pinecone/core-2020-05-10-deduplication huggingface dataset:
Role of mitogen-activated protein kinase in Zn-BC-AM PDT-induced apoptosis in nasopharyngeal carcinoma cells

Photodynamic therapy (PDT) with a recently developed photosensitizer Zn-BC-AM was found to effectively induce apoptosis in a welldifferentiated nasopharyngeal carcinoma (NPC) HK-1 cell line. Sustained activation of p38 mitogen-activated protein kinase (MAPK) and cjun N-terminal kinase (JNK) as well as a transient increase in activation of extracellular signal-regulated kinase (ERK) were observed immediately after Zn-BC-AM PDT. A commonly used p38 MAPK/JNK pharmacological inhibitor PD169316 was found to reduce PDTinduced apoptosis of HK-1 cells. PD169316 also prevented the loss of Bcl-2 and Bcl-xL in PDT-treated HK-1 cells. However, inhibition of JNK with SP600125 had no effect on Zn-BC-AM PDT-induced apoptosis while inhibition of ERK with PD98059 or p38 MAPK with SB203580 significantly increased Zn-BC-AM PDT-induced apoptosis. Further study showed that knockdown of the p38b isoform with siRNA also increased Zn-BC-AM PDT-induced apoptosis, indicating that the anti-apoptotic effect of PD169316 in PDT-treated HK-1 cells was probably independent of p38 MAPK or JNK activation. Taken together, the results suggest that inhibition of p38b and ERK may enhance the therapeutic efficacy of Zn-BC-AM PDTon NPC cells. It should be noted that data only based on the use of PD169316 should be interpreted in caution. Copyright © 2010 John Wiley & Sons, Ltd.postprin
Natural Helicobacter infection modulates mouse intestinal muscularis macrophage responses

Helicobacter species are common laboratory pathogens which induce intestinal inflammation and disease in susceptible mice. Since in vitro studies indicate that Helicobacter products activate macrophages, we hypothesized that in vivo Helicobacter infection regulates the inflammatory response of intestinal muscularis macrophages from C57Bl/6 mice. Helicobacter hepaticus infection increased surface expression of macrophage markers F4/80, CD11b, and MHC-II within whole intestinal muscle mounts. However, constitutive cytokine and chemokine production by macrophages isolated from infected mice significantly decreased compared to macrophages from uninfected mice despite no detectable bacterial products in the cultures. In addition, muscularis macrophages from infected mice up-regulated FIZZ-1 and SK-1 gene expression, suggesting the macrophages had an anti-inflammatory phenotype. Corresponding with increased anti-inflammatory gene expression, macrophages from infected mice were more phagocytic but did not produce cytokines after stimulation with LPS and IFN-γ or immune complexes and IL-4. Therefore, the presence of Helicobacter infection matures intestinal muscularis macrophages, modulating the constitutive macrophage response to become more antiinflammatory and resistant to secondary stimulation
Magnesium deficiency up-regulates Myod expression in rat skeletal muscle and C2C12 myogenic cells.

Magnesium (Mg) deficiency induces the production of free radicals, increases cytosolic ionized calcium concentration, and modulates the function of skeletal muscle in rats. The present study examined the effects of Mg deficiency on the gene expression of molecules related to myogenesis in the gastrocnemius muscle as well as in C2C12 myogenic cells. Ingestion of an Mg-deficient diet resulted in a lower weight of the gastrocnemius muscle and higher concentration of muscular TBARSs, an index of oxidative stress. Mg deficiency also enhanced the expression of Myod and myogenin. In vivo effects of Mg deficiency on myogenic gene expression were partially reproduced in in vitro C2C12 cells; expression of Myod was up-regulated by a mixed culture of myoblasts and myotubes with Mg-deficient medium, which related to the simultaneous up-regulation of Myhc IIb, a myotube-specific protein. The culture with Mg-deficient medium did not increase the gene transcript level of HO-1, another marker of oxidative stress, suggesting that Mg deficiency-induced Myod expression does not result from oxidative stress. Furthermore, oxidative stress induced by hydrogen peroxide did not increase Myod expression, whereas the expression of Myod, myogenin and Myhc IIb was decreased by oxidative stress from the initial phase of differentiation. The effects of Mg deficiency depended on the stages of myogenesis; myoblast culture in Mg-deficient differentiation medium did not affect the expression of Myod and Myhc IIb. The present study revealed stage-dependent effects of Mg deficiency on myogenesis
In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17-beta-estradiol analogue in breast adenocarcinoma cells

