An agarose gel electrophoretic method for analysis of sulfated glycosaminoglycans of cultured cells

Agarose disc gel electrophoresis has been adapted to achieve the separation of the major sulfated glycosaminoglycans produced by cells in culture. By use of buffers containing barium ion, mixtures of chondroitin sulfate, dermatan sulfate, and heparan sulfate are well resolved into discrete bands. The technique can be used preparatively as well as analytically to separate quantities of glycosaminoglycans up to a milligram in a 6-mm diameter gel.     

Surface charge of endothelial cells estimated from electrophoretic mobility

The surface charge density of endothelial cells was estimated from cell electrophoresis. Cultured endothelial cells from the bovine pulmonary artery were suspended in saline and placed in the lumen of a glass capillary. A voltage was applied across the capillary ends and the velocity imparted to the cells was measured with a microscope. Erythrocyte mobility was also measured. The mobility in (micron/s)/(V/cm) was 0.74 +/- 0.08 for endothelial cells and 1.03 +/- 0.15 for erythrocytes. Charge density in esu/cm2 was calculated as 2.62 x 10(4) and 0.91 x 10(4) for endothelial and red cells, respectively. Removal of sialic acid did not affect the mobility of endothelial cells, but it reduced that of red cells to near zero. Endothelial cell mobility decreased either with ionic strength or calcium concentration. Our results strongly suggest that the surface charge of endothelial cells is dependent on sulfated glycosaminoglycans.     

Enzymatic lysis of microbial cells

Cell wall lytic enzymes are valuable tools for the biotechnologist, with many applications in medicine, the food industry, and agriculture, and for recovering of intracellular products from yeast or bacteria. The diversity of potential applications has conducted to the development of lytic enzyme systems with specific characteristics, suitable for satisfying the requirements of each particular application. Since the first time the lytic enzyme of excellence, lysozyme, was discovered, many investigations have contributed to the understanding of the action mechanisms and other basic aspects of these interesting enzymes. Today, recombinant production and protein engineering have improved and expanded the area of potential applications. In this review, some of the recent advances in specific enzyme systems for bacteria and yeast cells rupture and other applications are examined. Emphasis is focused in biotechnological aspects of these enzymes.     

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. I. Characterization of different pools of sulfated glycosaminoglycans.

"Fibroblast-like" cells from the intimal layer of bovine aorta were grown in culture. The formation, composition, molecular weight and turnover rate of different pools of glycosaminoglycans were investigated in cultures incubated in the presence [35S]sulfate or [14C]glucosamine. The newly synthesized glycosaminoglycans are distributed into an extracellular pool (37 - 58%), a cell-membrane associated or pericellular pool (23 - 33%), and an intracellular pool (19 - 30%), each pool exhibiting a characteristic distribution pattern of chondroitin sulfate, dermatan sulfate, heparan sulfate and hyaluronate. The distribution pattern of the extracellular glycosaminoglycans resembles closely that found in bovine aorta. A small subfraction of the pericellular pool - tentatively named "undercellular" pool--has been characterized by its high heparan sulfate content. The intracellular and pericellular [35S]glycosaminoglycan pools reach a constant radioactivity after 8-12 h and 24 h, respectively, whereas the extracellular [35S]glycosaminoglycans are secreted into the medium at a linear rate over a period of at least 6 days. The intracellular glycosaminoglycans are mainly in the process of degradation, as indicated by their low molecular weight and by their half-life of 7 h, but intracellular dermatan sulfate is degraded more rapidly (half-life 4-5 h) than intracellular chondroitin sulfate and heparan sulfate (half-life 7-8 h). Glycosaminoglycans leave the pericellular pool with a half-life of 12-14 h by 2 different routes: about 60% disappear as macromolecules into the culture medium, and the remainder is pinocytosed and degraded to a large extent. Extracellular and at least a part of the pericellular glycosaminoglycans are proteoglycans. Even under dissociative conditions (4M guanidinium chloride) their hydrodynamic volume is sufficient for partial exclusion from Sepharose 4B gel. The existence of topographically distinct glycosaminoglycan pools with varying metabolic characteristics and differing accessibility for degradation requiresa reconsideration and a more reserved interpretation of results concerning the turnover rates of glycosaminoglycans as determined in arterial tissue.     

