748,439 research outputs found

    Novel 5-oxo-hexahydroquinoline derivatives: design, synthesis, in vitro P-glycoprotein-mediated multidrug resistance reversal profile and molecular dynamics simulation study

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    Overexpression of the efflux pump P-glycoprotein (P-gp) is one of the important mechanisms of multidrug resistance (MDR) in many tumor cells. In this study, 26 novel 5-oxo-hexahydroquinoline derivatives containing different nitrophenyl moieties at C-4 and various carboxamide substituents at C-3 were designed, synthesized and evaluated for their ability to inhibit P-gp by measuring the amount of rhodamine 123 (Rh123) accumulation in uterine sarcoma cells that overexpress P-gp (MES-SA/Dx5) using flow cytometry. The effect of compounds with highest MDR reversal activities was further evaluated by measuring the alterations of MES-SA/Dx5 cells' sensitivity to doxorubicin (DXR) using MTT assay. The results of both biological assays indicated that compounds bearing 2-nitrophenyl at C-4 position and compounds with 4-chlorophenyl carboxamide at C-3 demonstrated the highest activities in resistant cells, while they were devoid of any effect in parental nonresistant MES-SA cells. One of the active derivatives, 5c, significantly increased intracellular Rh123 at 100 mu M, and it also significantly reduced the IC50 of DXR by 70.1% and 88.7% at 10 and 25 mu M, respectively, in MES-SA/Dx5 cells. The toxicity of synthesized compounds against HEK293 as a noncancer cell line was also investigated. All tested derivatives except for 2c compound showed no cytotoxicity. A molecular dynamics simulation study was also performed to investigate the possible binding site of 5c in complex with human P-gp, which showed that this compound formed 11 average H-bonds with Ser909, Thr911, Arg547, Arg543 and Ser474 residues of P-gp. A good agreement was found between the results of the computational and experimental studies. The findings of this study show that some 5-oxo-hexahydroquinoline derivatives could serve as promising candidates for the discovery of new agents for P-gp-mediated MDR reversal

    Biological effects of cigarette smoke in cultured human retinal pigment epithelial cells.

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    The goal of the present study was to determine whether treatment with cigarette smoke extract (CSE) induces cell loss, cellular senescence, and extracellular matrix (ECM) synthesis in primary human retinal pigment epithelial (RPE) cells. Primary cultured human RPE cells were exposed to 2, 4, 8, and 12% of CSE concentration for 24 hours. Cell loss was detected by cell viability assay. Lipid peroxidation was assessed by loss of cis-parinaric acid (PNA) fluorescence. Senescence-associated ß-galactosidase (SA-ß-Gal) activity was detected by histochemical staining. Expression of apolipoprotein J (Apo J), connective tissue growth factor (CTGF), fibronectin, and laminin were examined by real-time PCR, western blot, or ELISA experiments. The results showed that exposure of cells to 12% of CSE concentration induced cell death, while treatment of cells with 2, 4, and 8% CSE increased lipid peroxidation. Exposure to 8% of CSE markedly increased the number of SA-ß-Gal positive cells to up to 82%, and the mRNA expression of Apo J, CTGF, and fibronectin by approximately 3-4 fold. Treatment with 8% of CSE also increased the protein expression of Apo J and CTGF and the secretion of fibronectin and laminin. Thus, treatment with CSE can induce cell loss, senescent changes, and ECM synthesis in primary human RPE cells. It may be speculated that cigarette smoke could be involved in cellular events in RPE cells as seen in age-related macular degeneration

    Monitoring the South African National Antiretroviral Treatment Programme, 2003-2007: the IeDEA Southern Africa collaboration.

