Home About us Contact
|
Home About us Contact | ||
|
|
||
Specific Inhibition (specific + inhibition)
Selected AbstractsResearch Article: Effective and Specific Inhibition of the CD40,CD154 Costimulatory Interaction by a Naphthalenesulphonic Acid DerivativeCHEMICAL BIOLOGY & DRUG DESIGN, Issue 4 2010Emilio Margolles-Clark Costimulatory interactions are important regulators of T-cell activation and, hence, promising therapeutic targets in autoimmune diseases as well as in transplant recipients. Following our recent identification of the first small-molecule inhibitors of the CD40,CD154 costimulatory protein,protein interaction (J Mol Med 87, 2009, 1133), we continued our search within the chemical space of organic dyes, and we now report the identification of the naphthalenesulphonic acid derivative mordant brown 1 as a more active, more effective, and more specific inhibitor. Flow cytometry experiments confirmed its ability to concentration-dependently inhibit the CD154(CD40L)-induced cellular responses in human THP-1 cells at concentrations well below cytotoxic levels. Binding experiments showed that it not only inhibits the CD40,CD154 interaction with sub-micromolar activity, but it also has considerably more than 100-fold selectivity toward this interaction even when compared to other members of the tumor necrosis factor superfamily pairs such as TNF-R1,TNF-,, BAFF-R(CD268),BAFF(CD257/BLys), OX40(CD134),OX40L(CD252), RANK(CD265),RANKL(CD254/TRANCE), or 4-1BB(CD137),4-1BBL. There is now sufficient structure-activity relationship information to serve as the basis of a drug discovery initiative targeting this important costimulatory interaction. [source] Specific inhibition of transforming growth factor-,2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotidesFEBS JOURNAL, Issue 8 2001Zhong-Jian Shen To elucidate the role of endogenous transforming growth factor (TGF)-,2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S-ODNs) complementary to regions in mRNA of TGF-,2 were synthesized and examined their effects on TGF-,2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S-ODNs were designated for three different target regions in the mRNA of TGF-,2. Among several antisense S-ODN analyzed, an oligonucleotide (AS-11) complementary to the translation initiation site of mRNA of TGF-,2 demonstrated a selective and strong inhibitory effect on TGF-,2 production in osteoblast cells. Other antisense S-ODNs which were designated for other regions in mRNA of TGF-,2 and one- or three-base mismatched analogs of AS-11 showed little or much less antisense activities than AS-11. Therefore, the most effective target site in mRNA of TGF-,2 is at the initiation codon region. The antisense effects of AS-11 were observed without reduction of levels of mRNA of TGF-,2. Furthermore, the inhibition of TGF-,2 expression by antisense S-ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF-,2 in osteoblast cells. [source] Hepatitis B and C virus coinfection: A novel model system reveals the absence of direct viral interference,HEPATOLOGY, Issue 1 2009Pantxika Bellecave Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been associated with severe liver disease and frequent progression to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests reciprocal replicative suppression of the two viruses, or viral interference. However, interactions between HBV and HCV have been difficult to study due to the lack of appropriate model systems. We have established a novel model system to investigate interactions between HBV and HCV. Stable Huh-7 cell lines inducibly replicating HBV were transfected with selectable HCV replicons or infected with cell culture,derived HCV. In this system, both viruses were found to replicate in the same cell without overt interference. Specific inhibition of one virus did not affect the replication and gene expression of the other. Furthermore, cells harboring replicating HBV could be infected with cell culture,derived HCV, arguing against superinfection exclusion. Finally, cells harboring replicating HBV supported efficient production of infectious HCV. Conclusion: HBV and HCV can replicate in the same cell without evidence for direct interference in vitro. Therefore, the viral interference observed in coinfected patients is probably due to indirect mechanisms mediated by innate and/or adaptive host immune responses. These findings provide new insights into the pathogenesis of HBV,HCV coinfection and may contribute to its clinical management in the future. (HEPATOLOGY 2009.) [source] Participation of protein kinase C , isoform and extracellular signal-regulated kinase in neurite outgrowth of GT1 hypothalamic neuronsJOURNAL OF NEUROCHEMISTRY, Issue 6 2002Youngshik Choe Abstract In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform-selective inhibitors and transfection-based expression of enhanced green fluorescence protein (EGFP)-fused PKC isoforms. 