells and panaxydol treatment was found to significantly inhibit the proliferation of human lung cancer cells (A549) and human pancreatic cancer cells (AsPC-1 and MIA PaCa-2), of which AsPC-1 cells were most sensitive to its treatment. DNA flow cytometric analysis indicated that panaxydol blocked cell cycle progression at the G1 phase in A549 cells, which accompanied by a parallel reduction of protein expression of cyclin-dependent kinase (CDK) 2, CDK4, CDK6, cyclin D1 and cyclin E. CDK inhibitors (CDKIs), such as p21CIP1/WAF1 and p27KIP1, were gradually upregulated after panaxydol treatment at the protein levels. Furthermore, panaxydol induced the activation of p53 in A549 cells. In addition, panaxydol also induced apoptosis of AsPC-1 and MIA PaCa-2 cells, as shown by accumulation of subG1 and apoptotic cell populations. Panaxydol triggered the activation of caspase-3, -8, -9 and the cleavage of poly (ADP-ribose) polymerase (PARP). Reduction of mitochondrial transmembrane potential by panaxydol was determined by staining with dihexyloxacarbocyanine iodide. Furthermore, panaxydol suppressed the levels of anti-apoptotic proteins, XIAP and Bcl-2, and increased the levels of proapoptotic proteins, Bax and Bad. In addition, panaxydol inhibited the activation of Akt and extracellular signal-regulated kinase (ERK) and activated the p38 mitogen-activated protein kinase kinase (MAPK). Our results suggest that panaxydol is an anti-tumor compound that causes p53-mediated cell cycle arrest and apoptosis via mitochondrial apoptotic pathway in various cancer cells.

Keywords: apoptosis, cancer, G1 arrest, panaxydol

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Authors: Man-Ho Oh

Abstract:

Membrane localized Leucine-Rich-Repeat Receptor-Like Kinases (LRR-RLKs) play crucial roles in plant growth and abiotic/biotic stress responses in higher plants including Arabidopsis and Brassica species. Among several Receptor-Like Kinases (RLKs), Leucine-Rich-Repeat Receptor-Like-Kinases (LRR-RLKs) are the major group of genes that play crucial roles related to growth, development and stress conditions in plant system. Since it is involved in several functional roles, it seems to be very important to investigate their roles in higher plants. We are particularly interested in brassinosteroid (BR) signaling, which is mediated by the BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor kinase and its co-receptor, BRI1-ASSOCIATED KINASE 1 (BAK1). Autophosphorylation of receptor kinases is recognized to be an important process in activation of signaling in higher plants. Although the plant receptors are generally classified as Ser/Thr protein kinases, many other receptor kinases including BRI1 and BAK1 are shown to autophosphorylate on Tyr residues in addition to Ser/Thr. As an interesting result, we determined that several 14-3-3 regulatory proteins bind to BRI1-CD and are phosphorylated by several receptor kinases in vitro, suggesting that BRI1 is critical for diverse signaling.

Keywords: autophosphorylation, brassinosteroid, BRASSINOSTEROID INSENSITIVE 1, BRI1-ASSOCIATED KINASE 1, Leucine-Rich-Repeat Receptor-Like Kinases (LRR-RLKs)

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Authors: Réka Kovács, György Téglásy, Szilvia Boros

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Introduction: Aim of the Study: Low body fat percentage frequently occurs in aesthetic sports. Because of the unrealistic expectations, their quantity and quality of nutrition intake are inadequate. This can be linked to several health issues which appear in blood samples (iron, ferritin, creatine kinase, etc.). Our retrospective study aimed to investigate the connection between body composition (InBody 770 monitor) and blood samples test results among elite adolescent (14-18 years) and adult (19-28 years) aesthetic athletes. Methods: Data collection happened between 01.08.2022. and 15.08.2022 in National Institute for Sports Medicine, Budapest. The final group consisted of 111 athletes (n=111; adolescents: n=68, adults: n=43). We used descriptive statistics, a two-sample t-test, and correlation analysis with Microsoft Office Excel 2007 software. Our results were considered significant if p<0,05. Results: In 33,3% (37/111) we found low body fat percentage (girls and women: <12%, boys and men: <8%) and in 64% (71/111) high creatine kinase levels. Differences were found mainly in the adolescent group. We found a correlation between body weight and total cholesterol, visceral fat and triglyceride, hematocrit and iron-linking capacity, moreover body fat percentage and ferritin levels. Discussion: It is important to start education about sports nutrition at an early age. The connection between low body fat percentage, serum iron, triglyceride, and ferritin levels refers to the fact that the nutrition intake of the athletes is inadequate. High blood concentrations of creatine kinase may show a lack of proper recovery, which is essential to improve health and performance.

Keywords: body fat percentage, creatine kinase, recovery, sports nutrition

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Authors: Monthon Tangjitmungman

Abstract:

Numerous diseases have been linked to oxidative stress, in which a disproportion of free radicals in the body leads to tissue or cell damage. Polyphenols are the most abundant antioxidants found in plants, and they are highly effective at scavenging oxidative free radicals. Due to the presence of phenolic compounds in Caesalpinia sappan has been discovered to have antioxidant activity. It has several health benefits, the most important of which is preventing cardiovascular and cancer diseases. This study aimed to determine the phenolic content and antioxidant activity of C. sappan extract using a variety of antioxidant assays. The extract of C. sappan was made using a mixture of solvents (ethyl alcohol: water in ratio 8:2). The total phenolic content of C. sappan extract was determined and expressed as gallic acid equivalents using the Folin-Cioucalteu method (GAE). The antioxidant activity of C. sappan extract was assessed using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay and the ABTS radical scavenging capacity assay. An association was found between antioxidant activity and total phenol content. The antioxidant activity of C. sappan extract was also determined by DPPH and ABTS assays. The IC50 values for C. sappan extract from DPPH and ABTS assays were 54.48 μg/mL ± 0.545 and 25.46 μg/mL ± 0.790, respectively, in the DPPH assay. In the DPPH assay, vitamin C was used as a positive control, whereas Trolox was used as a positive control in the ABTS assay. In conclusion, C. sappan extract contains a high level of total phenolics and exhibits significant antioxidant activity. Nevertheless, more research should be done on the antioxidant activity, such as SOD and ROS scavenging assays and in vivo experiments, to determine whether the compound has antioxidant activity.

Keywords: ABTS assay, antioxidant activity, Caesalpinia sappan, DPPH assays, total phenolic content

Procedia PDF Downloads 384