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    OBJECTIVES: To introduce the combined South African cohorts of the International epidemiologic Databases to Evaluate AIDS Southern Africa (IeDEA-SA) collaboration as reflecting the South African national antiretroviral treatment (ART) programme; to characterise patients accessing these services; and to describe changes in services and patients from 2003 to 2007. DESIGN AND SETTING: Multi-cohort study of 11 ART programmes in Gauteng, Western Cape, Free State and KwaZulu-Natal. SUBJECTS: Adults and children (<16 years old) who initiated ART with > or =3 antiretroviral drugs before 2008. RESULTS: Most sites were offering free treatment to adults and children in the public sector, ranging from 264 to 17,835 patients per site. Among 45,383 adults and 6,198 children combined, median age (interquartile range) was 35.0 years (29.8-41.4) and 42.5 months (14.7-82.5), respectively. Of adults, 68% were female. The median CD4 cell count was 102 cells/microl (44-164) and was lower among males than females (86, 34-150 v. 110, 50-169, p<0.001). Median CD4% among children was 12% (7-17.7). Between 2003 and 2007, enrolment increased 11-fold in adults and 3-fold in children. Median CD4 count at enrolment increased for all adults (67-111 cells/microl, p<0.001) and for those in stage IV (39-89 cells/microl, p<0.001). Among children <5 years, baseline CD4% increased over time (11.5-16.0%, p<0.001). CONCLUSIONS: IeDEA-SA provides a unique opportunity to report on the national ART programme. The study describes dramatically increased enrolment over time. Late diagnosis and ART initiation, especially of men and children, need attention. Investment in sentinel sites will ensure good individual-level data while freeing most sites to continue with simplified reporting

    Receptor Interacting Protein 3 is Required for Arsenite-mediated Necroptosis

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    Arsenic compounds such as sodium arsenite (SA) and arsenic trioxide (ATO) are toxic to human. Primarily, we pursued to outline the cell death modes caused by arsenic compounds and to address what proteins would be responsible for arsenite-induced cytotoxicity. Both SA and ATO substantially exhibited cytotoxic activity in L929 cells. Necrostatin-1 (Nec-1) treatment significantly protected cell death mediated by arsenic compounds, suggesting that cells are committed to die in a programmed necrotic way. A geldanamycin analog DMAG destabilized receptor interacting protein 3 (RIP3) and concomitantly protected cells from SA toxicity. Using interfering RNAs, we eventually found that RIP3 was responsible for its antagonizing effects on SA. Therefore, it is proposed that arsenic compounds execute necroptotic cell death of L929 via a RIP3 dependent pathway

    Effect of Aspirin and Salicylic Acid on LPA Induced Differentiation of P19 Stem Cells into Cardiomyocytes

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    The use of stem cell-based therapy in conjunction with existing medical interventions, to target complications caused by coronary artery disease (CAD) is not fully examined. In parallel, the role of lysophosphatidic acid (LPA); an important endogenous bioactive phospholipid, has shown cardioprotective characteristics at low physiological concentrations, providing a potential for future treatment plans. In addition, studies have indicated the promise of aspirin (ASA)/ salicylic acid (SA) or LPA to induce and promote cardiac differentiation of SCs in various models. Therefore, in this project, we investigated the effects of ASA/SA in the presence or absence of LPA to induce the differentiation of the murine P19 teratocarcinoma stem cell line into cardiomyocytes. Routine cell culture was undertaken using P19 stem cells cultured in complete α-minimal essential medium (α-MEM). In the first instance, the protocol was optimised to ensure that efficient and reproducible differentiation was achieved. Embryoid bodies (EB) were formed by seeding cells and left to aggregate over a period of 2 days in ultra-low attachment 96-well plates, to establish differentiation. P19 stem cells were pre-incubated for 1 hour with ASA and SA at varying concentrations (0.1mM, 0.3mM, 1mM and 3mM) and selective NFκB inhibitor (0.1nM CAY10470) were pre-incubated 1 hour prior to adding LPA (5µM). Control cells were cultured in complete α-MEM alone. 6-8 EBs were isolated and seeded into 12-well tissue culture plates and cultured for 6 days. Western blotting was used to confirm differentiation, examining for the expression of ventricular myosin light chain (MLC-1v), relative to β-actin. To determine the potential mechanism through which differentiation may be induced, changes in phosphorylation of activated NFκB and IκB were determined. Optimisation of the differentiation protocol revealed that 1 x 104 cells grown for 2 days, produced consistent EBs sizes which ranged between 350-450µm in diameter. These EBs efficiently differentiated into cardiomyocytes. Differentiation was consistently achieved using LPA (5µM) and at selected concentrations of ASA (0.3 -1mM, at day 3) and SA (1mM, at day 3). Maximal expression of MLC-1v in ASA/SA conditions was seen at 1mM. However, LPA induced differentiation was inhibited by both in combination treatment with ASA and SA, despite both inducing differentiation independently. Analysis of phosphorylated and native proteins associated with the NFκB complex was successfully detected. These initial studies indicated substantial expression of phospho NFκB in LPA, SA and ASA treated cells and increases were seen at the 6-9-hour time points. The expression of phospho IκB in LPA treated cells peaked at 10-15 mins, while ASA/SA treated cells showed phospho IκB peaking at a later time point (3 hours). In conclusion, the experiments conducted in this thesis have shown that both ASA/SA and LPA induced cardiomyocyte differentiation. However, when ASA or SA are used in combination with LPA, an antagonistic effect is seen, preventing LPA to induce differentiation

