11 research outputs found
Chloroquine reduces arylsulphatase B activity and increases chondroitin-4-sulphate: implications for mechanisms of action and resistance
<p>Abstract</p> <p>Background</p> <p>The receptors for adhesion of <it>Plasmodium falciparum</it>-infected red blood cells (RBC) in the placenta have been identified as chondroitin-4-sulphate (C4S) proteoglycans, and the more sulphate-rich chondroitin oligosaccharides have been reported to inhibit adhesion. Since the anti-malarial drug chloroquine accumulates in lysosomes and alters normal lysosomal processes, the effects of chloroquine on the lysosomal enzyme arylsulphatase B (ASB, N-acetylgalactosamine-4-sulphatase), which removes 4-sulphate groups from chondroitin-4-sulphate, were addressed. The underlying hypothesis derived from the recognized impairment of attachment of parasite-infected erythrocytes in the placenta, when chondroitin-4-sulphation was increased. If chloroquine reduced ASB activity, leading to increased chondroitin-4-sulphation, it was hypothesized that the anti-malarial mechanism of chloroquine might derive, at least in part, from suppression of ASB.</p> <p>Methods</p> <p>Experimental methods involved cell culture of human placental, bronchial epithelial, and cerebrovascular cells, and the <it>in vitro </it>exposure of the cells to chloroquine at increasing concentrations and durations. Measurements of arylsulphatase B enzymatic activity, total sulphated glycosaminoglycans (sGAG), and chondroitin-4-sulphate (C4S) were performed using <it>in vitro </it>assays, following exposure to chloroquine and in untreated cell preparations. Fluorescent immunostaining of ASB was performed to determine the effect of chloroquine on cellular ASB content and localization. Mass spectrometry and high performance liquid chromatography were performed to document and to quantify the changes in chondroitin disaccharides following chloroquine exposure.</p> <p>Results</p> <p>In the human placental, bronchial epithelial, and cerebrovascular cells, exposure to increasing concentrations of chloroquine was associated with reduced ASB activity and with increased concentrations of sGAG, largely attributable to increased C4S. The study data demonstrated: 1) decline in ASB activity following chloroquine exposure; 2) inverse correlation between ASB activity and C4S content; 3) increased content of chondroitin-4-sulphate disaccharides following chloroquine exposure; and 4) decline in extent of chloroquine-induced ASB reduction with lower baseline ASB activity. Confocal microscopy demonstrated the presence of ASB along the cell periphery, indicating extra-lysosomal localization.</p> <p>Conclusions</p> <p>The study data indicate that the therapeutic mechanism of chloroquine action may be attributable, at least in part, to reduction of ASB activity, leading to increased chondroitin-4-sulphation in human placental, bronchial epithelial, and cerebrovascular cells. In vivo, increased chondroitin-4-sulphation may reduce the attachment of <it>P. falciparum</it>-infected erythrocytes to human cells. Extra-lysosomal localization of ASB and reduced impact of chloroquine when baseline ASB activity is less suggest possible mechanisms of resistance to the effects of chloroquine.</p
Analysis of procainamide-derivatised heparan sulphate disaccharides in biological samples using hydrophilic interaction liquid chromatography mass spectrometry
Glycosaminoglycans (GAGs) are a family of linear heteropolysaccharides made up of repeating disaccharide units that are found on the surface and extracellular matrix of animal cells. They are known to play a critical role in a wide range of cellular processes including proliferation, differentiation and invasion. To elucidate the mechanism of action of these molecules, it is essential to quantify their disaccharide composition. Analytical methods that have been reported involve either chemical or enzymatic depolymerisation of GAGs followed by separation of non-derivatised (native) or derivatised disaccharide subunits and detection by either UV/fluorescence or MS. However, the measurement of these disaccharides is challenging due to their hydrophilic and labile nature. Here we report a pre-column LC-MS method for the quantification of GAG disaccharide subunits. Heparan sulphate (HS) was extracted from cell lines using a combination of molecular weight cutoff and anion exchange spin filters and digested using a mixture of heparinases I, II and III. The resulting subunits were derivatised with procainamide, separated using hydrophilic interaction liquid chromatography and detected using electrospray ionisation operated in positive ion mode. Eight HS disaccharides were separated and detected together with an internal standard. The limit of detection was found to be in the range 0.6–4.9 ng/mL. Analysis of HS extracted from all cell lines tested in this study revealed a significant variation in their composition with the most abundant disaccharide being the non-sulphated ∆UA–GlcNAc. Some structural functional relationships are discussed demonstrating the viability of the pre-column method for studying GAG biolog