12- O -Tetradecanoylphorbol-13-acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingolTM(a PKC,-selective inhibitor), but not by rottlerinTM (a PKC,-selective inhibitor), indicating that PKC, may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and ,-tubulin. To define the differential localization of PKC isoforms, EGFP-tagged PKC,, PKC,, and PKC, were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKC,-EGFP to growth cones and cell,cell adhesion sites, PKC,-EGFP to the nucleus, and PKC,-EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKC, (PKC,-Cat-EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKC, enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKC,-Cat-EGFP alone increased the neurite-outgrowth and doxycycline treatment further augmented the number of neurite-containing cells. We also examined the involvement of the extracellular signal-regulated kinase (ERK) MAP kinase in TPA-induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKC, with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase-1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKC, isoform-specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway. [source] Cleaved high molecular weight kininogen inhibits tube formation of endothelial progenitor cells via suppression of matrix metalloproteinase 2JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2010Y. WU Summary.,Background and objective:,Endothelial progenitor cells (EPCs) contribute to postnatal neovascularization, thus promoting wide interest in their therapeutic potential in vascular injury and prevention of their dysfunction in cardiovascular diseases. Cleaved high molecular weight kininogen (HKa), an activation product of the plasma kallikrein-kinin system (KKS), inhibits the functions of differentiated endothelial cells including in vitro and in vivo angiogenesis. In this study, our results provided the first evidence that HKa is able to target EPCs and inhibits their tube forming capacity. Methods and results:,We determined the effect of HKa on EPCs using a three-dimensional vasculogenesis assay. Upon stimulation with vascular endothelial growth factor (VEGF) alone, EPCs formed vacuoles and tubes, and differentiated into capillary-like networks. As detected by gelatinolytic activity assay, VEGF stimulated secretion and activation of matrix metallopeptidase 2 (MMP-2), but not MMP-9, in the conditioned medium of 3D culture of EPCs. Specific inhibition or gene ablation of MMP-2, but not MMP-9, blocked the vacuole and tube formation by EPCs. Thus, MMP-2 is selectively required for EPC vasculogenesis. In a concentration-dependent manner, HKa significantly inhibited tube formation by EPCs and the conversion of pro-MMP-2 to MMP-2. Moreover, HKa completely blocked the association between pro-MMP-2 and ,v,3 integrin, and its inhibition of MMP-2 activation was dependent on the presence of ,v,3 integrin. In a purified system, HKa did not directly inhibit MMP-2 activity. Conclusions:,HKa inhibits tube forming capacity of EPCs by suppression of MMP-2 activation, which may constitute a novel link between activation of the KKS and EPC dysfunction. [source] Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 3 2009Mary-Clare Miller Objective A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen-degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1-MMP), in synovial pannus invasiveness. Methods The expression and localization of MT1-MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1-MMP was analyzed by 3-dimensional (3-D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, or GM6001. The effect of adenoviral expression of a dominant-negative MT1-MMP construct lacking a catalytic domain was also examined. Results MT1-MMP was highly expressed at the pannus,cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3-D collagen matrix in an MT1-MMP,dependent manner. Invasion was blocked by TIMP-2 and GM6001 but not by TIMP-1. Invasion was also inhibited by the overexpression of a dominant-negative MT1-MMP, which inhibits collagenolytic activity and proMMP-2 activation by MT1-MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1-MMP,dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix. Conclusion MT1-MMP serves as an essential collagen-degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1-MMP,dependent invasion may represent a novel therapeutic strategy for RA. [source] Combined analysis of intracellular signalling and immunophenotype of human peripheral blood basophils by flow cytometry: a proof of conceptCLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2007D. G. Ebo Summary Background The signal transduction pathways and control mechanisms involved in IgE-mediated basophil activation remain incompletely understood. Objectives To investigate whether basophilic intracellular signal transduction and immunophenotype can be analysed simultaneously by flow cytometry. Methods Basophils in whole blood were stimulated with anti-IgE and latex antigen at various concentrations and during different time courses. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) as a representative of the intracellular signal transduction pathway and surface expression of CD63 was assessed simultaneously flow cytometrically. The effect of pre-incubation with IL-3 was assessed. Results Stimulation of the basophils with anti-IgE and allergen induces a rapid phosphorylation of p38 MAPK that peaks between 1 and 5 min and returns to baseline levels after 60 min. In contrast, CD63 up-regulation demonstrates a maximal but more continuous expression that peaks approximately 5 min later than phosphorylation of p38 MAPK. Specific inhibition of p38 MAPK reduced or almost completely abrogated up-regulation of CD63. Pre-incubation of the basophils with IL-3 produces a rapid p38 MAPK phosphorylation over basal levels, but this was weaker and shorter than for anti-IgE stimulation. Pre-incubation of the basophils with IL-3 did not potentiate anti-IgE-induced phosphorylation of p38 MAPK and did affect spontaneous or IgE-mediated CD63 up-regulation. Conclusions This study provides the proof that the flow cytometer allows an integrated analysis of basophilic intracellular signalling and immunophenotyping. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy. Capsule summary This study is the first to provide evidence for a combined analysis of basophilic intracellular signalling and immunophenotyping by flow cytometry. Owing to its technical simplicity, the low number of cells required and rapid analysis, the technique seems promising for use in the clinic as a diagnostic tool or to monitor therapy. [source] Fibrinogen-CD11b/CD18 interaction activates the NF-,B pathway and delays apoptosis in human neutrophilsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2003Carolina Rubel Abstract The regulation of neutrophil half-life by members of the coagulation cascade is critical for the resolution of the inflammatory response. We have demonstrated that soluble fibrinogen (sFbg) delays human neutrophil (PMN) apoptosis through a mechanism that involves CD11b interactions, and phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase,1/2 (ERK1/2). Since NF-,B is a key element in the regulation of apoptotic mechanisms in several immune cells, we investigated whether NF-,B is involved in the control of PMN survival by sFbg. We showthat sFbg triggers inhibitor protein ,B (I,B-,) degradation and NF-,B activation. Furthermore, pharmacological inhibition of NF-,B abrogates sFbg effects on apoptosis. In addition, specific inhibition of MAPK ERK1/2 significantly reduces NF-,B translocation by sFbg, suggesting a relationship between ERK1/2 and NF-,B activation. Similar results are obtained when granulocytic-differentiated HL-60 cells are treated with sFbg, making this model highly attractive for integrin-induced gene expression studies. It can be concluded that NF-,B participates in the prevention of apoptosis induced by sFbg with the participation of MAPK ERK1/2. These results shed light on the molecular mechanisms that control human granulocyte apoptosis, and suggest that NF-,B regulation may be of benefit for the resolution of the inflammatory response. [source] PRECLINICAL STUDY: Ecstasy-induced oxidative stress to adolescent rat brain mitochondria in vivo: influence of monoamine oxidase type AADDICTION BIOLOGY, Issue 2 2009Ema Alves ABSTRACT The administration of a neurotoxic dose of 3,4-methylenedioxymethamphetamine (MDMA; ,ecstasy') to the rat results in mitochondrial oxidative damage in the central nervous system, namely lipid and protein oxidation and mitochondrial DNA deletions with subsequent impairment of the correspondent protein expression. Although these toxic effects were shown to be prevented by monoamine oxidase B inhibition, the role of monoamine oxidase A (MAO-A) in MDMA-mediated mitochondrial damage remains to be evaluated. Thus, the aim of the present study was to clarify the potential interference of a specific inhibition of MAO-A by clorgyline, on the deleterious effects produced by a binge administration of a