2-Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low
oral bioavailability, several promising analogues of 2-methoxyestradiol have been developed. In this study, the in vitro influence of the
compound, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19), a non-commercially available 17-b-estradiol analogue, was tested on
the breast adenocarcinoma MCF-7 cell line. The in vitro influence of 24 h exposure to 0.18 mM of C19 on MCF-7 cells was evaluated on
cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization-optical transmitted light differential
interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and
compromised cell density in C19-treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes,
were also observed in C19-treated samples. An increase in the number of cells present in the sub-G1 fraction, as well as a reduction in
mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in
aggresome formation was observed in C19-treated cells. C19 induced both apoptosis and autophagy in MCF-7 cells.The Medical Research Council of South Africa (AK076; AL343), the Cancer Association of South Africa (AK246), RESCOM, School of Medicine (Faculty of Health Sciences, University of Pretoria), the National Research Foundation of South Africa (AL239) and the Struwig-Germeshuysen Cancer Research Trust of South Africa (AN074). The Bioinformatics and Computational Biology Unit (University of Pretoria) contributed to the in silico-design of the compound.http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1099-0844hb2013ay201
Downregulation of Pgc-1α expression by tea leaves and their by-products.

Previous studies indicate that muscle Pgc-1α expression governs the proportion of muscle fibre types. As a first step in using diet to manipulate the proportion of muscle fibre types by using Pgc-1α expression, the present study investigates the modulation of Pgc-1α expression by feedstuffs. A luciferase-based Pgc-1α reporter construct (Pgc-1α(-2553)-luc) that contains the mouse Pgc-1α promoter (-2553 to +78 bp) was prepared. A screen of ethanol extracts from 33 feedstuffs indicated that oolong tea and roasted green tea extracts decreased Pgc-1α(-2553)-luc expression in C2C12 myoblasts. The transcriptional repression of Pgc-1α by tea leaf extracts was reproduced in hepatic HepG2 cells. We further examined the effects of the alcohol extracts of tea waste and its silage on Pgc-1α transcription; the tea waste silage extract inhibited Pgc-1α transcription. Treatment with the extracts of raw tea leaves, tea waste and tea waste silage effectively decreased Pgc-1α mRNA levels during myogenesis of myosatellite cells. The present results suggest that tea leaves and their by-products could be used to modulate proportions of muscle fibre types
Enhancement of RANKL-induced MITF-E expression and osteoclastogenesis by TGF-β

Microphthalmia-associated transcription factor (MITF) is a transcription factor that is expressed in limited types of cells, including osteoclasts, but the expression and role of MITF during osteoclastogenesis have not been fully elucidated. The expression of the MITF-E isoform but not that of the MITF-A isoform was induced in response to differentiation stimulation towards osteoclasts by receptor activator of NF-κB ligand (RANKL) in both RAW264.7 cells and primary bone marrow cells. The RANKL-induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells was inhibited in RAW264.7 cells expressing siRNA for MITF-E. Transforming growth factor-β (TGF-β) enhanced RANKL-induced MITF-E expression and -TRAP positive multinucleated cell formation. In particular, TGF-β potentiated the formation of larger osteoclasts. The expression levels of NFATc1, TRAP and CtsK, genes related to osteoclast development and activity, were concurrently enhanced by TGF-β in the presence of RANKL. Furthermore, the expression of dendritic cell-specific transmembrane protein (DC-STAMP), Itgav, Itga2, Itga5, Itgb1, Itgb3 and Itgb5, genes related to cell adhesion and fusion, were up-regulated by co-treatment with TGF-β. In particular, the regulatory expression of Itgav and Itgb5 in response to RANKL with or without TGF-β resembled that of MITF-E. Because MITF is involved in cell fusion in some cell systems, these results imply a role for MITF-E as an enhancer of osteoclastogenesis and that RANKL-induced levels of both MITF-E mRNA and of MITF-dependent gene expression are enhanced by treatment with TGF-β
An Ex-Vivo Model using Human Osteoarthritic Cartilage Demonstrates the Release of Bioactive Insulin-like growth factor- 1 from a Collagen-Glycosaminoglycan Scaffold.