    Histone deacetylase inhibitor vorinostat suppresses the growth of uterine sarcomas in vitro and in vivo

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    <p>Abstract</p> <p>Background</p> <p>Uterine sarcomas are very rare malignancies with no approved chemotherapy protocols. Histone deacetylase (HDAC) inhibitors belong to the most promising groups of compounds for molecular targeting therapy. Here, we described the antitumor effects of suberoylanilide hydroxamic acid (SAHA; vorinostat) on MES-SA uterine sarcoma cells <it>in vitro </it>and <it>in vivo</it>. We investigated effects of vorinostat on growth and colony forming ability by using uterine sarcoma MES-SA cells. We analyzed the influence of vorinostat on expression of different HDACs, p21<sup>WAF1 </sup>and activation of apoptosis. Finally, we examined the antitumor effects of vorinostat on uterine sarcoma <it>in vivo</it>.</p> <p>Results</p> <p>Vorinostat efficiently suppressed MES-SA cell growth at a low dosage (3 μM) already after 24 hours treatment. Decrease of cell survival was even more pronounced after prolonged treatment and reached 9% and 2% after 48 and 72 hours of treatment, respectively. Colony forming capability of MES-SA cells treated with 3 μM vorinostat for 24 and 48 hours was significantly diminished and blocked after 72 hours. HDACs class I (HDAC2 and 3) as well as class II (HDAC7) were preferentially affected by this treatment. Vorinostat significantly increased p21<sup>WAF1 </sup>expression and apoptosis. Nude mice injected with 5 × 10<sup>6 </sup>MES-SA cells were treated for 21 days with vorinostat (50 mg/kg/day) and, in comparison to placebo group, a tumor growth reduction of more than 50% was observed. Results obtained by light- and electron-microscopy suggested pronounced activation of apoptosis in tumors isolated from vorinostat-treated mice.</p> <p>Conclusions</p> <p>Our data strongly indicate the high therapeutic potential of vorinostat in uterine sarcomas.</p

    Ανθεκτικότητα βιο-υµενικών κυττάρων Salmonella Typhimurium και Staphylococcus aureus υπό συνθήκες µονο- και µεικτής καλλιέργειας σε υποθανάτιες συγκεντρώσεις χλωριούχου βενζαλκονίου , υπεροξικού οξέος και υποχλωριώδους νατρίου

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    Στην παρούσα µελέτη µελετήθηκε η ανθεκτικότητα βιο-υµενικών κυττάρων Salmonella Typhimurium (ST) και Staphylococcus aureus (SA) (3 στελέχη ανά είδος), όταν αυτά αφέθηκαν να σχηµατίσουν βιο-υµένια είτε σε συνθήκες µόνο ή µεικτής καλλιέργειας σε επιφάνεια ανοξείδωτου χάλυβα (SS), σε τρία διαφορετικά απολυµαντικά (χλωριούχο βενζαλκόνιο BC, υπεροξικό οξύ ΡΑ και υποχλωριώδες νατρίου SH). Τα αποτελέσµατα της µελέτης έδειξαν ότι οι συνθήκες µεικτής καλλιέργειας οδήγησαν σε µείωση του πληθυσµού των βιο-υµενικών κυττάρων (0.6 και 1.1 log cfu/cm2 για ST και SA, αντίστοιχα), σε σύγκριση µε τις συνθήκες µονο-καλλιέργειας. Φάνηκε ότι το BC ήταν το πιο αποτελεσµατικό απολυµαντικό στις δύο περιπτώσεις βιο-υµενικών κυττάρων. Το PA ήταν πιο αποτελεσµατικό στην απολύµανση ST βιο-υµενικών κυττάρων και στις δύο συνθήκες καλλιέργειας. Ιn this study, the resistance of sessile cells of Salmonella Typhimurium (ST) and Staphylococcus aureus (SA) (3 strains per species), when these were left to form biofilms under either monoor dual-species conditions on a stainless steel surface, to 3 different disinfectants (benzalkonium chloride BC, paracetic acid PA and sodium hypochlorite SH) was investigated. Results showed that dual-species conditions seem to lead to a reduction in the number of sessile cells (0.6 and 1.1 log cfu/cm2 for ST and SA, respectively), compared to mono-species conditions. Regarding the disinfection resistance, in general BC was found to be more effective in both mono-and dual-species biofilm communities. PA was more effective against ST biofilm cells under both conditions