neurotoxic dose of MDMA (10 mg MDMA/kg of body weight, intraperitoneally, every 2 hours in a total of four administrations) to an adolescent rat model. The parameters evaluated were mitochondrial lipid peroxidation, protein carbonylation and expression of the respiratory chain protein subunits II of reduced nicotinamide adenine dinucleotide dehydrogenase (NDII) and I of cytochrome oxidase (COXI). Considering that hyperthermia has been shown to contribute to the neurotoxic effects of MDMA, another objective of the present study was to evaluate the body temperature changes mediated by MDMA with a MAO-A selective inhibition by clorgyline. The obtained results demonstrated that the administration of a neurotoxic binge dose of MDMA to an adolescent rat model previously treated with the specific MAO-A inhibitor, clorgyline, resulted in synergistic effects on serotonin- (5-HT) mediated behaviour and body temperature, provoking high mortality. Inhibition of MAO-A by clorgyline administration had no protective effect on MDMA-induced alterations on brain mitochondria (increased lipid peroxidation, protein carbonylation and decrease in the expression of the respiratory chain subunits NDII and COXI), although it aggravated MDMA-induced decrease in the expression of COXI. These results reinforce the notion that the concomitant use of MAO-A inhibitors and MDMA is counter indicated because of the resulting severe synergic toxicity. [source] Identification of direct and indirect targets of the Gln3 and Gat1 activators by transcriptional profiling in response to nitrogen availability in the short and long termFEMS YEAST RESEARCH, Issue 5 2006Bart Scherens Abstract Nitrogen catabolite repression (NCR) consists in the specific inhibition of transcriptional activation of genes encoding the permeases and catabolic enzymes needed to degrade poor nitrogen sources. Under nitrogen limitation or rapamycin treatment, NCR genes are activated by Gln3 or Gat1, or by both factors. To compare the sets of genes responding to rapamycin or to nitrogen limitation, we used DNA microarrays to establishing the expression profiles of a wild type strain, and of a double gln3,,gat1, strain, grown on glutamine, after addition of rapamycin, on proline, or after a shift from glutamine to proline. Analysis of microarray data revealed 392 genes whose expression was dependent on the nitrogen source quality. 91 genes were activated in a GATA factor-dependent manner in all growth conditions, suggesting a direct role of Gln3 and Gat1 in their expression. Other genes were only transiently up-regulated (stress-responsive genes) or down-regulated (genes encoding ribosomal proteins and translational factors) upon nitrogen limitation, and this regulation was delayed in a gln3,,gat1, strain. Repression of amino acid and nucleotide biosynthetic genes after a nitrogen shift did not depend on Gcn4. Several transporter genes were repressed as a consequence of enhanced levels of NCR-responsive permeases present at the plasma membrane. [source] Comparative analysis of the self-incompatibility (S -) locus region of Prunus mume: identification of a pollen-expressed F-box gene with allelic diversityGENES TO CELLS, Issue 3 2003Tetsuyuki Entani Background: Self-incompatibility (SI) in the Solanaceae, Rosaceae and Scrophulariaceae is gametophytically controlled by a single polymorphic locus, termed the S -locus. To date, the only known S -locus product is a polymorphic ribonuclease, termed S -RNase, which is secreted by stylar tissue and thought to act as a cytotoxin that degrades the RNA of incompatible pollen tubes. However, understanding how S -RNase causes S -haplotype specific inhibition of pollen tubes has been hampered by the lack of a cloned pollen S -determinant gene. Results: To identify the pollen S -determinant gene, we investigated the genomic structure of the S -locus region of the S1 - and S7 -haplotypes of Prunus mume (Japanese apricot), and identified 13 genes around the S-RNase gene. Among them, only one F-box gene, termed SLF (S -locus F-box), fulfilled the conditions for a pollen S -determinant gene: (i) together with the S-RNase gene, it is located within the highly divergent genomic region of the S -locus, (ii) it exhibits S -haplotype specific diversity among three analysed S -haplotypes, and (iii) it is specifically expressed in pollen, but not in the styles or leaves. Conclusion: The results indicate that SLF is a prime candidate for the pollen S -determinant gene of SI. [source] RGS9-2 mediates specific inhibition of agonist-induced internalization of D2 -dopamine receptorsJOURNAL OF NEUROCHEMISTRY, Issue 3 2010Jeremy Celver J. Neurochem. (2010) 114, 739,749. Abstract Regulator of G protein signaling 9-2 (RGS9-2), a member of the RGS family of GTPase accelerating proteins, is expressed specifically in the striatum, a brain region involved in controlling movement, motivation, mood and addiction. RGS9-2 can be found co-localized with D2 -class dopamine receptors in medium spiny striatal neurons and altered functioning of both RGS9-2 and D2 -like dopamine receptors have been implicated in schizophrenia, movement disorders and reward responses. Previously we showed that RGS9-2 can specifically co-localize with D2 -dopamine receptors (D2R). Here we provide further evidence of the specificity of RGS9-2 for regulating D2R cellular functions: the expression of RGS9-2 inhibits dopamine-mediated cellular internalization of D2R, while the expression of another RGS protein, RGS4, had no effect. In addition, the agonist-mediated internalization of the G protein coupled delta opioid receptor was unaffected by RGS9-2 expression. We utilized mutant constructs of RGS9-2 to show that the RGS9-2 DEP (for Disheveled, EGL-10, Pleckstrin homology) domain and the GTPase accelerating activity of RGS9-2 were necessary for mediating specific inhibition of D2R internalization. [source] Acute Ethanol Inhibits Extracellular Signal,Regulated Kinase, Protein Kinase B, and Adenosine 3,:5,-Cyclic Monophosphate Response Element Binding Protein Activity in an Age- and Brain Region,Specific MannerALCOHOLISM, Issue 4 2005L Judson Chandler Background: As little as a single episode of exposure of the developing brain to ethanol can result in developmental neuropathology and mental retardation. Extracellular signal,regulated kinases (ERKs), protein kinase B (PKB), and adenosine 3,:5,-cyclic monophosphate response element binding protein (CREB) are messenger molecules that play important roles in neuronal plasticity and survival. This study was undertaken to examine the effects of acute ethanol on ERK, PKB, and CREB activation in the brain. Methods: Immunoblot analysis was used to determine the effects of a 1-hr exposure of ethanol on levels of phospho-ERC in primary cortical cultures and in the cerebral cortex, hippocampus, and cerebellum of postnatal day 5 (PN5), postnatal day 21 (PN21), and adult rats. Results: In cortical cultures, ethanol (100 mM) significantly reduced activity-dependent activation of phospho-ERK, phospho-PKB, and phospho-CREB by approximately 50%. In PN5 rats, ethanol (3.5 g/kg) inhibited both phospho-ERK and phospho-PKB in the cerebral cortex and hippocampus but was without effect in the cerebellum. A similar brain region,specific inhibition of phospho-ERK was observed in PN21 rats, whereas in adult rats, ethanol inhibited phospho-ERK in all three brain regions. In contrast, ethanol had no effect on phospho-PKB in either PN21 or adult rats. Without exception, ethanol inhibited phospho-CREB in an identical brain region, and age-dependent manner as was observed for phospho-ERK. Finally, administration of the NMDA antagonist MK-801 (0.5 mg/kg) to PN5 rats had no effect on phospho-ERK or phospho-PKB levels in any brain region. Conclusion: The results demonstrate that acute ethanol inhibits ERK/PKB/CREB signaling in brain. This inhibition occurs in an age- and brain region,specific manner, with inhibition of PKB restricted to a time during the brain growth-spurt period. Furthermore, the lack of effect of MK-801 suggests that inhibition of NMDA receptors is unlikely to play a major role in binge ethanol inhibition of ERK/PKB/CREB signaling in vivo. [source] Nitrogen and phosphorus availability limit N2 fixation in beanNEW PHYTOLOGIST, Issue 2 2000E. O. LEIDI Availability of nitrogen (N) and phosphorus (P) might significantly affect N2 fixation in legumes. The interaction of N and P was studied in common bean (Phaseolus vulgaris), considering their effects on nodulation and N2 fixation, nitrate reductase activity, and the composition of N compounds in xylem sap. The effect of N on the uptake of P by plants was estimated by analysing rhizospheric pH and P concentration in xylem sap and in plant shoots. Inoculated bean plants were grown in pots containing perlite/vermiculite in two experiments with different amounts of P and N. In a third experiment, bean plants were grown on two soil types or on river sand supplied with different concentrations of N. At harvest, shoot growth, number of nodules and mass, and nitrogenase activity were determined. Xylem sap was collected for the determination of ureides, amino acids, nitrate and phosphate concentration. At low nitrate concentration (1 mM), increasing amounts of P promoted both nodule formation and N2 fixation, measured as ureide