Following previous studies of verapamil reversal of chloroquine resistance in malaria and multi-drug resistance in cancer cells, the effect of verapamil was investigated on nifurtimox-resistant Trypanosoma cruzi in vitro and antimony-resistant Leishmania donovani in vitro and in vivo. Verapamil alone was not active against either parasite, but in combination with nifurtimox it reversed the drug resistance of T. cruzi and in combination with sodium stibogluconate reversed the drug resistance of L. donovani.
IgG contains two major glycosylation sites in the CH2 domains at which complex biantennary oligosaccharides are attached at asparagine residues. A current interest is based on the finding that, compared to normal individuals. RA patients have increased levels of IgG oligosaccharide side chains lacking galactose and terminating in Nacetylglucosamine [ 11. Not only is the agalactosylation of IgG detectable prior to the onset of RA. 121 but the effect is also associated with a highly progressive disease course 131. Consequently. I@ agalactosylation shows potential for use in the RA field.Currently these changes in IgG glycosylation are detected by chemical means or by using lectin-binding assays 141 whilst identification of oligosaccharides attached to a glycoprotein involves their release from the protein by chemical or enzymatic means followed by radioactive or fluorescent labelling and separation by gel filtration or HPLC [ I . 51. The availablity of the enzyme PNGase and the introduction of FACE 161 provide alternative means of releasing N-linked oligosaccharides and the separation of their fluorescent labelled derivatives by polyacrylamide gel electrophoresis.We have investigated the use of PNGase and FACE in a preliminary study of IgG oligosaccharides in rheumatoid arthritis.IgG was prepared from the sera of seven definite RA patients and five healthy normal individuals using the combined G251DEAE-C column procedure of Sumar et al. 131. Pooled human and bovine IgG preparations and other glycoproteins (ovalbumin. fetuin) were also used. 1-5 mg of glycoprotein were digested at pH 2 for 48 h. followed by treatment with PNGase at pH 5 for I6 h according to Takahashi et al. 141. The digests were then desalted on G25 columns and after concentration by freeze-drying the oligosaccharides present were labelled at their reducing ends using either ANTS or AMAC in the presence of NaCNBH3 according to Jackson 161. ANTS and AMAClabelled products were separated by non-SDS Laemmli polyacrylamide gel electrophoresis; AMAC-labelled products were also subjected to polyacrylamide gel electrophoresis in the presence of Tris/borate buffer 161. Gels were viewed and photographed under LJV light. Oligosaccharide s e e standards were prepared by ANTS and AMAC labelling of amylase digests of wheat starch.Initial experiments using the FACE technique showed that pooled human and bovine IgCs gave five/six ANTSlabelled oligosaccharide components in the range 7-10 CUE. The major components were of 7. 9 and 10 CUE and were of approximately equal intensity of fluorescence. These patterns differed significantly from those given by fetuin and ovalbumin. Control experiments showed that the IgG pattern was dependent on the presence of the immunoglobulin and the action of both enzymes. whilst fluorescent components of GLJE less than 5 were not derived from the glycoprotein. Similar experiments using AMAC labelling showed a range of fluorescent components in both the presence and absence of borate in the electrophoresis buffer. Of these, only a larg...
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