Biomimetic scaffolds hold great promise for therapeutic repair of cartilage but although most
scaffolds are tested with cells in vitro, there are very few ex vivo models (EVM) where adult
cartilage and scaffolds are co-cultured to optimize their interaction prior to in vivo studies.
This study describes a simple, non-compressive method that is applicable to mammalian or
human cartilage and provides a reasonable throughput of samples.
Rings of full depth articular cartilage slices were derived from human donors undergoing
knee replacement for osteoarthritis (OA) and a 3mm core of a collagen/glycosaminoglycan
biomimetic scaffold (Tigenix UK) inserted to create the EVM. Adult OA chondrocytes were
seeded into the scaffold and cultures maintained for up to 30 days.
EVM were stable throughout experiments and cells remained viable. Chondrocytes seeded
into the EVM attached throughout the scaffold and in contact with the cartilage explants. Cell
migration and deposition of ECM proteins in the scaffold was enhanced by growth factors
particularly if the scaffold was preloaded with growth factors.
This study demonstrates that the EVM represents a suitable model which has potential for a
testing a range of therapeutic parameters such as numbers/types of cell, growth factors or
therapeutic drugs before progressing to costly pre-clinical trials.

Significance
Pre clinical trials of biomaterials for cartilage repair are very costly and all too often studies
progress directly from in vitro studies using isolated cells to in vivo studies without
investigating the interaction between the target tissue and the scaffold. Our study uses viable
cartilage from adult human donors with osteoarthritis and therefore represents the exact
scenario that the scaffold is designed for. The system is cheap and simple to set up and is
suitable for a 48 well plate format meaning a reasonable throughput is obtainable. This lends
the model to therapeutic drug testing.The authors would like to kindly acknowledge funding from the EPSRC and Tigenix Ltd
(LM), Technology Strategy Board and Tigenix Ltd (JW) and the NIHR (DH).This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1002/cbf.3112
In Vivo Chaperone-Assisted Folding of α-1,6-Fucosyltransferase from Rhizobium sp.

3 pages, 3 figures.-- PMID: 12794864 [PubMed].-- Supplementary information (Materials and methods, 8 pages) available at: http://www.wiley-vch.de/contents/jc_2268/2003/z514_s.pdfGlycosyltransferases have become powerful tools for the synthesis of oligosaccharides through their strict control over the stereo- and regioselectivity of glycosidic bond formation. One major drawback of glycosyltransferases in synthesis is their limited availability. The cloning and overexpression of bacterial glycosyltransferases is one alternative to overcome this problem. However, when a\ud
heterologous protein is over-expressed in E. coli misfolding and aggregation happen frequently, driving the recombinant protein into inactive aggregates known as inclusion bodies (I.B.). One possible and attractive strategy to avoid their formation is to increase the cellular levels of molecular\ud
chaperones. Chaperonins are able to mediate ATP-dependent folding of polypeptides to the native state. GroEL from E. coli is the best characterized chaperonin and its function is dependent on the cochaperonin GroES.A.B was supported by a postdoctoral I3P contract of the European Social Found. This\ud
work was supported by the Spanish DGI (Grants BQU2001-1503 and PTR1995-0568-OP).Peer reviewe
Synthesis, inhibition properties and theoretical study of the new nanomolar trehalase inhibitor 1-thiatrehazolin: Towards a structural understanding of trehazolin inhibition

6 páginas, 2 figuras, 2 esquemas, 2 tablas.-- ChemBioChem Special Issue: EMBO Symposium: Chemistry Meets Biology.A new trehazolin analogue, 1-thiatrehazolin, has been synthesized from carbohydrate precursors by a highly efficient route based on our previously developed ketone/oxime ether reductive carbocyclization reaction for the construction of the cyclitol ring and an intramolecular nucleophilic displacement reaction for the construction of the thiazoline ring. 1-Thiatrehazolin is a very potent, slow, tight-binding trehalase inhibitor. On the basis of the experimental results, a structural model for trehalase inhibition by trehazolin and analogues is proposed, supported by theoretical calculations.Financial support by the Ministry of Science and Technology of Spain (project BQU2000-1501-C02-01) is gratefully acknowledged.Peer reviewe
Solution Structure of Domain 6 from a Self-Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