content in the xylem sap. However, at high nitrate concentration (10 mM), nodulation and N2 fixation did not improve with increased P supply. Glutamine and aspartate were the main organic N compounds transported in the xylem sap of plants grown in low nitrate, whereas asparagine was the dominant N compound in xylem sap from plants grown in high nitrate. Nitrate reductase activity in roots was higher than in shoots of plants grown with low P and high N. In both soils and in the sand experiment, increased application of N decreased nodule mass and number, nitrogenase activity and xylem ureides but increased the concentration of asparagine in xylem sap. Increasing P nutrition improved symbiotic N2 fixation in bean only at low N concentrations. It did not alleviate the inhibitory effect of high nitrate concentration on N2 fixation. A decrease in plant P uptake was observed, as indicated by a lower concentration of P in the xylem sap and shoots, correlating with the amount of N supplied. Simultaneously with the specific inhibition of N2 fixation, high nitrate concentrations might decrease P availability, thus inhibiting even further the symbiotic association because of the high P requirement for nodulation and N2 fixation. [source] The three-dimensional structure of MAP kinase p38,: different features of the ATP-binding site in p38, compared with p38,ACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Sangita B. Patel The p38 mitogen-activated protein kinases are activated in response to environmental stress and cytokines and play a significant role in transcriptional regulation and inflammatory responses. Of the four p38 isoforms known to date, two (p38, and p38,) have been identified as targets for cytokine-suppressive anti-inflammatory drugs. Recently, it was reported that specific inhibition of the p38, isoform is necessary and sufficient for anti-inflammatory efficacy in vivo, while further inhibition of p38, may not provide any additional benefit. In order to aid the development of p38,-selective compounds, the three-dimensional structure of p38, was determined. To do so, the C162S and C119S,C162S mutants of human MAP kinase p38, were cloned, expressed in Escherichia coli and purified. Initial screening hits in crystallization trials in the presence of an inhibitor led upon optimization to crystals that diffracted to 2.05,Å resolution and allowed structure determination (PDB codes 3gc8 and 3gc9 for the single and double mutant, respectively). The structure of the p38, C162S mutant in complex with the same inhibitor is also reported (PDB code 3gc7). A comparison between the structures of the two kinases showed that they are highly similar overall but that there are differences in the relative orientation of the N- and C-terminal domains that causes a reduction in the size of the ATP-binding pocket in p38,. This difference in size between the two pockets could be exploited in order to achieve selectivity. [source] Aurora A selective inhibitor MLN8237 suppresses the growth and survival of HTLV-1-infected T-cells in vitroCANCER SCIENCE, Issue 5 2010Mariko Tomita Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle. We have shown previously that Aurora A expression is increased aberrantly in human T-cell leukemia virus type 1 (HTLV-1)-infected T-cell lines and primary adult T-cell leukemia cells, and a pan-Aurora kinase inhibitor, which inhibits both Aurora A and Aurora B kinases, reduces viability and induces apoptosis in these cells. However, the specific effects of Aurora A inhibition on HTLV-1-infected T-cells are poorly understood. In this study, we addressed this question by comparing the effects of MLN8237, a selective inhibitor of Aurora A, on cell viability, cell cycle progression, and induction of apoptosis in HTLV-1-infected and -uninfected T-cell lines. MLN8237 reduced the viability of HTLV-1-infected T-cell lines within 24 h, but its effects on that of HTLV-1-uninfected T-cell lines were moderate. MLN8237 induced early apoptosis of HTLV-1-infected T-cell lines without induction of polyploidy. It induced p53 and p21 expression in HTLV-1-infected but not in -uninfected T-cell lines, suggesting that MLN8237-treated HTLV-1-infected T-cell lines exit from mitosis and activate a p53-dependent postmitotic G1 checkpoint, leading to G1 arrest followed by the induction of apoptosis. Our results suggest that specific inhibition of Aurora A kinase is a potentially useful therapeutic strategy in the treatment of adult T-cell leukemia and that further in vivo exploration is warranted. (Cancer Sci 2010; 101: 1204,1211) [source] Sonic hedgehog derived from human pancreatic cancer cells augments angiogenic function