8 research outputs found
Cyclin A/cdk2 regulates adenomatous polyposis coli-dependent mitotic spindle anchoring
No full textMutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-β-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis
Evidence of a conserved role for Chlamydia HtrA in the replication phase of the chlamydial developmental cycle
No full textIdentification of the HtrA inhibitor JO146 previously enabled us to demonstrate an essential function for HtrA during the mid-replicative phase of the Chlamydia trachomatis developmental cycle. Here we extend our investigations to other members of the Chlamydia genus. C. trachomatis isolates with distinct replicative phase growth kinetics showed significant loss of viable infectious progeny after HtrA was inhibited during the replicative phase. Mid-replicative phase addition of JO146 was also significantly detrimental to Chlamydia pecorum, Chlamydia suis and Chlamydia cavie. These data combined indicate that HtrA has a conserved critical role during the replicative phase of the chlamydial developmental cycle
Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events
No full textCyclin A/cdk2 has a role in progression through Sphase, and a large pool is also activated in G(2) phase. Here we report that this G(2) phase pool regulates the timing of progression into mitosis. Knock down of cyclin A by siRNA or addition of a specific cdk2 small molecule inhibitor delayed entry into mitosis by delaying cells in G(2) phase. The G(2) phase-delayed cells contained elevated levels of inactive cyclin B/cdk1. However, increased microtubule nucleation at the centrosomes was observed, and the centrosomes stained for markers of cyclin B/cdk1 activity. Both microtubule nucleation at the centrosomes and the phosphoprotein markers were lost with short-term treatment of the cdk1/2 inhibitor roscovitine but not the Mek1/2 inhibitor U0126. Cyclin A/cdk2 localized at the centrosomes in late G(2) phase after separation of the centrosomes but before the start of prophase. Thus G(2) phase cyclin A/cdk2 controls the timing of entry into mitosis by controlling the subsequent activation of cyclin B/cdk1, but also has an unexpected role in coordinating the activation of cyclin B/cdk1 at the centrosome and in the nucleus
Apoptosis is Induced in Chlamydia trachomatis-infected HEp-2 Cells by the Addition of a Combination Innate Immune Activation Compounds and the Inhibitor Wedelolactone
No full textProblem Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. Method of study Innate immune activation of HEp-2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. Results The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp-2 cells under these conditions. Conclusion This is the first time that induction of apoptosis has been reported in C. trachomatis-infected cells when treated with a combination of innate immune activators and wedelolactone. © 2010 John Wiley and Sons A/S
The cell surface glycoprotein CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor-mediated cell migration
Get PDFEpidermal growth factor (EGF) activation of the EGF receptor (EGFR) is an important mediator of cell migration, and aberrant signaling via this system promotes a number of malignancies including ovarian cancer. We have identified the cell surface glycoprotein CDCP1 as a key regulator of EGF/EGFR-induced cell migration. We show that signaling via EGF/EGFR induces migration of ovarian cancer Caov3 and OVCA420 cells with concomitant up-regulation of CDCP1 mRNA and protein. Consistent with a role in cell migration CDCP1 relocates from cell-cell junctions to punctate structures on filopodia after activation of EGFR. Significantly, disruption of CDCP1 either by silencing or the use of a function blocking antibody efficiently reduces EGF/EGFR-induced cell migration of Caov3 and OVCA420 cells. We also show that up-regulation of CDCP1 is inhibited by pharmacological agents blocking ERK but not Src signaling, indicating that the RAS/RAF/MEK/ERK pathway is required downstream of EGF/EGFR to induce increased expression of CDCP1. Our immunohistochemical analysis of benign, primary, and metastatic serous epithelial ovarian tumors demonstrates that CDCP1 is expressed during progression of this cancer. These data highlight a novel role for CDCP1 in EGF/ EGFR-induced cell migration and indicate that targeting of CDCP1 may be a rational approach to inhibit progression of cancers driven by EGFR signaling including those resistant to anti-EGFR drugs because of activating mutations in the RAS/ RAF/MEK/ERK pathway
Living With Paradoxes: Victims of Sexual Violence in Germany and the Conduct of Everyday Life
Get PDF[eng] The thesis describes the everyday life of victims of sexual violence in Germany, based upon the qualitative study of in-depth interviews. By means of the grounded analysis of their action system of practical coping with life on a daily basis - the Conduct of Everyday Life - five principle patterns have been detected, which characterize the victims' common practices and everyday strategies: hyperactivity, retreat, time-out, disclosure and commitment. Moreover, the fragility of their lives depends on paradoxes which reduce their freedom of choice. They represent social symptoms which influence both the individual and social visibility and invisibility of sexual violence.[spa] "Viviendo con Paradojas: Víctimas de Violencia Sexual y la Conducta de la Vida Cotidiana". La tesis describe la vida cotidiana de víctimas de violencia sexual en Alemania, basándose en un estudio cualitativo de entrevistas en profundidad. A través del análisis fundamentado de su sistema de acción para superar las dificultades cotidianas de la vida día tras día - la Conducta de la Vida Cotidiana -, se han podido detectar cinco patrones principales que suelen caracterizar los patrones comunes y estrategias cotidianos de las víctimas: hiperactividad, retiro, tiempo muerto, revelación y compromiso. Además, sus vidas parecen marcadas por paradojas que reducen sus opciones de elección y libertad y que actúan como síntomas sociales que influyen en la visibilidad e invisibilidad tanto individual como social de la violencia sexual
GREEN FLUORESCENT PROTEIN (GFP), SUATU SIGNAL PENANDA QUANTITATIF UNTUK MEMONITOR PROLIFERASI SEL
Get PDFBanyak penelitian sel kultur yang melibatkan sejumlah besar sampel untuk dianalisis, baik yang berhubungan dengan pertumbuhan sel atau toksisitas senyawa terhadap sel. Green Fluorescent Protein (GFP) adalah suatu protein yang secara alami dapat berfluorescence dan banyak digunakan pada berbagai aplikasi seperti penanda untuk ekspresi gena, produksi heterologous protein atau monitoring efisiensi transfeksi. Penelitian ini bertujuan untuk membandingkan tingkat akurasi, taraf kepercayaan dan reprodusibilitas GFP untuk memonitor pertumbuhan sel.Kurva baku dibuat dengan serial dilusi sel CHO-K1-EGFP dalam media 10% FCS di 96 well plate. Jumlah sel dalam tiap sumuran dihubungkan dengan signal fluorescence. Untuk memonitor pertumbuhan sel, signal fluoresensi dibandingkan dengan metode Trypan Blue Exclusion yang jumlah sel dalam tiap sumuran dihitung selama periode waktu tertentu (n=3). Untuk monitoring pertumbuhan sel, signal dari GFP memperlihatkan korelasi yang baik dengan jumlah sel dengan tingkat linieritas 0,9866 dalam kisaran jumlah sel 1250 – 1 x 105 sell/sumuran (standar error maksimum 11%). Metode ini terbukti memungkinkan pengukuran langsung signal fluoresensi sehingga mampu menurunkan kemungkinan kesalahan yang terjadi pada saat preparasi sel yang dapat mempengaruhi akurasi data yang diperoleh. Sekali klones permanen (stable clones) diperoleh klons ini dapat digunakan untuk banyak aplikasi.Kata Kunci: Green Fluorescent Protein (GFP), Fluorescence, proliferasi sel, Trypan Blue Exclusio