Group II intron self-splicing is essential for correct expression of organellar genes in plants, fungi, and yeast, as well as of bacterial genes. Self-excision of these autocatalytic introns from the primary RNA transcript is achieved in a two-step mechanism apparently analogous to the one of the eukaryotic spliceosome. The 2'-OH of a conserved adenosine (the branch point) located within domain 6 (D6) acts as the nucleophile in the first step of splicing. Despite the biological importance of group II introns, little is known about their structural organization and usage of metal ions in catalysis. Here we report the first solution structure of a catalytically active D6 construct encompassing the branch point and the neighboring helical regions from the mitochondrial yeast intron ai5. The branch adenosine is the single unpaired nucleotide, and, in contrast to the spliceosomal branch site, resides within the helix being partially stacked between the two flanking GU wobble pairs. We identified a novel prominent Mg2+ binding site in the major groove of the branch site. Importantly, Mg2+ addition does not impair stacking of the branch-adenosine, but in contrast rather strengthens the interaction with the flanking uridines, as shown by NMR- and fluorescence studies. This means, that domain 6 presents the branch adenosine in a stacked fashion to the core of group II introns upon folding to the active conformation
Exchange Rate Constants of Invisible Protons in Proteins Determined by NMR Spectroscopy

Although labile protons that are exchanging rapidly with those of the solvent cannot be observed directly, their exchange rate constants can be determined by indirect detection of scalar-coupled neighboring nuclei. We have used heteronuclear NMR spectroscopy to measure the exchange rate constants of labile protons in the side chains of lysine and arginine residues in ubiquitin enriched in carbon-13 and nitrogen-15 at neutral pH. Exchange rate constants as fast as 40×103 s-1 were thus measured. These results demonstrate that NMR spectroscopy is a powerful tool for the characterization of lysine NH3+ and arginine NH groups in proteins at physiologically relevant pH values
Studies of the Structure of the N-Terminal domain from the Y4 receptor - a G protein-coupled receptor - and its Interaction with hormones from the NPY family

Binding of peptide hormones to G protein-coupled receptors is believed to be mediated through formation of contacts of the ligands with residues of the extracellular loops of family 1 GPCRs. Here we have investigated whether additional binding sites exist within the N-terminal domain, as studied in the form of binding of peptides from the neuropeptide Y (NPY) family to the N terminus of the Y4 receptor (N-Y4). The N-terminal domain of the Y4 receptor has been expressed in isotopically enriched form and studied by solution NMR spectroscopy. The peptide is unstructured in solution, whereas a micelle-associated helical segment is formed in the presence of dodecylphosphocholine (DPC) or sodium dodecylsulfate (SDS). As measured by surface plasmon resonance (SPR) spectroscopy, N-Y4 binds with approximately 50 M affinity to the pancreatic polypeptide (PP), a high-affinity ligand to the Y4 receptor, whereas binding to neuropeptide Y (NPY) and peptide YY (PYY) is much weaker. Residues critical for binding in PP and in N-Y4 have been identified by site-directed mutagenesis. The data indicate that electrostatic interactions dominate and that this interaction is mediated by acidic ligand and basic receptor residues. Residues of N-Y4 are likely to contribute to the binding of PP, and in addition might possibly also help to transfer the hormone from the membrane-bound state into the receptor binding pocket
Enzymatic Polymerization of Phosphonate Nucleosides

5'-O-Phosphonomethyl-2'-deoxyadenosine (PMdA) proved to be a good substrate of the Therminator polymerose. In this article, we investigated whether the A, C, T and U analogues of this phosphonate nucleoside (PMdN) series can function as substrates of natural DNA polymerases. PMdT and PMdU could only be polymerized enzymatically to a limited extent, Nevertheless, PMdA and PMdC could be incorporated into a DNA duplex with complete chain elongation by all the DNA polymerases tested. A mixed sequence of four nucleotides containing modified C T and A residues could be obtained with the Vent(exo(-)) and Therminator polymerases. The kinetic values for the incorporation of PMdA by Vent(exo-) polymerase were determined; a reduced K-VI value was found for the incorporation of PMdA compared to the natural substrate. Future polymerase directed evolution studies will allow us to select an enzyme with a heightened capacity to process these modified DNA building blocks into modified strands.status: publishe
From Kinase to Cyclase: An Unusual Example of Catalytic Promiscuity Modulated by Metal Switching

