Madin-Darby Canine Kidney Cell Line Research Articles

Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension

Adaptation of continuous cell lines to growth in suspension in a chemically defined medium has significant advantages for design and optimization in manufacturing of biologicals. In this work, changes in the protein expression level during a step-wise adaptation of an adherent Madin Darby canine kidney (MDCK) cell line to suspension growth were analyzed. Therefore, three cell line adaptations were performed independently. Two adaptations were monitored closely to characterize short term changes in protein expression levels after serum deprivation. In addition, initial stages of suspension growth were analyzed for both adaptations. The third adaptation involved MDCK suspension cells (MDCKSUS2) grown over an extended time period to achieve robust growth characteristics. Here, cells of the final stage of adaptation were compared with their parental cell line (MDCKADH). A combination of two dimensional differential gel electrophoresis for relative protein quantification and tandem mass spectrometry for protein identification enabled insights into cellular physiology. The two closely monitored cell line adaptations followed different routes regarding specific changes in protein expression but resulted in similar proteome profiles at the initial stages of suspension growth analyzed. Compared to the MDCKADH cells more than 90% of all changes in the protein expression level were identified after serum deprivation and were related to cytoskeletal structure, genetic information processing and cellular metabolism. Myosin proteins, involved in cellular detachment by actin-myosin contractile mechanisms were also differentially expressed. Interestingly, for both of the two adaptations, proteins linked for tumorigenicity, like lactoylglutathione lyase and sulfotransferase 1A1 were differentially expressed. In contrast, none of these proteins were differentially expressed for the MDCKSUS2 cell line. Overall, proteomic monitoring allowed identification of key proteins involved in adaptation from adherent to suspension growth. In addition, identified proteins related to tumorigenicity may represent markers to support cell clone selection at early stages of industrial cell line development.

An outbreak of influenza A(H3N2) in Alappuzha district, Kerala, India, in 2011.

Influenza is an RNA virus that belongs to the Orthomyxoviridae family. It causes a highly contagious acute respiratory illness, has been recognized since ancient times, and is a major health threat throughout the world. An outbreak of influenza-like illness (ILI) was reported from Alappuzha district of Kerala State between late June and July 2011. This investigation was conducted to determine the clinical picture, causative agents, and epidemiological characteristics of the illness. The World Health Organization (WHO)'s case definition for ILI was followed throughout the investigation. Nasal or throat swabs were collected from 204 suspected patients. Real-time reverse transcription polymerase chain reaction (RT-PCR)-based diagnosis was performed to detect influenza A and B viruses and their subtypes. Madin-Darby canine kidney (MDCK) cell line was used for virus isolation. One-step RT-PCR was performed to amplify the HA1 gene of influenza A(H3N2). The amplicons for the HA1 gene of influenza A(H3N2) were sequenced, and phylogenetic analysis was done. Analysis of the data revealed that 96 (47.05%) of the 204 respiratory specimens collected were influenza A(H3N2) and only 6 (2.94%) were A(H1N1)pdm09. Phylogenetic analysis revealed that the isolated A(H3N2) was closely related to the 2012-2013 northern hemisphere vaccine strain (A/Victoria/361/2011/H3N2). An influenza A(H3N2) outbreak was confirmed in Alappuzha district of Kerala state with a co-circulation of A(H1N1)pdm09. No substantial difference in the sequence was observed in the etiological agent, and the virus was found to be sensitive to oseltamivir.

The Selection and Assessment of a Low‐Efflux MDCK Cell Line for use in a High‐Throughput Permeability Screening Assay

ObjectivesTo establish a low‐efflux Madin‐Darby canine kidney cell line for potential use in a high throughput permeability screening assay.MethodsMDCK cells were subcloned by limiting dilution. Individual MDCK colonies were isolated and clones were expanded to cell culture plates and assessed for efflux potential. The permeability characteristics and corresponding efflux ratios were measured using a standard bidirectional permeability assay conducted using 96‐well permeable support plates.Results72 clones were initially screened for efflux potential. 8 clones demonstrating the lowest level of efflux were further cultured for continued evaluation. The 8 clones were further reduced in number to 2 by comparison of efflux between clones, where clones demonstrating the lowest efflux were maintained. One clone, whose efflux did not increase with longitudinal culture time, was selected for use in a high‐throughput permeability screening assay. The final clone showed limited efflux of common P‐gp substrates: loperamide, quinidine, dipyridamole; BCRP substrates: prazosin, topotecan, imatinib; and an MRP‐2 substrate: valsartan. Despite the lack of transport of multiple probe substrates efflux was observed for some compounds such as vinblastine and saquinavir. The final clone was further tested using a diverse set of molecules.ConclusionsA low efflux MDCK subclone (MDCK‐LE) was identified. The MDCK‐LE cells demonstrate low efflux for many representative substrates of efflux transporters, though the cells are not entirely a “no efflux” cell line as demonstrated by the efflux of vinblastine and saquinavir, they do delineate the permeability of structurally diverse molecules over a broad permeability range.

