Half-Sandwich Ir(III) and Os(II) Complexes of Pyridyl-Mesoionic Carbenes as Potential Anticancer Agents

A series of cationic chlorido arene-iridium(III) and arene-osmium(II) complexes with bidentate pyridyl functionalized mesoionic carbenes (MIC) of the 1,2,3-triazol-5-ylidene type have been prepared. The variations in the ligand structures include the position of the pyridyl substituent relative to the triazolylidene ring (N-wingtip vs C-wingtip), phenyl versus ethyl substituents, and incorporation of several functional groups at the phenyl substituents. Five complexes have been characterized by X-ray structural analysis. All complexes, including osmium(II) and ruthenium(II) analogues having a pyrimidyl in place of the pyridyl group, have been studied for their cytotoxic activity on a human cervical carcinoma HeLa cell line. Two of the compounds, Ir5 and Ir9, were the most cytotoxic with IC50 values of 7.33 μM and 2.01 μM, respectively. Examination of their cytotoxic effect on different cell lines revealed that they preferentially kill cancer over normal cells. The Ir5 and Ir9 compounds arrested cells in G2 and induced a dose-dependent increase in SubG0/G1 cell population. Apoptosis, as the primary mode of cell death, was confirmed by Annexin V/PI staining, detection of cleaved PARP, and caspases 3 and 7 activity upon treatment of HeLa cells with both compounds. The higher toxicity of Ir9 is probably due to its increased accumulation in the cells compared to Ir5. The role of glutathione (GSH) in the protection of cells against Ir5 and Ir9 cytotoxicity was confirmed by pretreatment of cells either with buthionine sulfoximine (inhibitor of GSH synthesis) or N-acetyl-cysteine (precursor in GSH synthesis)

Flavonoidi kao inhibitori Lck i Fyn kinaza

Phosphorylation of tyrosine residues constitutes a unique signaling pathway involved in regulation of most cellular processes responding to different extracellular stimuli. The enzymes that carry out this modification are tyrosine kinases. These enzymes enable the transfer of γ-phosphate from ATP to the phenol –OH group of tyrosine on protein substrates. Development of specific and potent protein kinase inhibitors is important not only for treatment of diseases, but also as a tool to investigate the physiological roles of protein kinases. Flavonoids are biologically active polyphenol compounds naturally occurring in many plants. They are recognized as inhibitors of Fyn and Lck protein kinases, two representatives of the Src family of non receptor kinases involved in T-cell signaling transport. In the described experiments, the inhibitory activity of flavonoids on Fyn and Lck kinases was monitored by the ELISA method. Myricetin showed the highest inhibitory effect, and no ATP-competitive mechanism of inhibition was observed on Fyn tyrosine kinase. The affinity of human Fyn and Lck for two different substrates, polypeptide polymer Poly Glu:Tyr (4:1) and peptide M3-01, was also tested.Tirozinska fosforilacija predstavlja jedinstveni mehanizam prijenosa signala primljenoga iz okoline do stanič ne jezgre koja će odgovoriti na podražaj. Velika skupina enzima odgovorna za provo|enje ove modifikacije poznata je pod jedinstvenim nazivom tirozinske kinaze. One omogućuju prijenos -fosfata ATP-a na –OH skupinu tirozina supstrata. Lck i Fyn su dvije nereceptorske Src tirozinske kinaze, koje su važne komponente regulacije aktivnosti T-limfocita. Rezultati ispitivanja flavonoida, biološki aktivnih polifenolnih spojeva prirodno prisutnih u mnogim biljkama, upućuju na njihov inhibitorni učinak na proteinske kinaze Fyn i Lck. Najveću inhibiciju pokazao je miricetin, a pri ispitivanju mehanizma inhibicije Fyn kinaze nije ustanovljena kompeticija s ATP-om. Ispitivanje potencijalne inhibicijske aktivnosti flavonoidnih spojeva prema kinazama Fyn i Lck, provedeno je ELISA metodom. Pri tome je uspore|en afinitet ispitivanih enzima prema supstratima s različitim brojem skupina podložnih fosforilaciji, odnosno prema polipeptidnome polimeru glutaminske kiseline i tirozina Poly Glu:Tyr (4:1) i M3-01 peptidu. Razvoj učinkovitih kinaznih inhibitora važan je ne samo za prevenciju i liječenje mnogih bolesti već i za bolje razumijevanje uloge proteinskih kinaza u organizmu

Isolation of MDCK cells with low expression of mdr1 gene and their use inmembrane permeability screening

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P-glycoprotein activity. Clone isolation via the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery

Grapevine Leafroll-Associated Virus 3 Replication in Grapevine Hosts Changes through the Dormancy Stage

Grapevine leafroll-associated virus 3 (GLRaV-3) is a graft-transmissible virus present in every viticultural region of the world and poses a large threat to grapevine production. Frequent coinfections with other viruses, the large number of grapevine varieties, the complexity of processes involved in plant response to virus infection, and the lack of studies on GLRaV-3 replication limit our knowledge of GLRaV-3 damaging effects and their background. In this study, five different inocula, one containing GLRaV-3 and others containing GLRaV-3 in combination with different grapevine viruses were green grafted to 52 different grapevine plants of four varieties to analyze the influence of the phenological stage and virus composition on GLRaV-3 replication. Relative concentration analysis by quantitative PCR conducted over a 16-month period revealed that other viruses as well as plant stage had a significant effect on GLRaV-3 replication and symptoms expression. The replication was most pronounced in the deep dormancy stage at the beginning of the infection, and the least at the exit of the dormancy stage. This study brings new insight into GLRaV-3 replication and discusses about viral interactions in one of the most economically important perennial plants, the grapevine

Claudins: Beyond Tight Junctions in Human IBD and Murine Models

Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity–dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium–induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and flow cytometry. The claudin expression pattern in uninflamed and inflamed colon varied between species and murine strains. In IBD and both animal models, diverse alterations in claudin expression by epithelial and inflammatory cells were recorded. Tissue mRNA levels for each studied claudin reflected changes within cell lineage and, at the same time, mirrored the ratio between various cell types. Based on the results of the study, it can be concluded that 1) claudins are not expressed exclusively by epithelial cells, but by certain types of cells of mesodermal origin as well ; 2) changes in the claudin mRNA level should be interpreted in the context of overall tissue alterations ; and 3) both IBD animal models that were analyzed can be used for investigating claudins as a therapy target, respecting their similarities and differences highlighted in this study