Quantification and gel electrophoretic analysis of proteins extracted from vascular endothelial cells

The inner walls of tubular organs and body cavities are lined with a sheet composed of cells which control the passage through the walls and thus are of considerable physiological and biochemical interest. They are difficult to prepare but their content can be extracted and analysed, using laminar extraction and sensitive bioluminescence methods. This has been shown for the endothelial cells which line the interior of blood vessels. Although it is dealing with small amounts of material, only a minor part of it is consumed in the analyses. The bioluminescence measurements can thus be combined with other analytical procedures such as protein assay and electrophoresis. The possibilities of extending the biochemical information is of interest in the efforts to clarify the pathogenesis of vascular diseases. Laminar elution of protein from endothelial cells of the rat aorta was traced by the simultaneous efflux of adenine nucleotides. These were determined in a bioluminescence assay after conversion to ATP, while the fluorescamine reaction was used for the quantification of protein. The elution patterns obtained by these two methods showed an initial peak with a common maximum. The new possibilities of detecting relevant biochemical changes were evidenced by the finding of a marked protein loss in the fractions containing the outflux from the endothelial cells of diabetic rats. Electrophoresis of proteins eluted from endothelial cells resulted in separation into a large number of bands, but no differences were detected in the electropherograms at comparisons between normal and diabetic rats. It remains to be clarified, whether the protein depletion of the endothelial sheet is due to a concomitant loss of cells or to a cytoplasmatic loss without profound changes of the cytoplasmatic composition.     

One-pot analysis of sulfated glycosaminoglycans

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.     

Interaction of sulfated glycosaminoglycans with lectins

The sulfated glycosaminoglycans, such as keratan sulfate and chitin sulfate having 3-hydroxy free N-acetyl-beta-D-glucosaminyl residues as constituents, reacted with wheat germ agglutinin and Solanum tuberosum agglutinin by sugar-specific interaction. The glycosaminoglycans showed different inhibitory activities to the hemagglutination reaction of these lectins and keratan sulfate and its modified products formed insoluble complexes with both of the lectins at pH 7.0 in physiological saline solutions (0.15 M NaCl). S. tuberosum agglutinin was precipitated within a particularly narrow concentration range of keratan sulfate, and the formation of a soluble complex was observed by gel chromatography. These interactions were specifically inhibited by N,N'-diacetylchitobiose but not by 2 M NaCl. The specific interactions of the glycosaminoglycans with S. tuberosum agglutinin were confirmed by their ultraviolet difference spectra with two peaks at 285 and 298 nm attributable to the tryptophan residues in the binding site of the agglutinin. It was also found that S. tuberosum agglutinin and wheat germ agglutinin have different binding specificities. The presence of sulfate groups in either keratan sulfate or chitin sulfate did not interfere with their specific interactions with S. tuberosum agglutinin as strongly as with wheat germ agglutinin. The N-acetylneuraminic acid residues in keratan sulfate were found to be receptor sites for wheat germ agglutinin but not for S. tuberosum agglutinin.     