Establishment of MDCK Stable Cell Lines Expressing TMPRSS2 and MSPL and Their Applications in Propagating Influenza Vaccine Viruses in Absence of Exogenous Trypsin.

We established two Madin-Darby canine kidney (MDCK) cell lines stably expressing human airway transmembrane protease: transmembrane protease, serine 2 (TMPRSS2) and mosaic serine protease large form (MSPL) which support multicycle growth of two H5 highly pathogenic avian influenza viruses (HPAIV) recombinant vaccines (Re-5 and Re-6) and an H9 avian influenza virus (AIV) recombinant vaccine (Re-9) in the absence of trypsin. Data showed that the cell lines stably expressed TMPRSS2 and MSPL after 20 serial passages. Both MDCK-TMPRSS2 and MDCK-MSPL could proteolytically cleave the HA of Re-5, Re-6, and Re-9 and supported high-titer growth of the vaccine without exogenous trypsin. Re-5, Re-6, and Re-9 efficiently infected and replicated within MDCK-TMPRSS2 and MDCK-MSPL cells and viral titer were comparable to the virus grown in MDCK cells with TPCK-trypsin. Thus, our results indicate a potential application for these cell lines in cell-based influenza vaccine production and may serve as a useful tool for HA proteolytic cleavage-related studies.

Analysis of cytokine production in a newly developed canine tracheal epithelial cell line infected with H3N2 canine influenza virus.

The Madin-Darby canine kidney (MDCK) cell line is typically used to analyze pathological features after canine influenza virus (CIV) infection. However, MDCK cells are not the ideal cell type, because they are kidney epithelial cells. Therefore, we generated an immortalized canine tracheal epithelial cell line, KU-CBE, to more reliably study immune responses to CIV infection in the respiratory tract. KU-CBE cells expressed the influenza virus receptor, α-2,3-sialic acid (SA), but not α-2,6-SA. KU-CBE and MDCK cells infected with H3N2 CIV demonstrated comparable virus growth kinetics. Gene expression levels of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, and interferon (IFN)-β were estimated in both KU-CBE and MDCK cells infected with CIV by real-time reverse transcription polymerase chain reaction (qRT-PCR). Of these cytokines, IL-4, IL-10, TNF-α, and IFN-β mRNAs were detected in both cell lines. Gene expression of IL-4, IL-10, and TNF-α was not significantly different in the two cell lines. However, MDCK cells exhibited a significantly higher level of IFN-β mRNA than KU-CBE cells at 18 h post infection. Additionally, the protein concentrations of these four cytokines were determined by enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants obtained from the two CIV-infected cell lines. MDCK cells produced significantly higher amounts of IL-4 and IFN-β than KU-CBE cells. However, KU-CBE cells produced a significantly higher amount of TNF-α than MDCK cells. These data indicated that the newly developed canine tracheal epithelial cells exhibited different cytokine production patterns compared to MDCK cells when infected with CIV. Inflammation of the respiratory tract of dogs induced by CIV infection may be attributed to the elevated expression level of TNF-α in canine tracheal epithelial cells.

Establishment of a functional secretory IgA transcytosis model system in vitro for functional food screening.

A characteristic mucosal immune response involves the production of antigen-specific secretory immunoglobulin A (SIgA) antibodies. In order to study transcytosis by mimicking the SIgA secretion and to screen for SIgA secretion-promoting substances, we developed a model system of a transfected Madin-Darby canine kidney (MDCK) cell line that expresses the human polymeric immunoglobulin receptor (pIgR). We thus isolated the human dIgA (dimeric IgA)/pIgA (polymeric IgA) complex as the binding ligands. In the present study, a recombinant vector encoding the human pIgR gene was constructed and infected into MDCK cells. Following reverse transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunofluorescence staining, we confirmed that pIgR was expressed in the transfectant MDCK-pIgR cells and was located at the basolateral side of the cell surface. We also confirmed the coexistence of the dIgA/pIgA complex in the IgA myeloma serum. The covalent dIgA/pIgA complex was then isolated from the serum of an IgA multiple myeloma patient using an ÄKTA purifier operation system with a HiPrep 16/60 Sephacryl S-300 HR column, in order to utilize the complex as transcytosis ligands for human pIgR. Finally, we confirmed the uptake of the isolated human dIgA/pIgA complex into MDCK-pIgR cells. We demonstrated that the human dIgA/pIgA complex was transcytosed into the apical side of the monolayer cells. Therefore, our MDCK-human pIgR cell transcytosis model is an operational system and can be used for screening functional food components that promote dIgA/pIgR transcytosis as well as SIgA secretion.

In-vitro validation of self designed siRNA targeting non-structural 1 gene of Influenza A virus

The genomic variability makes Influenza A virus (IAV) difficult to be con-trolled by existing vaccines or anti-influenza drugs. Viral gene targeting siRNA induces the RNAi mechanism in the host and silents the gene by cleaving mRNA. Objective was to develop siRNA targeting non-structural 1 gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence align-ment) of NS1 gene of IAV strains. To choose the most efficient siRNA, three levels screening method was developed. Ultimately one siRNA duplex was selected on the basis of its unique position in conserved region. siRNA effica-cy was confirmed in vitro on commonly used Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/2011(H3N2)] and Influenza A/pdmH1N1 [A/India/LKO2151/2012(H1N1)]. Of total 173 strains worldwide and 30 strains from India, 32 bp long (position 561 - 592) conserved region was identified. The longest ORF of NS1 gene was targeted by the selected siRNA, which showed 65.5% inhibition in replication of Influenza A/pdmH1N1 and 67.2% inhibition in replication of Influenza A/H3N2 at 48 hpi on MDCK cell line. This study shows that siRNA targeting NS1 may be quite effective in controlling IAV rep-lication so can be used as anti-IAV therapeutic agent.

Isolation of Canine parvovirus with a view to identify the prevalent serotype on the basis of partial sequence analysis.

Aim:The aim of this study was to isolate Canine parvovirus (CPV) from suspected dogs on madin darby canine kidney (MDCK) cell line and its confirmation by polymerase chain reaction (PCR) and nested PCR (NPCR). Further, VP2 gene of the CPV isolates was amplified and sequenced to determine prevailing antigenic type.Materials and Methods:A total of 60 rectal swabs were collected from dogs showing signs of gastroenteritis, processed and subjected to isolation in MDCK cell line. The samples showing cytopathic effects (CPE) were confirmed by PCR and NPCR. These samples were subjected to PCR for amplification of VP2 gene of CPV, sequenced and analyzed to study the prevailing antigenic types of CPV.Results:Out of the 60 samples subjected to isolation in MDCK cell line five samples showed CPE in the form of rounding of cells, clumping of cells and finally detachment of the cells. When these samples and the two commercially available vaccines were subjected to PCR for amplification of VP2 gene, a 1710 bp product was amplified. The sequence analysis revealed that the vaccines belonged to the CPV-2 type and the samples were of CPV-2b type.Conclusion:It can be concluded from the present study that out of a total of 60 samples 5 samples exhibited CPE as observed in MDCK cell line. Sequence analysis of the VP2 gene among the samples and vaccine strains revealed that samples belonged to CPV-2b type and vaccines belonging to CPV-2.

Oseltamivir Resistant Influenza A (H1N1) Virus Infection in Mumbai, India

Objectives: Oseltamivir is an important antiviral agent approved for treatment of infections caused by Influenza virus. The emergence of an Oseltamivir resistant seasonal Influenza A (H1N1) virus in 2007 in Norway and pandemic Influenza A (H1N1) virus in 2009 [Influenza A (H1N1) pdm 09] is of major concern. This study aimed for examining antiviral drug resistance of Influenza A viruses circulating during 2009 Influenza season in Mumbai. Methods: Nasopharyngeal swabs positive for Influenza A virus were inoculated on Madin-Darby canine kidney cell line for virus isolation. Molecular analysis of neuraminidase gene and matrix gene was carried out to detect known mutations contributing to resistance. Resistance to neuraminidase was assayed using a commercially available chemiluminescence based NA-Star assay kit. Results: Genotypically, a total of 47 isolates of Influenza A (H1N1) pdm 09 isolates were observed to harbor mutations known to confer resistance to Adamantane. However, no known mutations conferring resistance to neuraminidase (NA) inhibitors were detected. Resistance to Oseltamivir was observed in a single isolate of seasonal Influenza A (H1N1) isolate with an extremely high IC50 value of 1261 nM. Conversely, the isolate was sensitive to Adamantane. Phylogenetic analysis revealed that NA gene of this Oseltamivir-resistant isolate from Mumbai was antigenically related to human A (H1N1) A/Brisbane/59/2007 vaccine strain. Conclusions: Surveillance on drug susceptibility of circulating strains in Mumbai helped us to identify Oseltamivir resistance in seasonal Influenza virus. The study highlights the importance of continual surveillance on Influenza in India to ensure that antiviral prescribed by clinicians is effective when treating patients for Influenza infections.

IQGAP1 controls tight junction formation through differential regulation of claudin recruitment.

IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42-JNK pathway.

Transepithelial resistance in human bestrophin-1 stably transfected Madin–Darby canine kidney cells

Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin–Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first–fourth day) both cell lines showed the same or similar values ​​of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.

Construction and validation of a Gluc-GFP dual-labeled system

Objective To construct a Gaussia luciferase-green fluorescent protein （ Gluc-GFP ） dual-labeled system for the investigation of in vitro and in vivo pathogenesis of virus infections .Methods Two DNA fragments encoding G1-10 and G11 were split from the gene encoding GFP and then respectively inserted into pcDNA3.1 vector to construct the recombinant expression plasmids G1-10-pcDNA3.1 and G11-pcDNA3.1.The genes encoding Gluc and G11 were linked by a fusion PCR and then inserted into pcDNA3.1 vector to construct the fusion plasmid.The expression plasmids of Gluc-G11-pcDNA3.1 and G1-10-pcDNA3.1 were transfected into 293T cells.The expression of Gluc in the supernatants was measured and a fluorescence microscopy was used to observe cells with GFP fluorescence.To simplify the construction process, a Madin-Darby canine kidney （ MDCK ） cell line consistently express G1-10 was constructed by Lentivector Expression Systems.The dual-labeled system was constructed by transfecting G11-pcDNA3.1 plasmid into the MDCK cell line.The puromycin and red fluorescent protein （ RFP） were used for screening and validation.Results The feasibility of split-GFP strategy was confirmed by using fluorescence micro-scope.The expression of luciferase in the supernatants and cells with green fluorescence could be detected after co-transfection of G1-10-pcDNA3.1 and Gluc-G11-pcDNA3.1 plasmids into 293T cells.The construc-ted MDCK cell line could stably express G1-10.Conclusion The physiological activity of Gluc was not af-fected by creating Glu-G11 fusion protein.The MDCK cell line stably expressing G1-10 could simplify the construction of the dual-labeled system.The successfully constructed Gluc-GFP dual-labeled system would provide a useful tool for further investigation on the pathogenesis of virus infections. Key words: Dual-labeled system Gluc-GFP Molecular imaging Virus research

New technologies and challenges of novel virus detection.

The use of new cell substrates for the development of biologicals, particularly tumorigenic and tumor-derived cell lines, can pose a major regulatory challenge due to safety concerns related to the presence of novel viruses, latent and occult viruses including oncogenic viruses, and endogenous retroviruses, since these may not be detected by the currently recommended conventional assays. This report is a summary of our laboratory's experiences using advanced nucleic acid-based technologies to evaluate a Madin-Darby canine kidney (MDCK) cell line and the insect Sf9 cell line derived from Spodoptera frugiperda, and presents some ongoing efforts to address the challenges of novel virus detection.

Anomeric selectivity and influenza A virus inhibition study on methoxylated analogues of Pentagalloylglucose

Anomeric selectivity in galloylation of d-glucose and d-mannose with carboxylic acid was explored under steglich conditions. Base catalyst 4-dimethylaminopyridine favored the formation of alpha-anomers, while adding an acid and carbodiimide favored the formation of beta-anomers. Steric hindrance between α,β-unsaturated acid and C-2 OH stereochemistry (adjacent carbon to anomeric) influenced anomeric selectivity for both d-glucose and d-mannose. The influenza A virus inhibition activities of the synthesized compounds were evaluated in Madin–Darby canine kidney cell line using the cytopathic effect inhibition assay. All the synthetic methoxylated analogues showed more considerable activity against influenza A virus than their corresponding acids, which indicated the sugar core as key functionality for anti-viral activity. The activities of trimethoxy-cinnamic acid Pentagalloylglucose analogues, 3α, 3β, 4α, and 4β (IC50, 109.1μM, 134.4μM, 119.5μM, 111.1μM, respectively) were better than those of trimethoxy-benzoic acid Pentagalloylglucose analogues, 1-αβ and 2α, 2β (IC50, 209.8μM, 132.9μM, 161.2μM, respectively), which suggested that the double bond in cinnamic acid Pentagalloylglucose analogues makes the major contribution for influenza A virus inhibitory activity. Notably, several anomeric mixtures showed better activities than pure alpha or beta anomer and were almost two times more effective than Ribavirin, a clinically used anti-viral drug.

Abstract 601: A Biased AT1 Receptor Agonist Stimulates COX2 Expression In Intercalated Cells

We recently discovered a pathway by which angiotensin (Ang) II, acting via angiotensin type 1 receptors (AT1R), triggers expression of cyclo-oxygenase (COX)-2 in intercalated cells (IC) of the collecting duct (CD), generating vasodilator prostaglandins, and attenuating the development of hypertension. We found that AT1R-dependent activation of COX2 could be recapitulated in isolated medullary collecting ducts (IMCDs) and in the C11 clone of the Madin-Darby canine kidney (MDCK) cell line permanently transfected with the AT1AR. This cell line has many features resembling ICs including expression of V-ATPase B1. The robust increase in COX2 expression in C11 cells after AngII was blunted after p38 inhibition (5.1*±0.8 vs. 1.4*±0.4; p=0.014), while PKC and MEK1 inhibition had negligible effects. Ang II activation of AT1R triggers both canonical G-protein and β-arrestin pathways and each pathway may have distinct physiological consequences. In IMCDs, we found that stimulation of COX2 by Ang II requires β-arrestin2, as this response was abrogated in IMCDs from mice lacking β-arrestin2. We next tested the capacity for an AT1R β-arrestin-biased ligand, TRV120023, to activate COX2 in C11 cells. These biased ligands block G-protein signalling like conventional AT1R blockers (ARBs), while stimulating β-arrestin-dependent pathways. TRV120023 caused significant stimulation of COX2 at concentrations of 0.1μM-10μM, indicating that activation of AT1R-linked β-arrestin pathways alone is sufficient to stimulate COX2. Exposure of the IC cell line by TRV also triggered phosphorylation of ERK1/2. The p38 inhibitor SB203580 attenuated TRV-dependent stimulation of ERK1/2 and reduced COX2 expression. In summary, activation of β-arrestin with TRV120023 is sufficient to induce COX2 expression via a pathway involving ERK1/2 and p38 MAPK. This suggests that β-arrestin-biased AT1R agonists might have added beneficial effects compared to conventional ARBs by blocking detrimental G-protein-dependent pathways while preserving beneficial β-arrestin-dependent pathways, such as enhanced expression of COX2.

In silico designing of siRNA targeting PB 1 gene of Influenza A virus and in vitro validation

Genomic variability makes Influenza A virus (IAV) the least susceptible to existing vaccines or anti-influenza drugs. siRNA targeting viral gene silent the gene by cleaving mRNA. Objective: To develop siRNA targeting polymerase basic 1 (PB1) gene and to validate its efficiency in vitro. Materials and Methods: siRNA was designed rationally, targeting the most conserved region of PB1 gene of IAV strains. To choose the most efficient siRNA, three levels screening method was developed. One was selected due to its unique position in conserved region. siRNA efficacy was confirmed in vitro on Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/2011(H3N2)] and Influenza A/pdmH1N1 [A/India/LKO2151/2012(H1N1)]. Result and Conclusion: Total 147 strains worldwide and 43 Indian strains, when aligned, showed seven sets of conserved regions (> 30 bp stretch and < 5% mismatches). The longest ORF was targeted by the selected siRNA, which showed 57% inhibition in replication of Influenza A/pdmH1N1 and 60.6% inhibition in replication of Influenza A/H3N2 at 72 hpi and 48 hpi respectively on MDCK cell line. This study shows that siRNA targeting PB1 may be moderately effective in controlling IAV replication so can be used as anti-IAV therapeutic agent.