    Decidual natural killer cell interactions with trophoblasts are impaired in pregnancies at increased risk of preeclampsia.

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    Transformation of the uterine spiral arteries (SAs) during pregnancy is critical to support the developing fetus, and is impaired in some pregnancy disorders, including preeclampsia. Decidual natural killer (dNK) cells play a role in SA remodeling, although their interactions with fetal trophoblast remain unclear. A uterine artery Doppler resistance index (RI) in the first trimester of pregnancy can be used as a proxy measure of the extent of SA remodeling; we have used this technique to characterize dNK cells from pregnancies with normal (normal RI) and impaired (high RI) SA remodeling, which display least and highest risk of developing preeclampsia, respectively. We examined the impact of dNK cell secreted factors on trophoblast motility, chemoattraction, and signaling pathways to determine the contribution of dNK cells to SA transformation. We demonstrated that the chemoattraction of the trophoblast by dNK cells is impaired in pregnancies with high RI, as is the ability to induce trophoblast outgrowth from placental villous explants. These processes are dependent on activation of the extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase-Akt signaling pathways, which were altered in trophoblasts incubated with secreted factors from dNK cells from high RI pregnancies. Therefore, by characterizing pregnancies using uterine artery Doppler RI before dNK cell isolation, we have identified that impaired dNK-trophoblast interactions may lead to poor placentation. These findings have implications for pregnancy pathological conditions, such as preeclampsia

    Profiling Cell Surface Sialylation and Desialylation Dynamics of Immune Cells

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    Sialic acids (SAs) are a diverse family of naturally occurring 2-keto-3-deoxy-nononic acids that are involved in a wide range of biological processes, including early fetal development, cellular recognition, and utilization by microbes. While it is clear that cell surface SAs are highly involved in the immune system, the sialylation status of individual immune cells and functions are still unknown. In this study, I combined the newly developed LC-MS/MS methods with flow cytometry and confocal microscopy to systematically study the sialylation and desialylation dynamics of macrophages at different conditions. First, I developed an accurate LC-MS/MS method to quantify free SA in human plasma with isotope-labeled standard calibration and 3,4-diaminotoluene derivatization. This method is capable to distinguish SA analogus in complex biological samples, which paves the path for dynamic SAs research. Menwhile, another LC-MS/MS method with direct SAs quantification was developed for high throughtput analysis. This method does not require complicated sample preparation and can quantify SA at 2 ng/mL. Next, I performed globally profiling of sialylation status of Raw 264.7 macrophages by flow cytometry, confocal microscopy, and LC-MS/MS. Both flow cytometry and confocal microscopy showed the predominat of a-2,3 linked SAs on the cell surface, and increase of a-2,6 linked SAs after atorvastatin treatment. Moreover, LC-MS/MS showed total SA increased 3 times upon treatment. Further experiment indicated the correlation of a-2,6 linked SAs with cell apoptosis. Finally, I systematically examined the sialylation and desialylation profiles of THP-1 monocytes after differentiation and polarization. Both a-2,3 and a-2,6 linked SAs on the cell surface were decreased during diffrentiation, which was in accordance with the increased free SA in the medium and elevated activity of NEU1 sialidase. Meanwhile, the increase of SA expression during differentiation was evidenced by siaoglycoconjugates inside the cells and total SA in the cell lysate. Overall, the combined approach has bee successfully applied to profile SAs in the cell culture system. LC-MS/MS can accurately quantify SA in a high throughtput fashion. The SA linkages can be distinguished by flow cytometry and confocal microscopy with specific lectin labelings. The SA levels and linkages provide markers of cells at different status
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