Formation of chlorinated breakdown products during degradation of sunscreen agent, 2-ethylhexyl-4-methoxycinnamate in the presence of sodium hypochlorite
Study on the stability of supercritical fluid extracted rosemary (Rosmarinus Offcinalis L.) essential oil
The aim of this study was to examine the influence of storage conditions and duration on compo- sition and antioxidant activity of supercritical fluid (SCF) extracted essential oil of rosemary (Rosmarinus officinalis L.). Supercritical extraction was carried out sequentially by using SCF carbondioxide in the first two steps and with 5% ethanol as entrainer in the third step. The compositions of the extracts were determined by gas chromatography/mass spectrometry. The total phenolics were analyzed using Folin-Ciocalteau assay. Antioxidant activities of the extracts were tested by ß-carotene-linoleic acid bleaching method. The extracts stored at 4°C in the dark for 14 weeks were associated with slight changes in their composition. However, storage under indirect day light at room temperature caused considerable changes in the composi- tions of the oils due to the chemical transformations in some of their components. Both the total phenolic contents and the antioxidant activities were significantly decreased after storage. © Pleiades Publishing, Ltd., 2010.Çukurova ÜniversitesiACKNOWLEDGMENTS This work was financially supported by Çukurova University (Project no. FEF2007BAP12). The authors are thankful to Dr. Yesim Özogul from Faculty of Fisheries in Çukurova University for her support in providing their GC MS instrument for analysis
Impact of salt exposure on N-acetylgalactosamine-4-sulfatase (arylsulfatase B) activity, glycosaminoglycans, kininogen, and bradykinin
N -acetylgalactosamine-4-sulfatase (Arylsulfatase B; ARSB) is the enzyme that removes sulfate groups from the N-acetylgalactosamine-4-sulfate residue at the non-reducing end of chondroitin-4-sulfate (C4S) and dermatan sulfate (DS). Previous studies demonstrated reduction in cell-bound high molecular weight kininogen in normal rat kidney (NRK) epithelial cells when chondroitin-4-sulfate content was reduced following overexpression of ARSB activity, and chondroitinase ABC produced similar decline in cell-bound kininogen. Reduction in the cell-bound kininogen was associated with increase in secreted bradykinin. In this report, we extend the in vitro findings to in vivo models, and present findings in Dahl salt-sensitive (SS) rats exposed to high (SSH) and low salt (SSL) diets. In the renal tissue of the SSH rats, ARSB activity was significantly less than in the SSL rats, and chondroitin-4-sulfate and total sulfated glycosaminoglycan content were significantly greater. Disaccharide analysis confirmed marked increase in C4S disaccharides in the renal tissue of the SSH rats. In contrast, unsulfated, hyaluronan-derived disaccharides were increased in the rats on the low salt diet. In the SSH rats, with lower ARSB activity and higher C4S levels, cell-bound, high-molecular weight kininogen was greater and urinary bradykinin was lower. ARSB activity in renal tissue and NRK cells declined when exogenous chloride concentration was increased in vitro. The impact of high chloride exposure in vivo on ARSB, chondroitin-4-sulfation, and C4S-kininogen binding provides a mechanism that links dietary salt intake with bradykinin secretion and may be a factor in blood pressure regulation
Capillary Electrophoresis For Total Glycosaminoglycan Analysis
A capillary zone electrophoresis-laser-induced fluorescence detection (CZE-LIF) method was developed for the simultaneous analysis of disaccharides derived from heparan sulfate, chondroitin sulfate/dermatan sulfate, hyaluronan, and keratan sulfate. Glycosaminoglycans (GAGs) were first depolymerized with the mixture of GAG lyases (heparinase I, II, III and chondroitinase ABC and chondroitinase AC II) and GAG endohydrolase (keratinase II) and the resulting disaccharides were derivatized by reductive amination with 2-aminoacridone. Nineteen fluorescently labeled disaccharides were separated using 50 mM phosphate buffer (pH 3.3) under reversed polarity at 25 kV. Using these conditions, all the disaccharides examined were baseline separated in less then 25 min. This CZE-LIF method gave good reproducibility for both migration time (a parts per thousand currency sign1.03 % for intraday and a parts per thousand currency sign4.4 % for interday) and the peak area values (a parts per thousand currency sign5.6 % for intra- and a parts per thousand currency sign8.69 % for interday). This CZE-LIF method was used for profiling and quantification of GAG derivative disaccharides in bovine cornea. The results show that the current CZE-LIF method offers fast, simple, sensitive, reproducible determination of disaccharides derived from total GAGs in a single run.Wo