Enzymatic responses to radiation in cultured vascular endothelial and smooth muscle cells

Confluent monolayers of bovine aortic endothelial and smooth muscle cells were exposed to 0-5.0 Gy of 60Co gamma rays. From 0 to 72 hr after irradiation, the monolayer and culture medium were analyzed for cell (nuclei) number, DNA and protein content, the activities of angiotensin converting enzyme (ACE), lactate dehydrogenase (LDH), and superoxide dismutase (SOD), and LDH isoenzyme profile. Irradiated endothelial cells exhibited a time- and dose-dependent increase in cell detachment, decreased DNA and protein content and reduced ACE active per attached cell, increased LDH and SOD activities per microgram of DNA, and increased LDH activity in the culture medium. The latter was accompanied by a shift from LDH 1 to LDH 4 and 5. The release of LDH activity, observed after 0.5 Gy, was the most sensitive endothelial response, and occurred independent of or preceding cell detachment. Vascular smooth muscle cells contained two to three times more SOD activity than did endothelial cells and exhibited no significant responses to 5.0 Gy.     

Glucose reduces PDGF production and cell proliferation of cultured vascular endothelial cells

The effect of glucose on PDGF production and cell proliferation was studied on cultured bovine aortic endothelial cells. PDGF levels were measured using an enzyme-linked immunosorbent assay technique newly developed in our laboratory. The cell proliferation rate was determine on the basis of 3H-thymidine incorporation into cellular DNA. PDGF levels in culture medium were below the detection limit of the assay. However, PDGF levels were measurable in cultured endothelial cells at confluence. Both PDGF production and thymidine incorporation were significantly reduced in the endothelial cells cultured with high concentrations of glucose. These results suggest that reduced PDGF production and cell proliferation may be involved in altered vascular endothelial function in diabetics.     

Electroendosmosis correction for electrophoretic mobility determined in cells

Electroendosmosis is a complicating factor in gel electrophoresis. Determination of electroendosmotic mobility by the use of vitamin B₁₂ as a marker in agar and agarose gels at different concentrations revealed that electroendosmosis was not reduced to zero by extrapolation of observed mobility values to zero gel concentration. It is shown that a Ferguson plot of the observed values of electrophoretic mobilities yields the correct values for K_R; however the extrapolated values of electrophoretic mobility must still be corrected for electroendosmosis to obtain the true electrophoretic mobilities.     

Enzymatic lysis of staphylococcal cells and isolation of cell walls

Procedures for lyzing staphylococcal cells with the use of ultrasound, lysozyme and a lytic enzyme complex of Actinomyces recifensis var. lyticus, 2435 were compared. The lysis level was estimated by two parameters: lower optical density and protein yield percentage. It was found that ultrasound provided rather high levels of cell destruction reaching 60-68 per cent. The use of lysozyme enabled to destroy 16 per cent of the cells. The enzyme complex of strain 2435 showed high lytic activity with respect to the tested culture. For destroying dense staphylococcal suspensions it appeared necessary to study the effect of preliminary treatment of the cells with various chemical substances on their liability to the effect of the enzyme complex. It was demonstrated that treatment of the cells with 0.01-0.1 M cystein HCl solutions, 0.01-0.02 M sodium dodecylsulfate solutions or 0.05-0.5 M sodium hydroxide solutions increased 2.6-4.7-fold the cell liability to enzymatic hydrolysis. The studies enabled to develop conditions providing complete lysis of 10-percent staphylococcal cell suspension within 5 to 15 minutes under the effect of the lytic enzyme complex of strain 2435. A procedure for isolating cell walls was developed.     

Exposure to oxidized low-density lipoprotein reduces activable Ras protein in vascular endothelial cells

Oxidized low-density lipoprotein (ox-LDL) has been shown to alter the migratory and proliferative activities of the vascular endothelial cells (EC) in response to serum and growth factors. The mechanism underlying the antiproliferative effect of ox-LDL on vascular EC has not been fully elucidated. In this report, we show that exposure of vascular EC to ox-LDL results in a marked reduction of the membrane-associated Ras protein. Further study shows that in ox-LDL-treated EC, reduction of the membrane-associated Ras protein is correlated with a reduced amount of active Ras (Ras-guanosine triphosphate), indicating that the Ras signaling pathway is attenuated. The attenuation of the Ras signaling pathway in ox-LDL-treated EC may thus be responsible for the retarded response to the mitogenic stimulation of serum and growth factors.     

Synthesis of sulfated glycosaminoglycans by embryonic corneal epithelium

The primary corneal stroma is produced by the overlying epithelium. The endothelium appears between 4 and 5 days, fibroblasts at 6 days, and at 12 days the epithelium stratifies. We investigated the synthesis of glycosaminoglycan (GAG) by the epithelium during this developmentally significant period. The sulfated GAG synthesized by isolated 4–6-day-old corneal epithelia during the first 24 hr in vitro are entirely accountable for as chondroitin sulfates and heparan sulfates. Nearly 50% of the total sulfated GAG synthesized by epithelia on Millipore filters is lost to the medium, but only 30–40% is lost when frozen killed lens capsule or stroma is the substratum. Retention of isotope by the tissue is correlated with visible matrix polymerization. The relative amount of heparan sulfate synthesized by the developing epithelium 24 hr in vitro decreases from about 50% of the total sulfated GAG for 4-day-old epithelium to 12% for 12-day-old epithelium. A similar decrease in heparan sulfate synthesis occurs with time in culture. The relative amount of GAG identified as chondroitin sulfate and heparan sulfate is the same when ³H-glucosamine is used to label GAG as when ³⁵SO₄ is used. We conclude that the corneal epithelium produces only sulfated polysaccharides. Since hyaluronate is synthesized by whole 5-day-old corneas, it must be the product of the endothelium.     

Effect of cell substratum on lateral mobility of lipids in the plasma membrane of vascular endothelial cells

Bovine vascular endothelial cells can be maintained in a highly differentiated state in vitro, either by the addition of fibroblast growth factor (FGF) to the culture medium or by plating the cells on extracellular matrix (ECM)-coated dishes. Under these conditions the cells proliferate actively and at confluence form a tightly packed monolayer composed of nonoverlapping polarized cells. A fluorescence recovery after photobleaching method was used to determine the lateral mobility coefficient D of the lipophilic fluorescent probe, 5N-(hexadecanoyl)-aminofluorescein (HEDAF), in the basal and apical plasma membranes of endothelial cells under various culture conditions (cells on glass coverslips in the presence or absence of FGF, or cells plated on ECM in the exponential growth phase or at confluence). A heterogeneous distribution of lateral diffusion coefficients D was found in a given cell population. Nevertheless, for the basal membrane, a "mean" D value close to 2.0 x 10(-9) cm2/s was found for all the culture conditions. The "mean" D value of HEDAF in the apical pole was slightly higher when sparse cells were exposed to FGF (D = 2.2 x 10(-9) cm2/s) and was further enhanced when cells were growing or confluent on ECM-coated coverslips (D = 2.7 x 10(-9) cm2/s). On the other hand, when the cells were maintained in the absence of FGF on glass coverslips, similar "mean" D values were found in both cell poles (D = 2.0 x 10(-9) cm2/s). These results show that lateral mobility of lipids in endothelial plasmalemma varies in response to external factors such as FGF and the ECM.     

Lysosomal storage of sulfated glycosaminoglycans in renal interstitial cells of rats treated with tilorone

Summary This investigation provides histochemical evidence for lysosomal storage of sulfated glycosaminoglycans (GAGs) in the interstitial cells of the renal cortex and in macrophage-like cells of the renal medullary zones of rats chronically treated with the drug tilorone. This compound is known to interfere with lysosomal degradation of sulfated GAGs; therefore cells that develop GAG-storage can be assumed to be involved in the turnover of GAGs. In view of this consideration, the most remarkable and still unexplained finding was that the intrinsic interstitial cells in the papilla, which is known to be particularly rich in sulfated GAGs, did not show the cytological symptoms of lysosomal GAG-storage. The present findings may stimulate further studies focused on the cellular sites of turnover of the sulfated GAGs present in the renal medullary interstitium.