100 research outputs found

    Cytological and molecular identification of haemogregarina stepanowi in blood samples of the European pond turtle (emys orbicularis) from quarantine at Belgrade zoo

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    Blood smears stained with Diff Quick are the initial tool for cytological diagnosis of Haemogregarina spp. However, the development of sensitive and specific molecular methods enabled the detection and identification of parasites in the sample and to clarify the evolutionary relationships of adeleorinid parasites within the Apicomplexa. The current study was attempted in order to perform cytological investigation and molecular identification of the hemoparasites in thirty European pond turtles (Emys orbicularis) from the quarantine section at Belgrade Zoo, which have been found in poor health condition with massive skin hemorrhages, based on intraerythrocytic parasitic forms on hematological smears and 18S rDNA sequence, respectively. Different life cycle stages of the Haemogregarina sp. were noticed within the erythrocytes in the peripheral blood. Biochemical analysis indicated lower values of AST and iron in most of the infected turtles while hematological analysis showed a changed hematocrit value, a decrease in the number of red blood cells and low hemoglobin levels. Amplifications of the 18S rDNA sequence of Haemogregarina were detected in 30/30 (100%) turtles with clinical symptoms. The identity of PCR products was confirmed by direct DNA sequencing. Future research concerning H. stepanowi in Serbia should be applied to its definitive host-the leech

    ZD6474 reverses multidrug resistance by directly inhibiting the function of P-glycoprotein

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    P-glycoprotein (P-gp) pumps multiple types of drugs out of the cell, using energy generated from ATP, and confers multidrug resistance (MDR) on cancer cells. ZD6474 is an orally active, selective inhibitor of the vascular endothelial growth factor receptor, epidermal growth factor receptor, and rearranged during transfection tyrosine kinases. This study was designed to examine whether ZD6474 reverses P-gp-mediated MDR in cancer cells. Here, we show that clinically achievable levels of ZD6474 reverse P-gp-mediated MDR of the P-gp-overexpressing cell lines derived from breast cancer, MCF-7/adriamycin (ADR), and human oral epidermoid carcinoma, KBV200 to ADR, docetaxel, and vinorelbine. This ability to reverse the P-gp-mediated resistance is comparable to that of another frequently used reversal agent known as verapamil. ZD6474 itself moderately inhibits the proliferation of both MCF-7 and MCF-7/ADR cells with almost equal activity, but its inhibitory effect is not altered by co-incubation with verapamil, suggesting that ZD6474 may not be a substrate of P-gp. In addition, ZD6474 increases the intracellular accumulation of the P-gp substrate, rhodamine-123, and ADR, by enhancing the uptake and/or decreasing the efflux of these compounds in resistant cells. Further studies show that ZD6474 stimulates ATPase activity in a dose-dependent manner, which is required for the proper function of P-gp. In contrast, ZD6474 does not inhibit the expression level of P-gp. Our results suggest that ZD6474 is capable of reversing MDR in cancer cells by directly inhibiting the function of P-gp, a finding that may have clinical implications for ZD6474

    Multicentre phase II studies evaluating imatinib plus hydroxyurea in patients with progressive glioblastoma

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    Contains fulltext : 79699.pdf (publisher's version ) (Closed access)BACKGROUND: We evaluated the efficacy of imatinib mesylate in addition to hydroxyurea in patients with recurrent glioblastoma (GBM) who were either on or not on enzyme-inducing anti-epileptic drugs (EIAEDs). METHODS: A total of 231 patients with GBM at first recurrence from 21 institutions in 10 countries were enrolled. All patients received 500 mg of hydroxyurea twice a day. Imatinib was administered at 600 mg per day for patients not on EIAEDs and at 500 mg twice a day if on EIAEDs. The primary end point was radiographic response rate and secondary end points were safety, progression-free survival at 6 months (PFS-6), and overall survival (OS). RESULTS: The radiographic response rate after centralised review was 3.4%. Progression-free survival at 6 months and median OS were 10.6% and 26.0 weeks, respectively. Outcome did not appear to differ based on EIAED status. The most common grade 3 or greater adverse events were fatigue (7%), neutropaenia (7%), and thrombocytopaenia (7%). CONCLUSIONS: Imatinib in addition to hydroxyurea was well tolerated among patients with recurrent GBM but did not show clinically meaningful anti-tumour activity

    Overcoming ABCG2-mediated drug resistance with imidazo-[1,2-b]-pyridazine-based Pim1 kinase inhibitors

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    Purpose Multidrug efflux pumps such as ABCG2 confer drug resistance to a number of cancer types, leading to poor prognosis and outcome. To date, the strategy of directly inhibiting multidrug efflux pumps in order to overcome drug resistance in cancer has been unsuccessful. An alternative strategy is to target proteins involved in the regulation of multidrug efflux pump activity or expression. Pim1 kinase has been demonstrated to phosphorylate ABCG2, promote its oligomerisation and contribute to its ability to confer drug resistance. Methods In the present manuscript, imidazo-pyridazine-based inhibitors of Pim1 were examined for their ability to overcome ABCG2-mediated drug resistance. Drug efficacy was measured as a cytotoxic response or an effect on transport by ABCG2. Protein expression patterns were assessed using western immuno-blotting. Results The two Pim1 inhibitors increased the potency of flavopiridol, mitoxantrone, topotecan and doxorubicin, specifically in ABCG2-expressing cells. This effect was associated with an increase in the cellular accumulation of [3H]-mitoxantrone, suggesting direct impairment of the transporter. However, prolonged pre-incubation with the studied inhibitors greatly enhanced the effect on mitoxantrone accumulation. The inhibitors caused a significant time-dependent reduction in the expression of ABCG2 in the resistant cells, an effect that would improve drug efficacy. Conclusion Consequently, it appears that the Pim1 inhibitors display a dual-mode effect on ABCG2-expressing cancer cells. This may provide a powerful new strategy in overcoming drug resistance by targeting proteins that regulate expression of efflux pumps

    Interaction with the 5D3 Monoclonal Antibody Is Regulated by Intramolecular Rearrangements but Not by Covalent Dimer Formation of the Human ABCG2 Multidrug Transporter*

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    Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S–S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S–S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S–S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S–S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein