During a search for neuraminidase inhibitors derived from medicinal fungi, we found that the fermentation broth of Phellinus linteus exhibited potent neuraminidase inhibitory activity. Through bioassay-guided fractionation, two active compounds were purified from the ethyl acetate-soluble portion of the fermentation broth of P. linteus. These structures were identified as inotilone (1) and 4-(3,4-dihydroxyphenyl)-3-buten-2-one (2) by spectroscopic methods. Compounds 1 and 2 inhibited H1N1 neuraminidase activity with IC50 values of 29.1 and 125.6 microM, respectively, in a dose-dependent manner. They also exhibited an antiviral effect in a viral cytopathic effect reduction assay using MDCK cells. These results suggest that compounds 1 and 2 from the culture broth of P. linteus would be good candidates for the prevention and therapeutic strategies towards viral infections.
Cytopathogenic Effect, Viral ; Fermentation* ; Fungi ; Inhibitory Concentration 50 ; Madin Darby Canine Kidney Cells ; Neuraminidase*

canine adenovirus type 2 (CAV-2) vaccine candidate using the recently isolated Korean CAV-2 strain; we termed the vaccine APQA1701-40P and evaluated its safety and immunogenicity in dogs.MATERIALS AND METHODS: To generate the anti-CAV-2 vaccine, APQA1701 was passaged 40 times in MDCK cells growing in medium containing 5 mM urea and the virus was inactivated using 0.05% (volume per volume) formaldehyde. Two vaccines were prepared by blending inactivated APQA1701-40P with two different adjuvants; both were intramuscularly injected (twice) into guinea pigs. The safety and immunogenicity of the Cabopol-adjuvanted vaccine were evaluated in seronegative dogs. The humoral responses elicited were measured using an indirect enzyme-linked immunosorbent assay (I-ELISA), and via a virus neutralization assay (VNA).RESULTS: The new, inactivated CAV-2 vaccine strain, APQA1701-40P, lacked six amino acids of the E1b-19K protein. In guinea pigs, the Cabopol-adjuvanted vaccine afforded a slightly higher VNA titer and I-ELISA absorbance than an IMS gel-adjuvanted vaccine 4 weeks post-vaccination (p>0.05). Dogs inoculated with the former vaccine developed a significantly higher immune titer than non-vaccinated dogs.CONCLUSION: The Cabopol-adjuvanted, inactivated CAV-2 vaccine was safe and induced a high VNA titer in dogs.]]>
Adenoviruses, Canine ; Amino Acids ; Animals ; Dogs ; Enzyme-Linked Immunosorbent Assay ; Formaldehyde ; Guinea Pigs ; Madin Darby Canine Kidney Cells ; Urea ; Vaccines

PURPOSE: We tried to evaluate the effect of oxalate on the growth of distal renal tubular epithelial cells MATERIALS AND METHODS: Madin-Darby canine kidney(MDCK) cell line, derived from canine distal renal tubular epithelial cell, supplied from American Type Culture Collection was used in this study. RPMI 1640 containing 10mM HEPES-buffer, 100IU/ microliterpenicillin, 100 microgram/ microliterstreptomycin, 300 microgram/ microliterglutamine, and with or without 10% fetal bovine serum(FBS) was used as the culture media. And the cell survivals under various concentrations of ammonium oxalate were assessed by MTT assay. RESULTS: Exposure of MDCK cells to oxalate resulted in a concentration dependent suppression of the cell growth in both 10% FBS supplied media and FBS deprived media. CONCLUSIONS: From these observations we could conclude that oxalate is not a mere constituent of calcium oxalate stone, but oxalate might offer a good environment for calcium oxalate stone formation in the urinary tract via MDCK cellular damage.
Calcium Oxalate ; Cell Line ; Culture Media ; Epithelial Cells* ; Madin Darby Canine Kidney Cells ; Oxalic Acid ; Urinary Tract

The pathophysiology of glandular dysfunction in Sjögren's syndrome (SS) has not been fully elucidated. Previously, we reported the presence of autoantibodies to AQP-5 in patients with SS, which was associated with a low resting salivary flow. The purpose of this study was to investigate the presence of anti-AQP1 autoantibodies. To detect anti-AQP1 autoantibodies, cell-based indirect immunofluorescence assay was developed using MDCK cells that overexpressed human AQP1. By screening 112 SS and 52 control sera, anti-AQP1 autoantibodies were detected in 27.7% of the SS but in none of the control sera. Interestingly, the sera that were positive for anti-AQP1 autoantibodies also contained anti-AQP5 autoantibodies in the previous study. Different from anti-AQP5 autoantibodies, the presence of anti-AQP1 autoantibodies was not associated with the salivary flow rate. Although anti-AQP1 autoantibodies are not useful as a diagnostic marker, the presence of autoantibodies to AQP1 may be an obstacle to AQP1 gene therapy for SS.
Aquaporin 1 ; Autoantibodies* ; Fluorescent Antibody Technique ; Fluorescent Antibody Technique, Indirect ; Genetic Therapy ; Humans ; Madin Darby Canine Kidney Cells ; Mass Screening

OBJECTIVETo develop a LC-MS/MS method for determination of deferiprone in cell lysate and to study the potential interaction between deferiprone and hOCTs or hOAT1 transporters in vitro.

METHODSThe determination was performed on an Agilent Eclipse Plus C18 column(3.5 μm, 2.1 mm×50 mm).The gradient mobile phase was composed of solvent A:0.1% formic acid in water, and B:0.1% formic acid in acetonitrile. The mass spectrometer with an electrospray interface was operated in positive ion mode with multiple reaction monitoring (MRM) scan mode monitored the ion pair of deferiprone at m/z 140→96, or phenacetin at m/z 180→110. The effects of deferiprone on the accumulation of typical substrates of hOCTs and hOAT1 were evaluated by MDCK-hOCTs and MDCK-hOAT1 cells respectively. The accumulation of deferiprone was also investigated in MDCK-hOCTs cells and mock cells with or without typical inhibitors.

RESULTSThe standard curve was linear over the range of 5-300 nmol/L. The assay recovery of deferiprone was above 94%, and the intra-day precision (RSD) was less than 2.0%. The accumulation of MPP(+) in MDCK-hOCTs cells with 300 μmol/L deferiprone were 73.5%, 87.1% and 70.4%, respectively. The uptake of deferiprone in MDCK-hOCTs and mock cells did not show significant difference. Deferiprone of 100 μmol/L did not significantly affect the accumulation of 6-CF in MDCK-hOAT1 cell.

CONCLUSIONThe method is sensitivity and suitable for the determination of deferiprone in cell lysate. Deferiprone can significantly inhibit hOCT1 and hOCT3, but has no effects on hOCT2 and hOAT1. hOCTs may not play a major role in the transport of deferiprone.

Animals ; Chromatography, Liquid ; Dogs ; Humans ; Madin Darby Canine Kidney Cells ; Organic Anion Transporters ; drug effects ; Pyridones ; pharmacology ; Tandem Mass Spectrometry

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin-Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.
Cell Line* ; Collagenases ; Distemper Virus, Canine* ; Embryonic Structures ; Epithelial Cells ; Family Characteristics ; Fibroblasts ; Kidney* ; Madin Darby Canine Kidney Cells ; Retroviridae ; Telomerase

Canine adenovirus type 1 (CAV-1) infection results in hepatitis in dogs. In this study, we investigated the biologic and genetic characteristics of the CAV-1 vaccine strain (CAV1V) to improve quality control about CAV vaccine. The identity of CAV1V as CAV-1 was confirmed based on its cytopathic effects and the results of hemagglutination (HA) and immunofluorescence assays, and electron microscopy. The CAV1V strain reached 10(7.5) TCID(50)/mL in MDCK cells at 4 days post-inoculation and exhibited hemmagglutination activity of 256 U using guinea pig erythrocytes. Intranuclear fluorescence in the infected cells was observed and typical adenoviruses were observed in electon microscope. CAV1V strain was identified as a CAV-1 strain by nucleotide sequence analysis. In a comparison of the nucleotide sequences of the fiber genes of several CAV strains, CAV1V showed the highest similarity (99.8%) with the GLAXO strain, which was isolated in Canada. Our biological characterization of CAV1V will facilitate quality control of the canine hepatitis vaccine.
Adenoviridae ; Adenoviruses, Canine ; Animals ; Base Sequence ; Canada ; Dogs ; Erythrocytes ; Fluorescence ; Fluorescent Antibody Technique ; Guinea Pigs ; Hemagglutination ; Hepatitis ; Madin Darby Canine Kidney Cells ; Microscopy, Electron ; Quality Control

The purpose of the present study was to determine the role of inositol 1,4,5-trisphosphate receptor 3 (IP₃R3) in renal cyst development in autosomal dominant polycystic kidney disease (ADPKD). 2-aminoethoxy-diphenyl borate (2-APB) and shRNA were used to suppress the expression of IP₃R3. The effect of IP₃R3 on cyst growth was investigated in Madin-Darby canine kidney (MDCK) cyst model, embryonic kidney cyst model and kidney specific Pkd1 knockout (PKD) mouse model. The underlying mechanism of IP₃R3 in promoting renal cyst development was investigated by Western blot and immunofluorescence staining. The results showed that the expression level of IP₃R3 was significantly increased in the kidneys of PKD mice. Inhibiting IP₃R3 by 2-APB or shRNA significantly retarded cyst expansion in MDCK cyst model and embryonic kidney cyst model. Western blot and immunofluorescence staining results showed that hyperactivated cAMP-PKA signaling pathway in the growth process of ADPKD cyst promoted the expression of IP₃R3, which was accompanied by a subcellular redistribution process in which IP₃R3 was translocated from endoplasmic reticulum to intercellular junction. The abnormal expression and subcellular localization of IP₃R3 further promoted cyst epithelial cell proliferation by activating MAPK and mTOR signaling pathways and accelerating cell cycle. These results suggest that the expression and subcellular distribution of IP₃R3 are involved in promoting renal cyst development, which implies IP₃R3 as a potential therapeutic target of ADPKD.
Animals ; Dogs ; Mice ; Cysts/genetics* ; Inositol 1,4,5-Trisphosphate Receptors/pharmacology* ; Kidney/metabolism* ; Polycystic Kidney Diseases/metabolism* ; Polycystic Kidney, Autosomal Dominant/drug therapy* ; Madin Darby Canine Kidney Cells

The transcellular process of coumarin 6 (C6) loaded poly(ethyl ethylene phosphate)-co-poly (epsilon-caprolactone) (PEG-PCL) micelles on Madin-Darby Canine Kidney (MDCK) epithelial cells was investigated. C6 loaded PEG-PCL micelles were prepared using the thin-film hydration method. The size of the micelles was characterized by dynamic light scattering analysis using a Malvern Zetasizer Nano ZS. The critical micelle concentration (CMC) was determined by pyrene fluorescence probe method. And the transcellular process of the micelles on MDCK epithelial cells was investigated by using transmission electron microscope, laser confocal scanning microscope and Förster resonance energy transfer technology. It turned out that the size of PEG-PCL micelles was about 30 nm and CMC was 1.01 microg x mL(-1). PEG-PCL micelles were endocytosed in intact form and they could deliver hydrophobic drugs across the basolateral membrane of the epithelial cells. However, PEG-PCL is hardly being transported in micelle formation itself. The transportation of C6 by PEG-PCL micelles was through the transcellular pathway, yet not the paracellular pathway.
Animals ; Biological Transport ; Coumarins ; pharmacokinetics ; Dogs ; Drug Carriers ; Drug Delivery Systems ; Lactones ; chemistry ; Madin Darby Canine Kidney Cells ; Micelles ; Particle Size ; Polyethylene Glycols ; chemistry ; Thiazoles ; pharmacokinetics

OBJECTIVETo study the inhibitory activities of 3-O-β-chacotriosyl benzyl ursolate and its derivatives as potential new anti-influenza virus agents against the entry of H5N1 influenza viruses into the target cells.

METHODSFour target compounds were designed and synthesized, which were structurally related to the lead compound 3-O-β-chacotriosyl methyl ursolate (1). The inhibitory activities of these compounds were tested at a cellular level psuedovirus system targeting H5N1 influenza viruse entry.

RESULTS AND CONCLUSIONThe compounds 1b, 1c and 1d showed potent inhibitory activities against the entry of A/Thailand/Kan353/2004 pseudovirus into the target cells, and among them compound 1d showed the strongest inhibitory activity with an IC50 value of 0.96 ± 0.10 µmol/L. The structure-activity relationship analysis of these compounds indicated that when 17-COOH of ursolic acid was esterified, introduction of Me groups rather than aryl groups more strongly enhanced the inhibitory activity. Changing 17-COOH of ursolic acid into amide could increase the antiviral activity and decrease the cytotoxicity of the compounds in MDCK cells.

Animals ; Antiviral Agents ; chemistry ; Dogs ; Influenza A Virus, H5N1 Subtype ; drug effects ; physiology ; Madin Darby Canine Kidney Cells ; Structure-Activity Relationship ; Triterpenes ; chemistry ; Virus Internalization ; drug effects