“This is the pre-peer reviewed version of the following article: Sánchez-Moreno, I., Iturrate, L., Martín-Hoyos, R., Jimeno, M. L., Mena, M., Bastida, A. and García-Junceda, E. (2009) From Kinase to Cyclase: An Unusual Example of Catalytic Promiscuity Modulated by Metal Switching. ChemBioChem. 10, 225-229, which has been published in final form at http://www3.interscience.wiley.com/journal/121544668/abstract?CRETRY=1&SRETRY=0.”Evolution of new enzyme functions: We describe the promiscuous behaviour of the dihydroxyacetone (DHA) kinase from Citrobacter freundii strain CECT 4626. In addition to the transfer of the -phosphate of adenosine-5-triphosphate (ATP) to DHA, this ATP-dependent DHAK is able to catalyse the cyclization of FAD to yield riboflavin 4,5-cyclic phosphate (4,5-cFMN). This catalytic promiscuity is modulated by the divalent cation that forms the complex with the phosphorylated substrate.This work was supported by grants from Ministerio de Educación\ud
y Ciencia (CTQ2004-03523/BQU and CTQ2007-67403/BQU). I.\ud
S.-M and L. I were supported by a predoctoral fellowship from\ud
Comunidad de Madrid.Peer reviewe
Thermodynamic and computational studies on the binding of p53-derived peptides and peptidomimetic inhibitors to HDM2

Helix power: The binding interactions of linear and constrained beta-hairpin-shaped peptides with HDM2 were compared by using experimental and theoretical methods. The entropic advantages enjoyed by the constrained peptides were found to be largely offset by reduced enthalpic contributions to binding of the cyclic mimetics. Formation of hydrogen bonds upon helix folding could contribute significantly to the enhanced enthalpy observed in binding of the linear peptides.The human double minute 2 protein (HDM2) binds a short peptide derived from the N terminus of the tumor-suppressor protein, p53. This peptide (p53 residues 15-29) is flexible in free solution, but upon binding to HDM2 it folds into an amphipathic alpha-helical conformation. Three residues along one face of the p53 helix (Phe19, Trp23, and Leu26) dock into hydrophobic pockets on the surface of HDM2. A conformationally constrained cyclic beta-hairpin peptidomimetic of p53, with residues Phe1, 6-chloro-Trp3, and Leu4 in one strand of the beta-hairpin, was shown earlier to dock into the same pockets on HDM2. Here, we show by isothermal titration calorimetry that the entropy loss upon binding of the constrained peptide to HDM2 is, as would be expected, much lower (TDeltaS approximately 10 kcal mol(-1) at 300 K) than that for the linear peptide. However, the entropic advantage enjoyed by the constrained peptide is largely offset by a reduced enthalpic contribution, relative to the linear peptide, to binding of the cyclic mimetic. To explore the electronic nature of the interactions between the energetically important residues in each ligand and HDM2, hybrid quantum mechanical and electrostatic Poisson-Boltzmann computational studies were performed. The calculations reveal that significant stabilizing van der Waals interactions and polarization effects occur between the Trp side chain in each ligand and aromatic and aliphatic residues in HDM2. These stabilizing interactions are enhanced when a 6-chloro substituent is incorporated into the Trp, in agreement with the experimental studies. In addition, the calculations suggest that at least one stabilizing hydrogen bond is formed, between the Trp indole-NH in both ligands and HDM2. Other hydrogen-bonding interactions also arise, however, along the alpha-helical backbone of the linear peptide upon binding to HDM2, but are not mimicked in the constrained inhibitor-HDM2 complex. The formation of these hydrogen bonds upon helix folding could contribute significantly to the enhanced enthalpy observed in binding of the linear peptide to HDM2
Role of endonucleases XPF and XPG in nucleotide excision repair of platinated DNA and cisplatin/oxaliplatin cytotoxicity

Resistance of tumor cells to platinum anticancer agents poses a major problem in cancer chemotherapy. One of the mechanisms associated with platinum-based drug resistance is the enhanced capacity of the cell to carry out nucleotide excision repair (NER) on platinum-damaged DNA. Endonucleases XPF and XPG are critical components of NER, responsible for excising the damaged DNA strand to remove the DNA lesion. Here, we investigated possible consequences of down-regulation of XPF and XPG gene expression in osteosarcoma cancer cells (U2OS) and the impact on cellular transcription and DNA repair. We further evaluated the sensitivity of such cells toward the platinum anticancer drugs cisplatin and oxaliplatin.National Cancer Institute (U.S.) (Grant Number CA034992.)National University of Singapore.German Academic Exchange Service (DAAD