Engineering of Bismuth Oxide Nanoparticles to Induce Differential Biochemical Activity in Malignant and Nonmalignant Cells

The relationship between cytotoxicity and the levels of oxidative stress is reported with respect to annealing atmosphere in malignant 9L gliosarcoma tumor and nonmalignant Madin-Darby canine kidney cell lines.

Establishment of Optimized MDCK Cell Lines for Reliable Efflux Transport Studies

Madin-Darby canine kidney (MDCK) cells transfected with human MDR1 gene (MDCK-MDR1) encoding for P-glycoprotein (hPgp, ABCB1) are widely used for transport studies to identify drug candidates as substrates of this efflux protein. Therefore, it is necessary to rely on constant and comparable expression levels of Pgp to avoid false negative or positive results. We generated a cell line with homogenously high and stable expression of hPgp through sorting single clones from a MDCK-MDR1 cell pool using fluorescence-activated cell sorting (FACS). To obtain control cell lines for evaluation of cross-interactions with endogenous canine Pgp (cPgp) wild-type cells were sorted with a low expression pattern of cPgp in comparison with the MDCK-MDR1. Expression of other transporters was also characterized in both cell lines by quantitative real-time PCR and Western blot. Pgp function was investigated applying the Calcein-AM assay as well as bidirectional transport assays using (3) H-Digoxin, (3) H-Vinblastine, and (3) H-Quinidine as substrates. Generated MDCK-MDR1 cell lines showed high expression of hPgp. Control MDCK-WT cells were optimized in showing a comparable expression level of cPgp in comparison with MDCK-MDR1 cell lines. Generated cell lines showed higher and more selective Pgp transport compared with parental cells. Therefore, they provide a significant improvement in the performance of efflux studies yielding more reliable results.

Anti-plasmodial potential of crude alkaloidal extract of three plants used in traditional medicine in India

Malaria is one of the most important tropical diseases and the greatest cause of morbidity and mortality in India. The search for new antimalarial compounds has been necessitated by Plasmodium falciparum resistance to standard antimalarial drugs. Plants are important source of biologically active compounds and have potential for the development of novel antimalarial drugs. Since a number of alkaloids have been successfully used for the treatment of malaria since ancient time In this study the crude alkaloid extract of three young plants Carica papaya Linn. (Family: Caricaceae), Datura innoxia Mill. (Family: Solonaceae) and Ricinus communis Linn (Euphorbiaceae) were evaluated against Plasmodium falciparum 3D7. The mean inhibitory concentration (IC50), the mean cytotoxic concentration and the selectivity index were estimated. The cytotoxicity was estimated on Madin-Darby Canine Kidney (MDCK) cell line in maximum dose tested. The alkaloidal extract of C. papaya showed stronger antiplasmodial activity as compared to D. innoxia and R. communis, the IC50 values in the range of 28.35 to 93.17μg/mL. Results demonstrated alkaloids as the putative active compound showing promising antimalarial effect.

Evaluation of blood–brain barrier and blood–cerebrospinal fluid barrier permeability of 2-phenoxy-indan-1-one derivatives using in vitro cell models

2-Phenoxy-indan-1-one derivatives (PIOs) are a series of novel central-acting cholinesterase inhibitors for the treatment of Alzheimer's disease (AD). The adequate distribution of PIOs to the central nervous system (CNS) is essential for its effectiveness. However, articles related with their permeability in terms of CNS penetration across the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) have not been found. This study was undertaken to evaluate the in vitro BBB and BCSFB transport of PIOs using Madin-Darby canine kidney (MDCK), MDCK-MDR1 and Z310 cell line models. As a result, the transepithelial transport of PIOs did not differ between MDCK and MDCK-MDR1, and the result suggested that PIOs were not substrates for P-gp, which means that multidrug resistance (MDR) function would not affect PIOs absorption and brain distribution. High permeability of PIOs in Z310 was found and it suggested that PIOs had high brain uptake potential. The experiment also showed that PIOs had inhibitory effects on the MDR1-mediated transport of Rhodamine123 with an IC50 value of 40-54 μM. And we suggested that 5,6-dimethoxy-1-indanone might be the pharmacophoric moiety of PIOs that interacts with the binding site of P-gp.