of endothelial progenitor cellsCANCER SCIENCE, Issue 6 2008Madoka Yamazaki Hedgehog signaling is important in the pathogenesis of pancreatic cancer. Several recent observations suggest the involvement of sonic hedgehog (SHH) in postnatal neovascularization. We identified a novel role for SHH in tumor-associated angiogenesis in pancreatic cancer. Immunohistochemical analysis revealed that patched homolog 1 (PTCH1), both a receptor for and transcriptional target of hedgehog signaling, was expressed in a small fraction of endothelial cells within pancreatic cancer, but not in normal pancreatic tissue. When endothelial progenitor cells (EPC) isolated from human peripheral blood were cultured with supernatant from SHH-transfected 293 cells or pancreatic cancer cells, mRNA levels of vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 and angiopoietin-1 were significantly increased, whereas no such induction was observed in human umbilical vein endothelial cell (HUVEC) and human dermal microvascular endothelial cell (HMVEC). HUVEC tube formation was stimulated when cocultured with EPC, and preconditioning EPC with supernatant from KP-1 N pancreatic cancer cells highly expressing SHH significantly enhanced the effect. The effect was partially attenuated by specific inhibition of SHH with cyclopamine or a neutralizing antibody. These findings suggest that tumor-derived SHH can induce angiogenesis, and this is mediated by its effects on EPC specifically. Targeting SHH would be a novel therapeutic approach that can inhibit not only proliferation of cancer cells but also EPC-mediated angiogenesis. (Cancer Sci 2008; 99: 1131,1138) [source] Development and Biological Evaluation of a Novel Aurora A Kinase InhibitorCHEMBIOCHEM, Issue 3 2009Teresa Sardon Dr. Abstract Stop dividing: In the quest for antitumorigenic compounds, aurora A kinase has recently emerged as a potential drug target. In this paper three novel aurora inhibitors (shown in the illustration) have been tested for their biological activity in cultured cells. One of them (TC-28) appears to be a promising specific aurora A inhibitor in vivo. The aurora kinase family groups several serine/threonine kinases with key regulatory functions during cell division. The three mammalian members, aurora A, B and C, are frequently over-expressed in human tumors and the aurora A gene is located in a genomic region frequently amplified in breast and colon cancer. All these data have fuelled the idea that aurora kinases are promising targets for anticancer therapy. Indeed some inhibitory compounds are currently being evaluated in clinical trials. However, it was recently shown that mutations in the targeted kinase can confer resistance to a broad range of inhibitors and render patients resistant to treatments. Moreover, aurora A over-expression results in increased resistance to antimitotic agents. The development of new compounds targeting aurora A is therefore highly relevant. We describe here the synthesis of three novel aurora kinase inhibitors, TC-28, TC-34 and TC-107. We report their properties as aurora inhibitors in vitro and their effect on human tissue culture cell lines. Interestingly, our results show that TC-28 has properties compatible with the specific inhibition of aurora A, in vivo. [source] Differential effect of cholera toxin on CD45RA+ and CD45RO+ T cells: specific inhibition of cytokine production but not proliferation of human naive T cellsCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 2 2000K. Eriksson We have studied how cholera toxin (CT) and its non-toxic cell-binding B-subunit (CTB) affect the activation of pure human T cells in an anti-CD3-driven system. CT, as opposed to CTB, strongly suppressed the proliferative responses as well as cytokine production in CD4+ and CD8+ T cells. CT however, had a differential effect on naive and activated/memory T cell subsets. Costimulation through exogenous IL-2 or through CD28 cross-linking rescued the proliferation of CT-treated naive CD45RA+ T cells, but not of activated/memory CD45RO+ cells. IL-2 production and IL-2 receptor expression were markedly reduced by CT in all T cell fractions, i.e. also in CD45RA+ cells which had maintained proliferative responses. However, the proliferative responses of CT-treated CD45RA+ T cells were IL-2-dependent, as shown by blocking experiments using anti-IL-2 antibodies. These results indicate (i) that CTB has no cytostatic effect on human T cells, (ii) that CT affects proliferation and cytokine production by two different signal pathways, and (iii) that CT might interact with a signal pathway generated through or influenced by CD45. [source] | ||||||||||||||||